DR1: Am J Cardiovasc Drugs. 2001;1(6):411-20. Pluripotential mechanisms of cardioprotection with HMG-CoA reductase inhibitor therapy. Rosenson RS. Preventive Cardiology Center, Division of Cardiology, Department of Medicine and Preventive Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA. r-rosenson@northwestern.edu Treatment with hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been accompanied by a reduced risk of cardiovascular events. Rapid onset of clinical benefit and weak correlations between plasma low density lipoprotein-cholesterol levels and coronary lumen change or cardiovascular events indicates that nonlipid mechanisms are involved in this beneficial effects with HMG-CoA reductase inhibitors. Furthermore, more rapid onset of clinical benefit with HMG-CoA reductase inhibitors in patients with acute coronary syndromes or acute myocardial infarction than in those with stable coronary heart disease suggest that HMG-CoA reductase inhibitors facilitate repair of ruptured or ulcerated atherosclerotic plaque, facilitate plaque stabilization and/or reduce thrombus formation on ruptured plaques. Treatment with HMG-CoA reductase inhibitors improved endothelial dysfunction in patients with hypercholesterolemia and this improvement in endothelial function was not correlated with reduction in total serum cholesterol levels. Similarly, reduction in endothelial pre-proendothelin mRNA expression and endothelin synthesis and blood pressure lowering with HMG-CoA reductase inhibitors occurred independent of lipid-lowering. Finally, HMG-CoA reductase inhibitors increased endothelial nitric oxide levels i.e. upregulated endothelial nitric oxide synthetase expression via post-transcriptional mechanisms and prevented its down-regulation by oxidized LDL-C. HMG-CoA reductase inhibitors have been shown to modulate the immune response by inhibiting activation of immune-competent cells such as macrophages, and antigen presentation to macrophages by T cells. Treatment with HMG-CoA reductase inhibitors can reduce expression, production and circulating levels of chemokines (monocyte chemoattractant protein-1) and proinflammatory cytokines [tumor necrosis factoralpha, interleukin (IL)-6 and IL-1beta]. HMG-CoA reductase inhibitors reduced inflammation in human atheroma: significantly fewer macrophages and T cells, less oxidized LDL-C and higher collagen content. In addition, treatment with HMG-CoA reductase inhibitor led to decreased cell death within the atheroma. Treatment with these agents also reduced expression of inducible cellular adhesion molecules, decreased secretion of metalloproteinases by macrophages, reduced vascular smooth muscle cell apoptosis. Lastly, HMG-CoA reductase inhibitors appear to have important effects on the thrombogenesis: reduced expression of tissue factor production and activity; increased production of tissue factor package inhibitor; decreased platelet thrombus formation and improved fibrinolysis as a result of lowered plasminogen activator inhibitor-1 levels. As the pluripotential cardioprotective mechanisms of HMG-CoA reductase inhibitors are further elucidated, it is envisaged that treatment with HMG-CoA reductase inhibitors will be initiated earlier and more frequently in patients with hypercholesterolemia. Publication Types: Review Review, Tutorial PMID: 14728000 [PubMed - indexed for MEDLINE] PR2: FASEB J. 2004 Jan;18(1):140-2. Epub 2003 Nov 20. Lovastatin inhibits Rho-regulated expression of E-selectin by TNFalpha and attenuates tumor cell adhesion. Nubel T, Dippold W, Kleinert H, Kaina B, Fritz G. University of Mainz, Institute of Toxicology, Division of Applied Toxicology, Mainz, Germany. E-selectin mediated cell-cell adhesion plays an important role in inflammatory processes and extravasation of tumor cells. Tumor necrosis factor-alpha (TNF-alpha) induces E-selectin gene and protein expression in primary human endothelial cells (HUVEC) and in an endothelial cell line (EA.hy-926). As shown by ELISA and FACS analyses, HMG-CoA reductase inhibitors (e.g., lovastatin) impair the TNF-alpha stimulated increase in E-selectin protein expression. Similar results were obtained for E-selectin mRNA expression and promoter activity, indicating that the effect of lovastatin is based on inhibition of gene expression. The effective inhibitory concentration of lovastatin was in a physiologically relevant range (IC50<0.1 microM). Lovastatin-mediated block of TNF-alpha induced E-selectin expression is due to inhibition of protein geranylgeranylation rather than farnesylation. Down-regulation of Rho signaling by coexpression of dominant-negative Rho mutants (i.e RhoA, RhoB and Rac) impaired TNF-alpha driven E-selectin gene expression, indicating Rho signaling to be essential for transcriptional activation of the E-selectin gene. Inhibition of E-selectin expression by lovastatin gives rise to a significant reduction in TNF-alpha stimulated adhesion of colon carcinoma cells to HUVEC. Furthermore, low concentration of lovastatin (i.e., < or =1 microM) attenuated TNF-alpha induced tumor cell invasion in vitro. The data support the view that statins might be clinically useful in protection against E-selectin mediated metastasis. PMID: 14630701 [PubMed - indexed for MEDLINE] NR3: J Environ Pathol Toxicol Oncol. 2003;22(2):75-92. Molecular regulation of cholesterol biosynthesis: implications in carcinogenesis. Singh RP, Kumar R, Kapur N. Department of Biotechnology, Indian Institute of Technology, Roorkee, India. rpsbsfbs@rurkiu.ernet.in Cholesterol synthesis was demonstrated to be mandatory for cellular growth and serves to supply one of the necessary building blocks for new membranes demanded by dividing cells during growth. The mevalonate pathway, which is regulated through a finely tuned mechanism, is responsible mainly for cholesterol enrichment to cells. Among the various steps, the production of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is the most critically regulated step catalyzed by HMG-CoA reductase. The ability of sterols to regulate both the transcriptional rates of the reductase gene and the degradative machinery for the reductase protein provides a multilevel system for controlling the expression of this enzyme. Much convincing evidence indicates that cells manifest a higher flux through the mevalonate pathway when proliferating than when they are in the cell cycle arrest condition; furthermore, tumors undergo deregulated cholesterogenesis mainly at the critical rate-controlling juncture (i.e., the reaction catalyzed by HMG-CoA reductase). The mevalonate component of the cholesterol biosynthesis plays a key role in controlling cell proliferation by generating prenyl intermediates, particularly farnesyl and geranyl-geranyl moieties. These isoprenoids covalently modify and thus modulate the biological activity of signal transducing proteins, such as that of the Ras superfamily. The prenylated Ras-mediated signal transduction pathway provides much of the molecular information needed to trigger cell proliferation. Therefore, depletion of mevalonate can block both the processing and the transforming activities of Ras, indicating that drugs such as lovastatin and compactin, which had previously been exploited for lowering cholesterol levels, may be useful chemotherapeutic agents for treating tumors harboring oncogenic Ras mutation. In addition, Ras prenylation, which provides much of the molecular information needed to trigger cell proliferation, represents an inviting target for the design of chemotherapeutic drugs that would interrupt such signaling events and arrest tumor cell proliferation. Publication Types: Review Review, Tutorial PMID: 14533871 [PubMed - indexed for MEDLINE] PR4: Development. 2003 Dec;130(23):5609-24. Epub 2003 Oct 1. The beta-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation. Zhang C, Basta T, Jensen ED, Klymkowsky MW. Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA. In Xenopus laevis, beta-catenin-mediated dorsal axis formation can be suppressed by overexpression of the HMG-box transcription factor XSOX3. Mutational analysis indicates that this effect is due not to the binding of XSOX3 to beta-catenin nor to its competition with beta-catenin-regulated TCF-type transcription factors for specific DNA binding sites, but rather to SOX3 binding to sites within the promoter of the early VegT- and beta-catenin-regulated dorsal-mesoderm-inducing gene Xnr5. Although B1-type SOX proteins, such as XSOX3, are commonly thought to act as transcriptional activators, XSOX3 acts as a transcriptional repressor of Xnr5 in both the intact embryo and animal caps injected with VegT RNA. Expression of a chimeric polypeptide composed of XSOX3 and a VP16 transcriptional activation domain or morpholino-induced decrease in endogenous XSOX3 polypeptide levels lead to an increase in Xnr5 expression, as does injection of an anti-XSOX3 antibody that inhibits XSOX3 DNA binding. These observations indicate that maternal XSOX3 acts in a novel manner to restrict Xnr5 expression to the vegetal hemisphere. PMID: 14522872 [PubMed - indexed for MEDLINE] PR5: Gene. 2003 Jul 17;312:173-9. Pneumocystis carinii STE11, an HMG-box protein, is phosphorylated by the mitogen activated protein kinase PCM. Vohra PK, Puri V, Kottom TJ, Limper AH, Thomas CF Jr. Thoracic Diseases Research Unit, 826 Stabile Building, Division of Pulmonary, Critical Care and Internal Medicine, Department of Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA. A pheromone-induced mitogen activated protein kinase (MAPK) pathway controls mating in fungi by regulating gene transcription. In the opportunistic fungus Pneumocystis carinii, we have identified a protein containing a high-mobility group (HMG) motif which is homologous to the transcriptional activators STE11 of Schizosaccharomyces pombe and STE12 of Saccharomyces cerevisiae. In fungi, this transcriptional activator functions in sexual development, filamentous growth, and pathogenicity. The fungal pheromone-activated MAPK phosphorylates the transcriptional activator to allow binding to pheromone-response elements in the promoter regions of certain genes. We have previously identified a P. carinii MAPK, PCM, which has significant homology to fungal MAPKs involved in mating. As an initial step in understanding the downstream molecules which interact with the PCM kinase, we have cloned a STE11 homologue in P. carinii. PCSTE11 has an open-reading frame of 1.5 kb which encodes a protein of 501 amino acids with a molecular weight of 56 kDa. Greatest homology was to S. pombe STE11 (52%). We have expressed a His-tag fusion of PCSTE11 and purified the protein with nickel affinity resin. PCM phosphorylates the purified protein indicating that PCSTE11 is associated with the MAPK cascade in P. carinii. PMID: 12909353 [PubMed - indexed for MEDLINE] DR6: Yeast. 2003 Jul 30;20(10):881-92. Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis. Wright R, Parrish ML, Cadera E, Larson L, Matson CK, Garrett-Engele P, Armour C, Lum PY, Shoemaker DD. University of Minnesota, Department of Genetics, Cell Biology and Development, 321 Church Street, 6-160 Jackson Hall, Minneapolis, MN 55455, USA. wrightr@umn.edu Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd. PMID: 12868057 [PubMed - indexed for MEDLINE] DR7: J Biomed Sci. 2003 Jul-Aug;10(4):396-405. HMG-CoA reductase inhibitors inhibit inducible nitric oxide synthase gene expression in macrophages. Huang KC, Chen CW, Chen JC, Lin WW. Department of Family Medicine, National Taiwan University Hospital, Taipei, Taiwan, ROC. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are potent inhibitors of cholesterol synthesis and have wide therapeutic use in cardiovascular diseases. Recent evidence, however, suggests that the beneficial effects of statins may extend beyond their action on serum cholesterol levels. In this study, we investigated the effects of lovastatin, pravastatin, atorvastatin and fluvastatin on macrophage formation of nitric oxide (NO) in murine RAW 264.7 cells. Stimulation of macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) resulted in inducible NO synthase (iNOS) expression, which was accompanied by a large amount of NO formation. At concentrations of 0.1-30 microM, statins can inhibit stimuli-induced NO formation and iNOS induction to different extents. This inhibition occurs at the transcriptional level, and displays potency in the order of lovastatin > atorvastatin > fluvastatin >> pravastatin. We found that LPS-induced I kappa B kinase and nuclear factor-kappa B (NF-kappa B) activation, as well as IFN-gamma-induced signal transducer and activator of transcription 1 (STAT1) phosphorylation, were reduced by lovastatin. Moreover, inhibition by lovastatin of NO production and kappa B activation was reversed by mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate. All these results suggest that inhibition of iNOS gene expression by statins can be attributed to interference with protein isoprenylation, which mediates both NF-kappa B and STAT1 activation in the upstream signaling pathways for iNOS gene transcription. Copyright 2003 National Science Council, ROC and S. Karger AG, Basel PMID: 12824699 [PubMed - indexed for MEDLINE] PR8: Nucleic Acids Res. 2003 May 1;31(9):2369-80. HMG box transcription factor TCF-4's interaction with CtBP1 controls the expression of the Wnt target Axin2/Conductin in human embryonic kidney cells. Valenta T, Lukas J, Korinek V. Institute of Molecular Genetics Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. Members of the Tcf/Lef family of the HMG box transcription factors are nuclear effectors of the Wnt signal transduction pathway. Upon Wnt signaling, TCF/LEF proteins interact with beta-catenin and activate transcription of target genes, while, in the absence of the Wnt signal, TCFs function as transcriptional repressors. All vertebrate Tcf/Lef transcription factors associate with TLE/Groucho-related co-repressors, and here we provide evidence for an interaction between the C-terminus of the TCF-4 HMG box protein and the C-terminal binding protein 1 (CtBP1) transcriptional co-repressor. Using Wnt-1-stimulated human embryonic kidney 293 cells, we show that CtBP1 represses the transcriptional activity of a Tcf/beta-catenin-dependent synthetic promoter and, furthermore, decreases the expression of the endogenous Wnt target, Axin2/Conductin. The CtBP1-mediated repression was alleviated by trichostatin A treatment, indicating that the CtBP inhibitory mechanism is dependent on the activity of histone deacetylases. PMID: 12711682 [PubMed - indexed for MEDLINE] NR9: Dev Biol. 2003 May 1;257(1):1-13. Pax6 autoregulation mediated by direct interaction of Pax6 protein with the head surface ectoderm-specific enhancer of the mouse Pax6 gene. Aota S, Nakajima N, Sakamoto R, Watanabe S, Ibaraki N, Okazaki K. Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, 565-0874, Suita, Osaka, Japan. aota@beri.or.jp The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. We identified a Pax6 protein-responsive element termed LE9 (52 bp in length) within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein formed in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activated LE9 synergistically with Pax6. A scanning mutagenesis experiment indicated that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins formed a complex when they were expressed together. Based on these findings, we propose a model in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein. PMID: 12710953 [PubMed - indexed for MEDLINE] PR10: Mol Endocrinol. 2003 Jul;17(7):1332-43. Epub 2003 Apr 3. Induction of the Sry-related factor SOX6 contributes to bone morphogenetic protein-2-induced chondroblastic differentiation of C3H10T1/2 cells. Fernandez-Lloris R, Vinals F, Lopez-Rovira T, Harley V, Bartrons R, Rosa JL, Ventura F. Departament de Ciencies Fisiologiques II, Campus de Bellvitge, Universitat de Barcelona, 08907 L' Hospitalet de Llobregat, Spain. Chondrogenesis leads to the formation of mature cartilage and generates initial skeletal elements that serve as templates for endochondral bone formation. Bone morphogenetic proteins (BMPs) are involved in several developmental and organogenetic processes and have been identified as key regulators in chondrogenesis. In the present study we sought to determine the transcriptional mechanisms contributing to the induction of chondrogenic markers by BMP-2. Time-course studies with BMP-2-stimulated C3H10T1/2 cells showed a dose-dependent appearance of Alcian-blue-positive material and up-regulated expression of type-II collagen mRNA. This last effect required new protein synthesis because addition of cycloheximide completely blocked the induction of type-II collagen mRNA. A region encompassing the chondrocyte-specific enhancer, localized in intron I of type-II collagen alpha1 chain (Col2a1) gene, is sufficient to confer BMP-2-dependent transcriptional induction of type-II collagen gene expression. Analysis of the expression levels of chondrogenic Sry-type high-mobility group (HMG) box proteins (SOX) transcription factors demonstrated a time-dependent induction of Sox6 expression by BMP-2 that correlated with the appearance of BMP-2- induced protein complexes bound to the chondrocyte-specific enhancer. Preincubation of nuclear extracts with SOX6 and SOX9 antibodies markedly reduced the intensity of these bands. Forced expression of SOX6 mimicked the BMP-2 effect, whereas coexpression of SOX9 promoted a synergistic interaction between both factors in transcription from the chondrocyte-specific enhancer. Moreover, overexpression of a SOX6 mutated form, devoid of its high-mobility group domain, was sufficient to prevent transcriptional induction of the chondrocyte-specific enhancer by BMP-2. Taken together, these results indicate that SOX6 is an important downstream mediator of BMP-2 signaling in chondrogenesis. PMID: 12677004 [PubMed - indexed for MEDLINE] DR11: Nucleic Acids Res. 2003 Mar 15;31(6):1693-703. Histone deacetylase inhibitors stimulate mitochondrial HMG-CoA synthase gene expression via a promoter proximal Sp1 site. Camarero N, Nadal A, Barrero MJ, Haro D, Marrero PF. Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain. The expression of mitochondrial HMG-CoA synthase in the colon has been correlated with the levels of butyrate present in this tissue. We report here that the effect of butyrate on mitochondrial HMG-CoA synthase gene expression is exerted in vivo at the transcriptional level, and that trichostatin A (TSA), a specific histone deacetylase inhibitor, also induces transcriptional activity and mRNA expression of the gene in human cell lines derived from colon carcinoma. Using chromatin immunoprecipitation assays, we show that histone deacetylase 1 (HDAC1) is associated with the endogenous mitochondrial HMG-CoA synthase promoter and that TSA induction correlates with hyperacetylation of H4 histone associated with the 5' flanking region of the gene. Overexpression of HDAC1 activity leads consistently to mitochondrial HMG-CoA synthase promoter hypoacetylation and reduces its transcriptional activity. The effect of butyrate and TSA maps to a single Sp1 site present in the proximal promoter of the gene, which is able to bind Sp1 and Sp3 proteins. Interestingly, the binding affinity of Sp1 and Sp3 proteins to the Sp1 site correlates with the TSA responsiveness of the promoter. Using a one-hybrid system (GAL4-Sp1 and GAL4-Sp3), we show that both proteins can mediate responsiveness to TSA in CaCo-2 cells employing distinct mechanisms. PMID: 12626711 [PubMed - indexed for MEDLINE] PR12: Eur J Pharmacol. 2003 Feb 21;462(1-3):15-23. Atorvastatin inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells. Bruemmer D, Yin F, Liu J, Kiyono T, Fleck E, Van Herle A, Graf K, Law RE. Division of Endocrinology, Diabetes and Hypertension and The Gonda (Goldschmied) Diabetes Center, David Geffen School of Medicine, University of California, Warren Hall, Suite 24-130, 900 Veteran Avenue, Los Angeles, CA 90095, USA. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to inhibit vascular smooth muscle cell growth, a key event in the pathogenesis of proliferative vascular diseases. The mechanism by which HMG-CoA reductase inhibitors exert their antiproliferative activity is not fully understood, especially their effect on DNA replication. We therefore investigated the effects of atorvastatin on minichromosome maintenance (MCM) protein 6 and 7 expression in vascular smooth muscle cells, two proteins essential for initiation of DNA replication. Stimulation of quiescent rat aortic vascular smooth muscle cells with fetal bovine serum induced MCM6 and MCM7 protein and mRNA expression, which was potently attenuated by atorvastatin in a dose-dependent fashion. Mevalonate completely abrogated the inhibitory effect on serum-induced MCM6 and MCM7 expression, demonstrating that biosynthesis of isoprenoids was likely the specific pathway blocked by atorvastatin. Transient transfection experiments revealed that atorvastatin inhibited MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. The MCM6 and MCM7 promoters contain several E2F sites critical for their transcriptional activation. Activity of a luciferase reporter plasmid containing four E2F elements was also strongly inhibited by atorvastatin. The inhibitory effect of atorvastatin on MCM6 and MCM7 was reversed by adenoviral-mediated overexpression of E2F, indicating that their downregulation by atorvastatin involves an E2F-dependent mechanism. These findings demonstrate that MCM proteins play an essential role during the proliferation of vascular smooth muscle cells and may provide a novel therapeutic target for proliferative vascular diseases. Inhibition of MCM6 and MCM7 expression by blocking E2F function may contribute importantly to the inhibition of vascular smooth muscle cell DNA synthesis by atorvastatin. PMID: 12591091 [PubMed - indexed for MEDLINE] PR13: Zoolog Sci. 2003 Jan;20(1):59-67. Expression pattern and transcriptional control of SoxB1 in embryos of the ascidian Halocynthia roretzi. Miya T, Nishida H. Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. The Sox family is a large group of transcription factors that are characterized by the presence of a DNA-binding HMG domain. We isolated HrSoxB1, an ascidian homolog of the Sox gene that belongs to the B1 subclass of the Sox family, from Halocynthia roretzi. Expression was initiated as early as the 8-cell stage. During cleavage stages, HrSoxB1 was expressed in three quarters of embryonic blastomeres but not in posterior-vegetal (B-line) blastomeres. Misexpression of mRNAs of HrPEM but not of macho-1, whose maternal mRNAs are localized to the posterior-vegetal cytoplasm of eggs and early embryos, repressed the anterior-vegetal expression of HrSoxB1. This result suggests that the zygotic expression of HrSoxB1 is controlled by the localized maternal mRNA. When HrSoxB1 was overexpressed in early embryos, ectopic expression of HrBra, a gene for a transcription factor expressed in notochord blastomeres, occurred in the most posterior blastomeres (B7.5), although these blastomeres did not eventually differentiate into notochord but developed into muscle, as they do in normal embryogenesis. In later embryogenesis, HrSoxB1 was specifically expressed in neural plate cells. However, overexpression of HrSoxB1 did not affect the expression of a neural plate marker gene, HrETR-1. PMID: 12560602 [PubMed - indexed for MEDLINE] NR14: Development. 2003 Mar;130(5):941-53. Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control. Ghislain J, Desmarquet-Trin-Dinh C, Gilardi-Hebenstreit P, Charnay P, Frain M. Unite 368 de l'Institut National de la Sante et de la Recherche Medicale, Ecole Normale Superieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France. Neural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. We have investigated the basis of the specific neural crest expression of Krox20 and identified a cis-acting enhancer element (NCE) located 26 kb upstream of the gene that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins and which responded to factors expressed throughout the neural crest. Consistent with this the NCE was strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevented the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organisation also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning. PMID: 12538520 [PubMed - indexed for MEDLINE] PR15: Arterioscler Thromb Vasc Biol. 2003 Jan 1;23(1):58-63. HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells. Dichtl W, Dulak J, Frick M, Alber HF, Schwarzacher SP, Ares MP, Nilsson J, Pachinger O, Weidinger F. Department of Internal Medicine, Division of Cardiology, Leopold-Franzens-University Innsbruck, Austria. wolfgang.dichtl@uibk.ac.at OBJECTIVE: Pleiotropic atheroprotective effects of HMG-CoA reductase inhibitors may be mediated on the level of vascular gene transcription. The aim of this study was to characterize the effects of statins on the activation of transcription factors known to regulate inflammation and cell proliferation/differentiation. METHODS AND RESULTS: Simvastatin, atorvastatin, and lovastatin (0.1 to 10 micro mol/L) inhibited the binding of nuclear proteins to both the nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) DNA consensus oligonucleotides in human endothelial and vascular smooth muscle cells as assessed by electrophoretic mobility shift assay (EMSA). The inhibitory effects of statins on NF-kappaB or AP-1-dependent transcriptional activity were examined by transient transfection studies. HMG-CoA reductase inhibitors upregulated IkappaB-alpha protein levels in endothelial cells and decreased c-Jun mRNA expression in smooth muscle cells as analyzed by Western and Northern blotting, respectively. Furthermore, statins inhibited DNA binding of hypoxia-inducible factor-1alpha. Downstream effects of statins included inhibition of plasminogen activator inhibitor-1 and vascular endothelial growth factor-A mRNA levels in endothelial cells. CONCLUSIONS: HMG-CoA reductase inhibitors downregulate the activation of transcription factors NF-kappaB, AP-1, and hypoxia-inducible factor-1alpha. These findings support the concept that statins have antiinflammatory and antiproliferative effects that are relevant in the treatment of atherosclerotic diseases. PMID: 12524225 [PubMed - indexed for MEDLINE] PR16: J Biol Chem. 2003 Feb 7;278(6):3742-50. Epub 2002 Nov 22. Mouse mu opioid receptor distal promoter transcriptional regulation by SOX proteins. Hwang CK, Wu X, Wang G, Kim CS, Loh HH. Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA. hwang025@umn.edu We have identified transcription factors that bind to specific sequences in 5'-distal promoter regulatory sequences of the mouse mu opioid receptor (mor) promoter using the yeast one-hybrid system. The sequence between -746 and -707 in mor distal promoter was used as the bait because it acts as a functional promoter element and binds several DNA-binding proteins. From an adult mouse brain cDNA library, five cDNA clones encoding three Sox gene family (Sry like high mobility group (HMG) box gene) transcriptional factors, mSOX18, mSOX21, and mSOX6, were isolated. Electrophoretic mobility shift assays confirmed the presence of a binding site for SOX proteins in the -731/-725 region. Additionally, we have also established that the flanking regions outside the core Sox-binding site play an essential role in high affinity binding. DNase I footprint analysis indicates that proteins from mouse brain interact with the Sox-binding site within the mor distal promoter. Finally, we demonstrated that overexpression of mSOX18 and/or mSOX21 was able to up-regulate mouse mor distal promoter activity in mor-expressing neuronal cells (NMB). These data indicate that SOX proteins might contribute to the transcriptional activity of the mor gene and suggest that mu opioid receptor could mediate some of the developmental processes in which SOX proteins are included. PMID: 12446692 [PubMed - indexed for MEDLINE] NR17: Int J Cardiol. 2002 Dec;86(2-3):153-68. New insights in the transcriptional activity and coregulator molecules in the arterial wall. de Nigris F, Lerman LO, Napoli C. Department of Medicine, University of Naples, PO Box 80131, Italy. A number of vascular diseases are associated with abnormal expression of genes that contribute to their pathophysiological and clinical manifestations, but at the same time offer potential therapeutic targets. One of the promising therapeutic approaches targets the pathophysiological pathways leading to aberrant gene activation, namely transcriptional activity and its molecular modulators (agonists, antagonists, coregulators, and nuclear receptors). The transcription factors can be divided into four classes (I-IV) classified by structural elements, like basic leucine zipper (bZIP) or basic helix-loop-helix (bHLH), which mediate their DNA binding activity but also determine the classes of drugs that can affect their activity. For example, statins modulate activation of the class-I transcription factor sterol responsive element-binding protein (SREBP), whose target genes including hydroxyl-methyl-glutaryl acetyl Coenzyme-A (HMG-CoA) reductase, HMG-CoA synthase, and the low-density lipoprotein receptor, all of which are involved in cholesterol and fatty acid metabolism. Similarly, insulin-like drugs target the nuclear receptor peroxisome-proliferator-activator-receptor (PPAR)-gamma (class-II), several anti-inflammatory drugs inhibit activation of nuclear factor kappa B (NFkappaB) (class-IV), while others (e.g. flavopiridol, rapamycin, and paclitaxel) target regulation of cell-cycle proteins. Increased understanding of the genetic and molecular basis of disease (e.g. transcriptional activity and its coregulation) will potentially enhance future diagnosis, treatment, and prevention of vascular diseases. Publication Types: Review PMID: 12419552 [PubMed - indexed for MEDLINE] NR18: Dev Biol. 2002 Sep 15;249(2):191-203. Molecular regulation of vertebrate early endoderm development. Shivdasani RA. Department of Adult Oncology and Cancer Biology, Dana-Faber Cancer Institute, Boston, MA 02115, USA. ramesh_shivdasani@dcfi.harvard.edu Detailed study of the ectoderm and mesoderm has led to increasingly refined understanding of molecular mechanisms that operate early in development to generate cellular diversity. More recently, a number of powerful studies have begun to characterize the molecular determinants of the endoderm, a germ layer previously neglected in developmental biology. Work in diverse model systems has converged on an integrated transcriptional and signaling pathway that serves to establish the vertebrate endoderm. A T-box transcription factor identified in Xenopus embryos, VegT, appears to function near the top of an endoderm-specifying transcriptional hierarchy. VegT activates and reinforces Nodal-related TGFbeta signaling and also induces expression of essential downstream transcriptional regulators, Mix-like paired-homeodomain and GATA factors. These proteins cooperate to regulate expression of a relay of HMG-box Sox-family transcription factors culminating with Sox 17, which may be an obligate mediator of vertebrate endoderm development. This review synthesizes findings in three vertebrate model organisms and discusses these genetic interactions in the context of the progressive acquisition of endodermal identity early in vertebrate development. Publication Types: Review PMID: 12221001 [PubMed - indexed for MEDLINE] PR19: Eur J Pharmacol. 2002 Jul 19;448(2-3):113-21. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase the binding activity and nuclear level of Oct-1 in mononuclear cells. Ortego M, Hernandez AG, Bustos C, Blanco-Colio LM, Hernandez-Presa MA, Tunon J, Egido J. Vascular Research Laboratory, Fundacion Jimenez Diaz, Avda Reyes Catolicos 2, 28040 Madrid, Spain. mortego@fjd.es 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are drugs very effective to decrease low-density lipoprotein (LDL) cholesterol. In addition, a number of studies suggest that statins have other beneficial clinical effects beyond cholesterol lowering. We recently reported that statins decrease nuclear factor kappa B (NF-kappaB) binding activity in monocytes and vascular smooth muscle cells. We now explored the effect of two different statins, simvastatin and atorvastatin, in the activation of the octamer transcription factor Oct-1 on the monocytic cell line THP-1. Oct-1 is a nuclear factor that represses the transcription of proinflammatory genes such as interleukin-8, CD11c/CD18, vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1). Low concentrations of both statins increased Oct-1 DNA binding activity (electrophoretic mobility shift assay) that was resolved into two specific bands. The upper one was supershifted by preincubation of nuclear extracts with anti-Oct-1 antibody. The lower one was supershifted by preincubation of nuclear extracts with an anti-Oct-2 antibody, also partially competed with 100 mol/l excess of cold activator protein-1 (AP-1) and attenuated by anti-c-Jun antibody. Both statins increased Oct-1 and Oct-2 nuclear protein levels (Western blot). In contrast, neither had any effect on PMA-differentiated cells, suggesting a distinct sensitivity between circulating monocytes and resident tissular macrophages. In addition, statins did not increase Oct-lipoprotein lipase binding activity that contains an Oct-1 binding element. The mRNA expression of interleukin-8, a chemokine containing Oct sites in its promoter, was diminished by statin pretreatment. Our results indicate that simvastatin and atorvastatin increase the activity of the transcriptional repressor Oct-1 in mononuclear cells, and could thus contribute to decrease the activation of these cells. These data suggest a possible novel mechanism supporting a certain anti-inflammatory effect of these two 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors. Copyright 2002 Elsevier Science B.V. PMID: 12144930 [PubMed - indexed for MEDLINE] DR20: Annu Rev Cell Dev Biol. 2002;18:345-78. Epub 2002 Apr 2. Proteolysis and sterol regulation. Hampton RY. Section of Cell and Developmental Biology, Division of Biology, University of California, San Diego, La Jolla 92093-0347, USA. rhampton@ucsd.edu The mammalian cell continuously adjusts its sterol content by regulating levels of key sterol synthetic enzymes and levels of LDL receptors that mediate uptake of cholesterol-laden particles. Control is brought about by sterol-regulated transcription of relevant genes and by regulated degradation of the committed step enzyme HMG-CoA reductase (HMGR). Current work has revealed that proteolysis is at the heart of each of these mechanistically distinct axes. Transcriptional control is effected by regulated cleavage of the membrane-bound transcription factor sterol regulatory element binding protein (SREBP), and HMGR degradation is brought about by ubiquitin-mediated degradation. In each case, ongoing cell biological processes are being harnessed to bring about regulation. The secretory pathway plays a central role in allowing sterol-mediated control of transcription. The constitutively active endoplasmic reticulum (ER) quality control apparatus is employed to bring about regulated destruction of HMGR. This review describes the methods and results of various studies to understand the mechanisms and molecules involved in these distinct but interrelated aspects of sterol regulation and the intriguing similarities that appear to exist at the levels of protein sequence and cell biology. Publication Types: Review PMID: 12142284 [PubMed - indexed for MEDLINE] NR21: Dev Biol. 2002 Aug 1;248(1):128-42. Dynamics of a developmental switch: recursive intracellular and intranuclear redistribution of Caenorhabditis elegans POP-1 parallels Wnt-inhibited transcriptional repression. Maduro MF, Lin R, Rothman JH. Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA. POP-1, a Tcf/Lef factor, functions throughout Caenorhabditis elegans development as a Wnt-dependent reiterative switch to generate nonequivalent sister cells that are born by anterior-posterior cell divisions. We have observed the interaction between POP-1 and a target gene that it represses as it responds to Wnt signaling. Dynamic observations in living embryos reveal that POP-1 undergoes Wnt-dependent nucleocytoplasmic redistribution immediately following cytokinesis, explaining the differential nuclear POP-1 levels in nonequivalent sister cells. In unsignaled (anterior) but not Wnt-signaled (posterior) sister cells, POP-1 progressively coalesces into subnuclear domains during interphase, coincident with its action as a repressor. While the asymmetric distribution of POP-1 in nonequivalent sisters apparently requires a 124-amino-acid internal domain, neither the HMG box nor beta-catenin interaction domains are required. We find that a transcriptional activator, MED-1, associates in vivo with the end-1 and end-3 target genes in the mesoderm (anterior sister) and in the endoderm (posterior sister) following the asymmetric cell division that subdivides the mesendoderm. However, in the anterior sister, binding of POP-1 to the end-1 and end-3 genes blocks their expression. In vivo, binding of POP-1 to the end-1 and end-3 targets (in the posterior sister) is blocked by Wnt/MAPK signaling. Thus, a Tcf/Lef factor represses transactivation of genes in an unsignaled daughter cell by abrogating the function of a bound activator. PMID: 12142026 [PubMed - indexed for MEDLINE] NR22: Atherosclerosis. 2002 Sep;164(1):15-26. Erratum in: Atherosclerosis. 2003 Jun;167(2):371. Sterol-regulatory element-binding protein (SREBP)-2 contributes to polygenic hypercholesterolaemia. Miserez AR, Muller PY, Barella L, Barella S, Staehelin HB, Leitersdorf E, Kark JD, Friedlander Y. Cardiovascular Genetics, Institute of Biochemistry and Genetics, Department of Clinical-Biological Sciences, University of Basel, Vesalgasse 1, Ch-4051, Basel, Switzerland. andre-r.miserez@unibas.ch Sterol-regulatory element-binding protein (SREBP)-2 is a key regulator of cholesterol. When cells are deprived of cholesterol, proteolytic cleavage releases the NH(2)-terminal domain of SREBP-2 that binds and activates the promoters of SREBP-2-regulated genes including the genes encoding the low-density lipoprotein (LDL) receptor, 3-hydroxymethyl-3-glutaryl-(HMG-)CoA-synthase, and HMG-CoA-reductase. Thus, SREPB-2 gene activation leads to enhanced cholesterol uptake and biosynthesis. A novel protein polymorphism (SREBP-2-595A/G) discovered in the regulatory domain of human SREBP-2 was investigated regarding its impact on cholesterol homeostasis. In human embryonic kidney (HEK)-293-cells, the cleavage-rate of the SREBP-2-595A-isoform was slightly decreased compared to that of the SREBP-2-595G-isoform. Since cleavage of SREBP-2 activates the LDL receptor-mediated uptake of plasma cholesterol, we hypothesized the LDL receptor-mediated uptake to be decreased in homozygous SREBP-2-595A-carriers and thus, plasma total cholesterol (TC) to be higher than in SREBP-2-595G-carriers. Multiple linear regression analysis of population samples from Switzerland (N=1334) and Israel (N=923) demonstrated a significant positive, gene dose-dependent association of the SREBP-2-595A-isoform with higher plasma TC (P=0.001). This cholesterol-modulating effect was present in hypercholesterolaemic (DeltaTC=1.05 mmol/l, 14.4%; P=0.002; N=477), but absent in normocholesterolaemic subjects (DeltaTC=0.06 mmol/l, 1.4%; P=0.334; N=1780). In summary, a slightly but constantly decreased cleavage-rate of the SREBP-2-595A-isoform compared to that of the SREBP-2-595G-isoform may lead to a reduced transcriptional activation of the LDL receptor-gene weakening the SREBP-mediated compensation mechanisms, and may, therefore, be a critical factor in the development of polygenic hypercholesterolaemia. PMID: 12119189 [PubMed - indexed for MEDLINE] DR23: J Biol Chem. 2002 Sep 6;277(36):32438-44. Epub 2002 Jun 13. Specificity of the stimulatory interaction between chromosomal HMGB proteins and the transcription factor Dof2 and its negative regulation by protein kinase CK2-mediated phosphorylation. Krohn NM, Yanagisawa S, Grasser KD. Department of Biotechnology, Institute of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that can contribute to transcriptional control by interaction with certain transcription factors. Using the transcription factor Dof2 and five different maize HMGB proteins, we have examined the specificity of the HMGB-transcription factor interaction. The HMG-box DNA binding domain of HMGB1 is sufficient for the interaction with Dof2. Although all tested HMGB proteins can interact with Dof2, the various HMGB proteins stimulate the binding of Dof2 to its DNA target site with different efficiencies. The HMGB5 protein is clearly the most potent facilitator of Dof2 DNA binding. Maximal stimulation of the DNA binding by the HMGB proteins requires association of HMGB and Dof2 prior to DNA binding. HMGB5 and Dof2 form a ternary complex with the DNA, but within the protein-DNA complex the interaction of HMGB5 and Dof2 is different from that in solution, as in contrast to the proteins in solution, they cannot be cross-linked with glutaraldehyde when bound to DNA. Phosphorylation of HMGB1 by protein kinase CK2 abolishes the interaction with Dof2 and the stimulation of Dof2 DNA binding. These findings indicate that transcription factors may recruit certain members of the HMGB family as assistant factors. PMID: 12065590 [PubMed - indexed for MEDLINE] DR24: Proc Natl Acad Sci U S A. 2002 May 28;99(11):7420-5. A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes. Hakimi MA, Bochar DA, Chenoweth J, Lane WS, Mandel G, Shiekhattar R. The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA. BRAF35, a structural DNA-binding protein, initially was identified as a component of a large BRCA2-containing complex. Biochemical analysis revealed the presence of a smaller core-BRAF35 complex devoid of BRCA2. Here we report the isolation of a six-subunit core-BRAF35 complex with the capacity to deacetylate histones, termed the BRAF-histone deacetylase complex (BHC), from human cells. BHC contains polypeptides reminiscent of the chromatin-remodeling complexes SWI/SNF and NuRD (nucleosome remodeling and deacetylating). Similar to NuRD, BHC contains an Mi2-like subunit, BHC80, and a PHD zinc-finger subunit as well as histone deacetylases 1/2 and an MTA-like subunit, the transcriptional corepressor CoREST. We show that BHC mediates repression of neuron-specific genes through the cis-regulatory element known as the repressor element 1 or neural restrictive silencer (RE1/NRS). Chromatin-immunoprecipitation experiments demonstrate the recruitment of BHC by the neuronal repressor REST. Expression of BRAF35 containing a single point mutation in the HMG domain of the protein abrogated REST-mediated transcriptional repression. These results demonstrate a role for core-BRAF35-containing complex in the regulation of neuron-specific genes through modulation of the chromatin structure. PMID: 12032298 [PubMed - indexed for MEDLINE] NR25: J Biol Chem. 2002 Jul 26;277(30):26804-7. Epub 2002 May 13. Differences in the regulation of the classical and the alternative pathway for bile acid synthesis in human liver. No coordinate regulation of CYP7A1 and CYP27A1. Bjorkhem I, Araya Z, Rudling M, Angelin B, Einarsson C, Wikvall K. Department of Pharmaceutical Biosciences, Division of Biochemistry, University of Uppsala, Box 578, S-751 23 Uppsala, Sweden. It has been reported that there is a coordinate regulation of sterol 27-hydroxylase (CYP27A1) and cholesterol 7alpha-hydroxylase (CYP7A1) in rats. Thus, the levels of the mRNA corresponding to these two enzymes were found to change in the same direction in rat liver and in isolated rat hepatocytes. In contrast, other groups have not seen such regulation of CYP27A1 in rabbit liver or in rat liver when using an activity assay. In the present work, the effect of bile acid treatment on human CYP27A1/luciferase reporter activity was studied in a transient transfection assay in human liver-derived HepG2 cells. Neither the endogenous 27-hydroxylase activity nor the CYP27A1/luciferase reporter activity were down-regulated by treatment of HepG2 cells with chenodeoxycholic acid or taurochenodeoxycholic acid. We also measured CYP27A1 mRNA and CYP7A1 mRNA in liver of humans subjected to treatment with chenodeoxycholic acid, ursodeoxycholic acid, hydroxymethylglutaryl (HMG)-CoA reductase inhibitor and a combination of HMG-CoA reductase inhibitor and cholestyramine. There was a 60-fold variation in the levels of CYP7A1 mRNA but only a 5-fold variation in the levels of CYP27A1 mRNA. There was no correlation between the two mRNA species. It is concluded that, in humans, there is little or no coordinate regulation of CYP7A1 and CYP27A1 at the transcriptional level, and that CYP27A1 is not subject to a negative feedback control by bile acids. The results underline that marked species differences may exist in mechanisms for control of synthesis of bile acids and cholesterol homeostasis. PMID: 12011083 [PubMed - indexed for MEDLINE] DR26: Gen Physiol Biophys. 2001 Dec;20(4):401-12. HMG-1 as regulatory trans-acting protein in the acute phase-induced expression of the rat liver haptoglobin gene. Grigorov I, Milosavljevic T, Cvetkovic I, Petrovic M. Molecular Biology Laboratory, Institute for Biological Research, Belgrade, Federal Republic of Yugoslavia. iligri@ibiss.bg.ac.yu Expression of the haptoglobin (Hp) gene is liver specific and acute phase (AP) responsive. It was previously shown that transcriptional induction process of the rat Hp gene during turpentine induced AP response has been mediated by the liver nucleoprotein p29 which was shown to be homologous to the HMG-1 chromatin-associated protein. The results presented in this report offered further evidence for the existence of structural and functional similarities between these two proteins implicating an involvement of HMG-1 in the regulation of the rat Hp gene transcription. By DNA binding assays we found the HMG-1 binding sites in the rat Hp gene cis-regulatory subelements A and C and revealed an increase in its DNA-binding after induction of AP response. In view of our previous and here shown data we assume that this increase could be a consequence of AP-induced release of HMG-1 from the chromatin and subsequent increase in its nuclear amount. PMID: 11989650 [PubMed - indexed for MEDLINE] NR27: Biol Chem. 2002 Feb;383(2):255-61. TCF: Lady Justice casting the final verdict on the outcome of Wnt signalling. Brantjes H, Barker N, van Es J, Clevers H. Department of Immunology, University Hospital Utrecht, The Netherlands. The Wnt signalling cascade plays an important role during embryonic patterning and cell fate determination and is highly conserved throughout evolution. Factors of the TCF/LEF HMG domain family (Tcfs) are the downstream effectors of this signal transduction pathway. Upon Wnt signalling, a cascade is initiated that results in the translocation of beta-catenin to the nucleus, where it interacts with Tcf to generate a transcriptionally active complex. This bipartite transcription factor is targeted to the upstream regulatory regions of Tcf target genes. In the absence of Wnt signals, beta-catenin is degraded in the cytoplasm via the ubiquitin-proteasome pathway. Several proteins are instrumental in achieving this tight regulation of beta-catenin levels in the cell, including adenomatous polyposis coli (APC), GSK3 beta, and Axin/Conductin. Deregulation of the Wnt signalling pathway is implicated in several forms of cancer, such as colon carcinoma and melanoma. This deregulation is achieved via mutation of APC, beta-catenin or Axin, resulting in elevated beta-catenin levels and the presence of constitutively active Tcf-beta-catenin complexes in the nucleus. The accompanying inappropriate activation of target genes is considered to be a critical, early event in this carcinogenesis. In addition to regulating beta-catenin levels, normal healthy cells have evolved a second level of regulation, by manipulating the activity of the Tcf proteins themselves. In the absence of Wnt signalling, Tcf complexes with several transcriptional repressor proteins ensuring active repression of Tcf target genes. In this review the dual role of Tcf proteins in the Wnt signalling cascade will be discussed. Publication Types: Review Review, Tutorial PMID: 11934263 [PubMed - indexed for MEDLINE] DR28: Crit Care Med. 2002 Jan;30(1 Supp):S36-S42. High-mobility group-I/Y proteins: Potential role in the pathophysiology of critical illnesses. Carvajal IM, Baron RM, Perrella MA. Pulmonary and Critical Care Division (IMC, RMB, MAP), Brigham and Women's Hospital, Boston, MA; and the Department of Medicine (MAP), Harvard Medical School, Boston, MA 02115. High-mobility group (HMG) proteins are architectural factors that have been shown to play a role in the transcriptional regulation of various mammalian genes. One family of HMG proteins, HMG-I/Y, is known to facilitate the initiation of gene transcription by modifying the conformation of DNA and recruiting transcription factors into an organized complex on transcriptional regulatory regions of specific genes. In many circumstances, the nuclear factor-kappaB family of transcription factors is involved in gene regulation that is mediated by HMG-I/Y. We will review the mechanisms by which HMG-I/Y proteins regulate gene transcription, give an overview of selected genes regulated by HMG-I/Y, summarize the potential roles of these genes in critical illnesses, and provide more detailed information about the role of HMG-I/Y in the regulation of nitric oxide synthase-2 during an inflammatory response, such as endotoxemia/sepsis. PMID: 11891405 [PubMed - as supplied by publisher] NR29: Development. 2002 Feb;129(4):993-1002. EGFR signalling inhibits Capicua-dependent repression during specification of Drosophila wing veins. Roch F, Jimenez G, Casanova J. University Museum of Zoology, Department of Zoology, University of Cambridge, Downing Street, Cambridge, UK, CB2 3EJ. roch@cict.fr Localised activation of the Ras/Raf pathway by Epidermal Growth Factor Receptor (EGFR) signalling specifies the formation of veins in the Drosophila wing. However, little is known about how the EGFR signal regulates transcriptional responses during the vein/intervein cell fate decision. We provide evidence that EGFR signalling induces expression of vein-specific genes by inhibiting the Capicua (Cic) HMG-box repressor, a known regulator of embryonic body patterning. Lack of Cic function causes ectopic expression of EGFR targets such as argos, ventral veinless and decapentaplegic and leads to formation of extra vein tissue. In vein cells, EGFR signalling downregulates Cic protein levels in the nucleus and relieves repression of vein-specific genes, whereas intervein cells maintain high levels of Cic throughout larval and pupal development, repressing the expression of vein-specific genes and allowing intervein differentiation. However, regulation of some EGFR targets such as rhomboid appears not to be under direct control of Cic, suggesting that EGFR signalling branches out in the nucleus and controls different targets via distinct mediator factors. Our results support the idea that localised inactivation of transcriptional repressors such as Cic is a rather general mechanism for regulation of target gene expression by the Ras/Raf pathway. PMID: 11861482 [PubMed - indexed for MEDLINE] NR30: Cancer Chemother Pharmacol. 2002 Jan;49(1):35-47. Enhanced antitumor activities of TZT-1027 against TNF-alpha or IL-6 secreting Lewis lung carcinoma in vivo. Natsume T, Koh Y, Kobayashi M, Fukumoto H, Takahashi F, Nakamura T, Ohe Y, Saijo N, Nishio K. Pharmacological Research Department, Teikoku Hormone Mfg., Co. Ltd., Kawasaki-shi, Kanagawa, Japan. PURPOSE: TZT-1027, an antimicrotubule agent that inhibits the polymerization of tubulin, shows potent antitumor activity in various transplantable tumor models in vivo. The high antitumor activity of TZT-1027 prompted us to speculate that this compound may have a mode of action other than its antimicrotubule and antimitotic activities. To elucidate the interaction of antitumor cytokines with TZT-1027 in tumors in vivo, we examined the antitumor activity of this agent against various cytokine gene-transfected Lewis lung carcinoma (LLC) cells inoculated into C57BL/6 mice. METHODS: In vitro growth inhibition was evaluated using the MTT assay, and in vivo activity was evaluated in subcutaneous models in C57BL/6 mice. The status of the vasculature in tumor tissues was evaluated immunohistochemically using anti-CD31 antibody. We used a cDNA macroarray to examine the gene expression profiles in tumor tissues removed from mice. RESULTS: TZT-1027 at 3 mg/kg showed potent antitumor activity in Mock (LLC-Neo cells) inoculated mice with a T/C% value of 16%. TZT-1027 at 3 mg/kg showed more potent antitumor activity in LLC-TNF cells and LLC-IL6 cells with T/C% values of 4% and 3%, respectively. TZT-1027 treatment destroyed the tumor vasculature as well as tumor cells in LLC-TNF and LLC-IL6 tissues of mice treated with TZT-1027. The LLC-TNF and LLC-IL6 tissues of mice treated with TZT-1027 had in common the independent alteration of the non-histone chromosomal protein HMG-14 and transcription factor 1 for heat shock gene. Focusing on the gene regulation related to angiogenesis, the alteration in transcriptional factors such as ets family genes and homeobox family genes was remarkable. CONCLUSIONS: These factors are candidates as determinants of the enhanced TZT-1027 antitumor activity in relation to these cytokines. PMID: 11855751 [PubMed - indexed for MEDLINE] NR31: Anticancer Res. 2001 Sep-Oct;21(5):3221-8. Transcriptional profiling of cell lines derived from an orthotopic pancreatic tumor model reveals metastasis-associated genes. Tarbe N, Evtimova V, Burtscher H, Jarsch M, Alves F, Weidle UH. Roche Diagnostics GmbH, Pharma Research, Penzberg, Germany. In order to identify genes associated with metastasis of ductal pancreatic adenocarcinoma we investigated pancreatic tumor cell lines derived from an orthotopic pancreatic tumor model in SCID mice. Transcriptional profiling (Affymetrix Gene Chip Technology) was performed with cell lines derived from the primary tumor and metastatic lesions such as mesentery, liver and lungs. We scored for genes commonly deregulated in the cell lines derived from the metastatic lesions. Of 7070 genes investigated, 59 (0.83%) were found to be deregulated in the cell lines derived from the metastatic lesions. We grouped these genes into different categories such as transcription, translation, cytoskeleton, cell adhesion, chromosome instability, tumor suppressor genes, enzymes and "others". The most remarkable features of the system are the up-regulation of high mobility group protein HMG-I (Y), twenty-one ribosomal proteins, GAPDH and the laminin receptor in the cell lines derived from the metastatic lesions, whereas tumor suppressor genes such as maspin and RB1 were down-regulated. Inhibition or reconstitution of the activity of these targets are an emerging strategy for inhibition of metastasis in this system. PMID: 11848476 [PubMed - indexed for MEDLINE] NR32: J Lipid Res. 2002 Jan;43(1):82-9. Tissue-specific transcriptional regulation of the cholesterol biosynthetic pathway leads to accumulation of testis meiosis-activating sterol (T-MAS). Tacer KF, Haugen TB, Baltsen M, Debeljak N, Rozman D. Institute of Biochemistry, Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia. Lanosterol 14alpha-demethylase (CYP51) produces follicular fluid meiosis-activating sterol (FF-MAS), which is converted further to testis meiosis-activating sterol (T-MAS). MAS are intermediates in the cholesterol biosynthetic pathway, with the ability to trigger resumption of oocyte meiosis in vitro. In contrast to the liver, where pre- and post-MAS genes are upregulated coordinately at the level of transcription by a cholesterol feedback mechanism through sterol regulatory element-binding proteins (SREBP), regulation differs in the testis. Genes encoding pre-MAS enzymes [HMG-CoA synthase (SYN), HMG-CoA reductase (RED), farnesyl diphosphate synthase (FPP), squalene synthase (SS), and CYP51] are upregulated during sexual development of the testis, although not all genes are turned on at the same time. Furthermore, two post-MAS genes, C-4 sterol methyl oxidase and sterol Delta(7)-reductase, are expressed at low levels and are not upregulated either in rat or human. This transcriptional discrepancy seems to be SREBP independent. Besides cAMP/cAMP-responsive element modulator, other unknown transcription factors control expression of individual cholesterogenic genes during spermatogenesis. HPLC analysis shows an 8-fold increase in T-MAS during development of rat testis whereas MAS is barely detectable in livers of the same animals.We propose that the lack of a coordinate transcriptional control over the cholesterol biosynthetic pathway contributes importantly to overproduction of the signaling sterol T-MAS in testis. PMID: 11792726 [PubMed - indexed for MEDLINE] DR33: Crit Care Med. 2002 Jan;30(1 Suppl):S36-42. High-mobility group-I/Y proteins: potential role in the pathophysiology of critical illnesses. Carvajal IM, Baron RM, Perrella MA. Pulmonary and Critical Care Division, Brigham and Women's Hospital, Boston, MA 02115, USA. High-mobility group (HMG) proteins are architectural factors that have been shown to play a role in the transcriptional regulation of various mammalian genes. One family of HMG proteins, HMG-I/Y, is known to facilitate the initiation of gene transcription by modifying the conformation of DNA and recruiting transcription factors into an organized complex on transcriptional regulatory regions of specific genes. In many circumstances, the nuclear factor-kappa B family of transcription factors is involved in gene regulation that is mediated by HMG-I/Y. We will review the mechanisms by which HMG-I/Y proteins regulate gene transcription, give an overview of selected genes regulated by HMG-I/Y, summarize the potential roles of these genes in critical illnesses, and provide more detailed information about the role of HMG-I/Y in the regulation of nitric oxide synthase-2 during an inflammatory response, such as endotoxemia/sepsis. Publication Types: Review Review, Tutorial PMID: 11782559 [PubMed - indexed for MEDLINE] NR34: FASEB J. 2002 Feb;16(2):258-60. Epub 2001 Dec 28. Effects of plant stanol esters on LDL receptor protein expression and on LDL receptor and HMG-CoA reductase mRNA expression in mononuclear blood cells of healthy men and women. Plat J, Mensink RP. Department of Human Biology, Maastricht University, 6200 MD, Maastricht, The Netherlands. J.Plat@HB.UNIMAAS.NL Functional foods enriched with plant stanols lower atherogenic LDL cholesterol concentrations. This effect is caused at least partly by a decreased intestinal cholesterol absorption. It has been suggested that LDL production is reduced after plant stanol consumption, but it is unknown whether LDL receptor expression is affected and contributes to the LDL-lowering effect of plant stanols. Markers for endogenous cholesterol synthesis (lathosterol) increase, but it is not known how this higher cholesterol synthesis is regulated at the molecular level. In a double-blind placebo controlled trial, we have found that compared with the control group (N=15), daily consumption of 3.8-4.0 g plant stanol esters (N=29) for 8 weeks increased LDL receptor mRNA levels in human mononuclear blood cells by 43% (P=0.003). LDL receptor protein concentrations on the surface of monocytes and T lymphocytes increased by 37% (P=0.003) and 25% (P=0.013), suggesting an increased translation. This increased protein expression appeared to be functional, since changes in serum LDL cholesterol correlated negatively with changes in LDL receptor mRNA levels (r=-0.361; P=0.015) and changes in LDL receptor protein expression in monocytes (r=-0.440; P<0.001) and T lymphocytes (r=-0.307; P=0.018). Based on these results, we suggest that the higher LDL receptor expression contributed to a lowered LDL formation along the apoB cascade. Whole body cholesterol synthesis increased, as indicated by the rise in serum cholesterol-standardized lathosterol concentrations, but the 34% increased HMG-CoA reductase mRNA concentrations did not reach statistical significance. Nor did it correlate significantly with changes in serum cholesterol-standardized lathosterol concentrations. This suggests that HMG-CoA reductase may be only partly regulated at a transcriptional level. Publication Types: Clinical Trial Controlled Clinical Trial PMID: 11772951 [PubMed - indexed for MEDLINE] DR35: J Biol Chem. 2002 Mar 1;277(9):7157-64. Epub 2001 Dec 17. HMGB1 and HMGB2 cell-specifically down-regulate the p53- and p73-dependent sequence-specific transactivation from the human Bax gene promoter. Stros M, Ozaki T, Bacikova A, Kageyama H, Nakagawara A. Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic. stros@ibp.cs The recently cloned gene p73 is a close homologue of p53, which is a crucial tumor suppressor gene for preventing the malignant transformation of cells by inducing cell cycle arrest and apoptosis. Previous reports have shown that architectural DNA-bending/looping chromosomal proteins HMGB1 and HMGB2 (formerly known as HMG1 and HMG2), which function in a number of biological processes including transcription and DNA repair, interact in vitro with p53 and stimulate p53 binding to DNA containing p53 consensus sites. Here, we report that HMGB1 physically interacts with two splicing variants of p73, alpha and beta (pull-down assay), and enhances binding of p73 to specific cognate DNA sites (gel-shift assay). Both HMG box domains of HMGB1, A and B, interact with p73alpha. Association of HMGB1 with p73, like the demonstrated ability of HMGB1 to stimulate p73 binding to different p53-responsive elements, requires the oligomerization region and/or region between DNA-binding domain and oligomerization domain of p73 (residues 312-381). Transient transfections revealed that ectopically expressed or endogenous HMGB1 and HMGB2 (antisense strategy) significantly inhibit in vivo both p73alpha/beta- and p53-dependent transactivation from the Bax gene promoter (and much less from Mdm2 and p21(waf1) promoters) in p53-deficient SAOS-2 cells. In contrast, HMGB1 and HGMB2 stimulate p73- or p53-dependent transactivation in p53-deficient H1299 cells, irrespective of the promoter used. Our results suggest that ubiquitously expressed HMGB1 and HMGB2 have potential to cell- and promoter-specifically down- or up-regulate in vivo transcriptional activity of different members of the p53 family. A possible mechanism of HMGB1-mediated modulation of p73- and p53-dependent transactivation is discussed. PMID: 11748232 [PubMed - indexed for MEDLINE] PR36: EMBO J. 2001 Dec 17;20(24):7197-208. A POP-1 repressor complex restricts inappropriate cell type-specific gene transcription during Caenorhabditis elegans embryogenesis. Calvo D, Victor M, Gay F, Sui G, Luke MP, Dufourcq P, Wen G, Maduro M, Rothman J, Shi Y. Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. In Caenorhabditis elegans, histone acetyltransferase CBP-1 counteracts the repressive activity of the histone deacetylase HDA-1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box-containing protein, POP-1, through an unknown mechanism. In this study, we show that CBP-1, HDA-1 and POP-1 converge on end-1, an initial endoderm-determining gene. In the E lineage, an essential function of CBP-1 appears to be the activation of end-1 transcription. We further identify a molecular mechanism for the endoderm-suppressive effect of POP-1 in the MS lineage by demonstrating that POP-1 functions as a transcriptional repressor that inhibits inappropriate end-1 transcription. We provide evidence that POP-1 represses transcription via the recruitment of HDA-1 and UNC-37, the C.elegans homolog of the co-repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate-determining gene during development. Furthermore, they identify a strategy by which concerted actions of histone deacetylases and other co-repressors ensure maximal repression of inappropriate cell type-specific gene transcription. PMID: 11742996 [PubMed - indexed for MEDLINE] NR37: Arterioscler Thromb Vasc Biol. 2001 Dec;21(12):1896-901. Downregulation of angiotensin II type 1 receptor by hydrophobic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in vascular smooth muscle cells. Ichiki T, Takeda K, Tokunou T, Iino N, Egashira K, Shimokawa H, Hirano K, Kanaide H, Takeshita A. Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan. ichiki@cardiol.med.kyushu-u.ac.jp 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, so-called statins, reduce the relative risk of a major coronary event by lowering the serum cholesterol level. In addition, statins may confer beneficial effects by cholesterol-lowering independent mechanisms, which are incompletely characterized. Because angiotensin II (Ang II) plays crucial roles in the pathogenesis of cardiovascular diseases, we examined the effect of statins on the expression of the Ang II type 1 receptor (AT(1)-R) in cultured vascular smooth muscle cells (VSMCs). Cerivastatin and fluvastatin reduced the AT(1)-R mRNA and the AT(1)-R protein levels; however, pravastatin lacked this effect. Cerivastatin and fluvastatin suppressed the AT(1)-R promoter activity measured by luciferase assay but did not affect AT(1)-R mRNA stability, suggesting that the suppression occurs at the transcriptional level. Coincubation of VSMCs with mevalonate or geranylgeranyl pyrophosphate but not with farnesyl pyrophosphate reversed the cerivastatin-induced AT(1)-R downregulation. Overexpression of dominant-negative Rho A also suppressed AT(1)-R mRNA expression. Treatment with cerivastatin for 24 hours reduced the calcium response of VSMCs to Ang II. Taken together, statins downregulate AT(1)-R expression through a mevalonate-dependent, geranylgeranyl pyrophosphate-dependent, and Rho A-dependent manner and attenuate the biological function of Ang II. Downregulation of AT(1)-R may contribute to the cholesterol-independent beneficial effect of statins on the cardiovascular system. PMID: 11742861 [PubMed - indexed for MEDLINE] DR38: Mol Cell. 2001 Nov;8(5):1063-73. Comment in: Mol Cell. 2001 Nov;8(5):932-3. An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF. Stefanovsky VY, Pelletier G, Hannan R, Gagnon-Kugler T, Rothblum LI, Moss T. Cancer Research Centre and Department of Medical Biology, Laval University, Hotel-Dieu de Quebec, 11 cote du Palais, G1R 2J6, Quebec, Canada. Ribosomal transcription in mammals is regulated in response to growth, differentiation, disease, and aging, but the mechanisms of this regulation have remained unresolved. We show that epidermal growth factor induces immediate, ERK1/2-dependent activation of endogenous ribosomal transcription, while inactivation of ERK1/2 causes an equally immediate reversion to the basal transcription level. ERK1/2 was found to phosphorylate the architectural transcription factor UBF at amino acids 117 and 201 within HMG boxes 1 and 2, preventing their interaction with DNA. Mutation of these sites inhibited transcription activation and abrogated the transcriptional response to ERK1/2. Thus, growth factor regulation of ribosomal transcription likely acts by a cyclic modulation of DNA architecture. The data suggest a central role for ribosome biogenesis in growth regulation. PMID: 11741541 [PubMed - indexed for MEDLINE] NR39: Biochim Biophys Acta. 2001 Oct 31;1533(3):220-32. Regulation of lipid metabolism and gene expression by fenofibrate in hamsters. Guo Q, Wang PR, Milot DP, Ippolito MC, Hernandez M, Burton CA, Wright SD, Chao Y. Department of Atherosclerosis and Endocrinology, Merck and Co., Inc., R80W250, P.O. Box 2000, Rahway, NJ 07065, USA. qiu_guo@merck.com Fenofibrate is a potent hypolipidemic agent that lowers plasma lipid levels and may thus decrease the incidence of atherosclerosis. Here we investigated the molecular mechanism of fenofibrate's hypolipidemic action by characterizing its in vivo effects on the expression of mRNAs and the activities of pivotal enzymes in cholesterol and triglyceride metabolism in the hamster. Treatment of hamsters with fenofibrate led to a dose-dependent reduction in serum cholesterol concentrations. Studies on the incorporation of [(14)C]acetate and [(14)C]mevalonate into cholesterol suggested that this effect occurs primarily through inhibition of cholesterol biosynthesis at steps prior to mevalonate. Fenofibrate decreased levels of hepatic enzyme activities and mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase. A potential mechanism for transcriptional regulation of these enzymes is via SREBP-2 that we found to be suppressed 2-fold by fenofibrate. Fenofibrate also lowered circulatory triglyceride levels. In keeping with the effect, we observed strong suppression of fatty acid synthase, acetyl-CoA carboxylase and apolipoprotein C-III mRNA and stimulation of lipoprotein lipase and acyl-CoA oxidase mRNA in the liver of fenofibrate-treated hamsters. These observations suggest that the effect of fenofibrate on triglyceride metabolism is likely to be a result of both decreased fatty acid synthesis and increased lipoprotein lipase and acyl-CoA oxidase gene expression in the liver. Surprisingly, alterations in lipoprotein lipase, acyl-CoA oxidase, acetyl-CoA carboxylase, and apolipoprotein C-III could not be observed in hamster hepatocytes incubated with fenofibric acid in vitro. These observations raise the possibility that changes in these genes may be secondary to the metabolic alterations occurring in animals but not in cultured cells and thus that the effect of fenofibrate on these genes may be indirect. PMID: 11731332 [PubMed - indexed for MEDLINE] NR40: Cancer Detect Prev. 2001;25(5):454-69. Novel human HALR (MLL3) gene encodes a protein homologous to ALR and to ALL-1 involved in leukemia, and maps to chromosome 7q36 associated with leukemia and developmental defects. Tan YC, Chow VT. Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge. We have identified and characterized the approximately 12-kb cDNA of a novel human gene (designated HALR for "homologous to ALR" and given the symbol MLL3 by the HUGO Gene Nomenclature Committee) for which open reading frame (ORF) encodes a predicted large hydrophilic nuclear protein comprising 4,025 amino acids with a calculated molecular mass of approximately 443 kD. Within the amino acid sequence of HALR were identified a SUVAR3-9, enhancer of zeste, trithorax (SET) domain, three plant homeodomain (PHD)-type zinc fingers, a high motility group (HMG)-1 box, a leucine-zipper-like pattern, two potential transactivating domains, several nuclear localization signals, and multiple nuclear receptor interaction signature motifs. Especially within the SET domain, PHD fingers and several other regions, the HALR protein exhibits significant similarity to ALR (acute lymphoblastic leukemia [ALL]-1 related), ALL-1/myeloid/lymphoid or mixed-lineage leukemia (ALL-1/MLL), and trithorax, evolutionarily conserved proteins that influence differentiation and development. Northern blot analysis demonstrated transcripts of approximately 11-12 kb, while reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that HALR is expressed in a wide range of human tissues and cancer cell lines. The HALR gene contains 46 exons, is estimated to span >101 kb, and is located on chromosome region 7q36. Terminal 7q deletions are common chromosomal aberrations encountered in hematological neoplasia and in holoprosencephaly 3, a midline embryonic defect involving forebrain development. We have also isolated the partial cDNA of the murine homologue of HALR, which displays high homology to its human counterpart. Taking into consideration its notable protein motifs, ubiquitous expression, evolutionary conservation and chromosomal position, HALR is likely to play a housekeeping role in transcriptional regulation, and may be involved in leukemogenesis and developmental disorders. PMID: 11718452 [PubMed - indexed for MEDLINE] NR41: Biochem J. 2001 Dec 1;360(Pt 2):363-70. Diverse effects of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase on the expression of VCAM-1 and E-selectin in endothelial cells. Rasmussen LM, Hansen PR, Nabipour MT, Olesen P, Kristiansen MT, Ledet T. Laboratory for Molecular Pathology, Institute of Pathology, University Hospital of Aarhus, Kommunehospitalet, DK-8000 Aarhus C, Denmark. lmras@dadlnet.dk The expression of monocyte adhesion molecules, such as VCAM-1 (vascular cell adhesion molecule-1) and E-selectin, on the surface of the endothelium is an important step in the initiation and progression of atherosclerotic lesions. We hypothesized that the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in endothelial cells could influence the expression of VCAM-1 and E-selectin. Using cultured human umbilical vein endothelial cells, we found that mevastatin (0.1-1 microM) significantly reduced the expression of VCAM-1 protein in cells activated by tumour necrosis factor-alpha (TNF-alpha) for 7 h. In contrast, TNF-alpha-induced E-selectin protein expression was augmented after mevastatin treatment. Mevastatin inhibited the mRNA expression of both VCAM-1 and E-selectin in TNF-alpha-stimulated endothelial cells. The activity of the transcription factor nuclear factor-kappa B, which is known to regulate the transcription of VCAM-1 and E-selectin, was significantly reduced after incubation with mevastatin. Analysis of the time-dependent variation in the TNF-alpha-induced expression of E-selectin, and estimation of the rate of surface disappearance of E-selectin together with measurement of the amounts of E-selectin molecules secreted, indicated that mevastatin inhibited the surface removal of E-selectin. This is compatible with the observed increase in E-selectin expression after statin treatment. All observed effects of mevastatin were reversed by mevalonate, the product of the HMG-CoA reductase reaction. In conclusion, inhibition of HMG-CoA reductase in endothelial cells attenuates VCAM-1 expression, but increases E-selectin expression, after cytokine induction. These diverse effects are associated with changes in the transcriptional regulation of the two adhesion molecule genes and modulation of the surface removal of E-selectin. PMID: 11716764 [PubMed - indexed for MEDLINE] PR42: Hypertension. 2001 Nov;38(5):1024-9. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors upregulate inducible NO synthase expression and activity in vascular smooth muscle cells. Kolyada AY, Fedtsov A, Madias NE. Department of Medicine, Tufts University School of Medicine, Division of Nephrology and the Tupper Research Institute, New England Medical Center, Boston, Massachusetts, USA. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase ameliorate atherosclerosis by both cholesterol-dependent and cholesterol-independent mechanisms. We examined whether HMG-CoA reductase inhibitors affect the expression and activity of inducible NO synthase (iNOS) in cultured rat aortic vascular smooth muscle (VSM) cells. Atorvastatin (34 to 68 micromol/L) markedly increased nitrite production, an increase that was essentially abrogated by the NO synthase inhibitor N(G)-monomethyl-L-arginine (500 micromol/L). Activity of iNOS, determined by the conversion of L-arginine to L-citrulline, increased 9-fold after atorvastatin treatment. Western blot and semiquantitative reverse transcriptase-polymerase chain reaction revealed that atorvastatin (34 to 68 micromol/L) strongly upregulated iNOS protein and mRNA levels, respectively. These concentrations of atorvastatin did not cause cytotoxicity, as judged by the cell survival rate. Similarly, simvastatin and lovastatin (34 micromol/L) caused robust upregulation of the iNOS protein level. Transfection experiments demonstrated that the -1034- to 88-bp human iNOS promoter was strongly induced by atorvastatin (34 micromol/L). Electromobility and supershift assays using a nuclear factor-kappaB (NF-kappaB) consensus oligonucleotide and nuclear extracts from VSM cells as well as transfection studies using an NF-kappaB reporter plasmid suggested that the transcriptional activation of the iNOS gene by atorvastatin is not mediated via the NF-kappaB pathway. We conclude that HMG-CoA reductase inhibitors potently upregulate iNOS expression and activity in VSM cells, at least in part, by transcriptional mechanisms that do not depend on transcription factor NF-kappaB. These effects might have important implications for the impact of HMG-CoA reductase inhibitors on atherosclerosis. PMID: 11711492 [PubMed - indexed for MEDLINE] NR43: Carcinogenesis. 2001 Oct;22(10):1727-31. Lovastatin-induced E2F-1 modulation and its effect on prostate cancer cell death. Park C, Lee I, Kang WK. Department of Medicine and Cancer Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, Korea. Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, induces growth arrest in a variety of cancer cell lines. Its mechanism of action, however, has not been completely elucidated. E2F-1 is thought to act as an oncogene and a tumour suppressor, with its action probably dependent upon the cellular context. We have shown in this study that transcriptional regulation and proteasomal degradation of E2F-1 are critical regulatory events in lovastatin-induced cell death. Accompanying this is a reduction in the E2F-1-regulated expression of cell cycle genes such as c-myc, cyclin D1, cyclin A and cyclin B1. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease in the S-phase cell population in response to lovastatin. Although expression of E2F-1 was reduced in three prostate cancer cell lines-PC-3, LNCaP and DU-145-the p21 and p27 protein levels were not increased in all the cell lines treated, suggesting that increase in p21 and p27 protein expression per se is not responsible for lovastatin-mediated down-regulation of E2F-1. The subsequent apoptotic death of these cells in the presence of lovastatin can be prevented by forced ectopic expression of E2F-1. Taken together, these facts imply that E2F-1 is the target of an HMG-CoA inhibitor and critical cell death mediator in prostate cancer cells. PMID: 11577016 [PubMed - indexed for MEDLINE] PR44: Biochem Biophys Res Commun. 2001 Sep 21;287(2):493-500. SOX18 directly interacts with MEF2C in endothelial cells. Hosking BM, Wang SC, Chen SL, Penning S, Koopman P, Muscat GE. Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia. Recently, we demonstrated that mutations in the Sry-related HMG box gene Sox18 underlie vascular and hair follicle defects in the mouse allelic mutants ragged (Ra) and RaJ. Ra mice display numerous anomalies in the homozygote including, oedema, peritoneal secretions, and are almost completely naked. Sox18 and the MADS box transcription factor, Mef2C, are expressed in developing endothelial cells. Null mutants in Sox18 and Mef2c display overlapping phenotypic abnormalities, hence, we investigated the relationship between these two DNA binding proteins. We report here the direct interaction between MEF2C and SOX18 proteins, and establish that these proteins are coexpressed in vivo in endothelial cell nuclei. MEF2C expression potentiates SOX18-mediated transcription in vivo and regulates the function of the SOX18 activation domain. Interestingly, MEF2C fails to interact or co-activate transcription with the Ra or RaJ mutant SOX18 proteins. These results suggest that MEF2C and SOX18 may be important partners directing the transcriptional regulation of vascular development. Copyright 2001 Academic Press. PMID: 11554755 [PubMed - indexed for MEDLINE] NR45: Hum Mol Genet. 2001 Aug 15;10(17):1785-92. Quantitative effects on gene silencing by allelic variation at a tetranucleotide microsatellite. Albanese V, Biguet NF, Kiefer H, Bayard E, Mallet J, Meloni R. Laboratoire de Genetique de la Neurotransmission, CNRS UMR C9923, Hopital de la Pitie Salpetriere, 83 Boulevard de l'hopital, 75013 Paris, France. Microsatellites are common repeated sequences, which are useful as genetic markers and lack any clearly established function. In a previous study we suggested that an intronic polymorphic TCAT repeat in the tyrosine hydroxylase (TH) gene, the microsatellite HUMTH01, may regulate transcription. The TH gene encodes the rate-limiting enzyme in the synthesis of catecholamines, and the microsatellite HUMTH01 has been used in genetic studies of neuropsychiatric and cardiovascular diseases, in which disturbances of catecholaminergic neurotransmission have been implicated. HUMTH01 alleles associated with these diseases act as transcriptional enhancers when linked to a minimal promoter and are recognized by specific nuclear factors. Here we show that allelic variations of HUMTH01 commonly found in humans have a quantitative silencing effect on TH gene expression. Two specific proteins, ZNF191, a zinc finger protein, and HBP1, an HMG box transcription factor, which bind the TCAT motif, were then cloned. Finally, allelic variations of HUMTH01 correlate with quantitative and qualitative changes in the binding by ZNF191. Thus, this repeated sequence may contribute to the control of expression of quantitative genetic traits. As the HUMTH01 core motif is ubiquitous in the genome, this phenomenon may be relevant to the quantitative expression of many genes in addition to TH. PMID: 11532988 [PubMed - indexed for MEDLINE] PR46: EMBO J. 2001 Aug 15;20(16):4500-11. Negative regulation of the Wnt-beta-catenin pathway by the transcriptional repressor HBP1. Sampson EM, Haque ZK, Ku MC, Tevosian SG, Albanese C, Pestell RG, Paulson KE, Yee AS. Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. In certain cancers, constitutive Wnt signaling results from mutation in one or more pathway components. The result is the accumulation and nuclear localization of beta-catenin, which interacts with the lymphoid enhancer factor-1 (LEF)/T-cell factor (TCF) family of HMG-box transcription factors, which activate important growth regulatory genes, including cyclin D1 and c-myc. As exemplified by APC and axin, the negative regulation of beta-catenin is important for tumor suppression. Another potential mode of negative regulation is transcriptional repression of cyclin D1 and other Wnt target genes. In mammals, the transcriptional repressors in the Wnt pathway are not well defined. We have previously identified HBP1 as an HMG-box repressor and a cell cycle inhibitor. Here, we show that HBP1 is a repressor of the cyclin D1 gene and inhibits the Wnt signaling pathway. The inhibition of Wnt signaling and growth requires a common domain of HBP1. The apparent mechanism is an inhibition of TCF/LEF DNA binding through a physical interaction with HBP1. These data suggest that the suppression of Wnt signaling by HBP1 may be a mechanism to prevent inappropriate proliferation. PMID: 11500377 [PubMed - indexed for MEDLINE] NR47: Gene. 2001 Jul 11;272(1-2):121-9. Characterization of an SRY-like gene, DSox14, from Drosophila. Sparkes AC, Mumford KL, Patel UA, Newbury SF, Crane-Robinson C. Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth, PO1 2DT, UK. We have characterized the DSox14 gene, a new member of the family of transcription factors related to the mammalian sex determining factor, SRY. It contains two exons and the intron is large for Drosophila at 2.8 kb. The encoded protein consists of 691 amino acids (72 kDa) and includes an HMG box domain, which is closely related to the mouse Sox4 DNA binding domain. Expression of the DSox14 HMG box domain in vitro shows that it binds the sequence AACAAT with a K(d) of 190 nM, generating a bend angle of 48.6 degrees. At higher protein concentrations, a second HMG box binds at the recognition sequence, increasing the bend angle by 5 degrees. DSox14 is variably expressed throughout development as three alternative transcripts but not at all during the 1st and 2nd larval instars. The several mRNA transcripts are produced primarily from different transcriptional start sites. Analysis of the expression of DSox14 mRNAs during early development shows that they are maternally contributed at a low level and ubiquitously expressed during embryogenesis. The widespread pattern of expression suggests that DSox14 affects a large number of target genes. PMID: 11470517 [PubMed - indexed for MEDLINE] NR48: Cardiovasc Res. 2001 Aug 1;51(2):283-93. Simvastatin reduces reperfusion injury by modulating nitric oxide synthase expression: an ex vivo study in isolated working rat hearts. Di Napoli P, Antonio Taccardi A, Grilli A, Spina R, Felaco M, Barsotti A, De Caterina R. Laboratory of Experimental Cardiology, Department of Clinical Sciences and Bioimaging, "G. d'Annunzio" University of Chieti, Ospedale S. Camillo de Lellis, Via Forlanini, 50, 66100, Chieti, Italy. OBJECTIVE: We tested the hypothesis of beneficial effects of the 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA)-reductase inhibitor simvastatin in a model of ischemia-reperfusion, and investigated potential mechanisms. METHODS: Isolated working rat hearts were subjected to 15 min global ischemia and 22-180 min reperfusion in the presence or absence of simvastatin (10-100 microM). We evaluated creatinephosphokinase and nitrite levels in coronary effluent, heart weight changes, microvascular permeability (extravasation of fluoresceine-labeled albumin), ultrastructural alterations, and the expression of endothelial (e) and inducible (i) nitric oxide synthase (NOS) (by reverse-transcribed polymerase chain reaction and Western blotting) in the presence or absence of the transcriptional inhibitor actinomycin-D. RESULTS: Simvastatin (25 microM) significantly reduced myocardial damage and vascular hyperpermeability, concomitant with a reduction in endothelial and cardiomyocyte lesions. Protection became less evident at 50 microM and reverted to increased damage at 100 microM. At 25 microM, simvastatin significantly increased eNOS mRNA and protein compared with untreated hearts, probably due to a post-transcriptional regulation since unaltered by animal pretreatment with actinomycin D. Simvastatin also significantly decreased iNOS mRNA and protein, as well as nitrite production after ischemia-reperfusion. The addition of the NOS inhibitor N(varpi)-nitro-L-arginine methylester (L-NAME, 30 microM) to 25 microM simvastatin-treated hearts significantly reduced cardioprotection against ischemia-reperfusion. CONCLUSIONS: In this model, in the absence of perfusing granulocytes, the acute administration of a pharmacologically relevant simvastatin concentration reduces ischemia-reperfusion injury and prevents coronary endothelial cell and cardiomyocyte damage by cholesterol-independent, NO-dependent mechanisms. PMID: 11470468 [PubMed - indexed for MEDLINE] NR49: EMBO J. 2001 Jul 2;20(13):3506-17. Spt16-Pob3 and the HMG protein Nhp6 combine to form the nucleosome-binding factor SPN. Formosa T, Eriksson P, Wittmeyer J, Ginn J, Yu Y, Stillman DJ. Department of Biochemistry, University of Utah School of Medicine, 50 N. Medical Drive Rm 211, Salt Lake City, UT 84132, USA. Tim.Formosa@hsc.utah.edu Yeast Spt16/Cdc68 and Pob3 form a heterodimer that acts in both DNA replication and transcription. This is supported by studies of new alleles of SPT16 described here. We show that Spt16-Pob3 enhances HO transcription through a mechanism that is affected by chromatin modification, since some of the defects caused by mutations can be suppressed by deleting the histone deacetylase Rpd3. While otherwise conserved among many eukaryotes, Pob3 lacks the HMG1 DNA-binding motif found in similar proteins such as the SSRP1 subunit of human FACT. SPT16 and POB3 display strong genetic interactions with NHP6A/B, which encodes an HMG1 motif, suggesting that these gene products function coordinately in vivo. While Spt16-Pob3 and Nhp6 do not appear to form stable heterotrimers, Nhp6 binds to nucleosomes and these Nhp6-nucleosomes can recruit Spt16-Pob3 to form SPN-nucleosomes. These complexes have altered electrophoretic mobility and a distinct pattern of enhanced sensitivity to DNase I. These results suggest that Spt16-Pob3 and Nhp6 cooperate to function as a novel nucleosome reorganizing factor. PMID: 11432837 [PubMed - indexed for MEDLINE] NR50: J Clin Invest. 2001 Jun;107(11):1423-32. Statin-induced inhibition of the Rho-signaling pathway activates PPARalpha and induces HDL apoA-I. Martin G, Duez H, Blanquart C, Berezowski V, Poulain P, Fruchart JC, Najib-Fruchart J, Glineur C, Staels B. Departement d'Atherosclerose, UR 545 Institut National de la Sante et de la Recherche Medicale (INSERM), Institut Pasteur de Lille and Faculte de Pharmacie, Universite de Lille II, Lille, France. Statins are inhibitors of the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. In addition to reducing LDL cholesterol, statin treatment increases the levels of the antiatherogenic HDL and its major apolipoprotein apoA-I. Here, we investigated the molecular mechanisms of apoA-I regulation by statins. Treatment with statins increased apoA-I mRNA levels in human HepG2 hepatoma cells, and this effect was reversed by the addition of mevalonate, implicating HMG-CoA reductase as the relevant target of these drugs. Pretreatment with Actinomycin D abolished the increase of apoA-I mRNA, indicating that statins act at the transcriptional level. Indeed, statins increased the human apoA-I promoter activity in transfected cells, and we have identified a statin response element that coincides with a PPARalpha response element known to confer fibrate responsiveness to this gene. The statin effect could be abolished not only by mevalonate, but also by geranylgeranyl pyrophosphate, whereas inhibition of geranylgeranyl transferase activity or treatment with an inhibitor of the Rho GTP-binding protein family increased PPARalpha activity. Using dominant negative forms of these proteins, we found that Rho A itself mediates this response. Because cotreatment with statins and fibrates activated PPARalpha in a synergistic manner, these observations provide a molecular basis for combination treatment with statins and fibrates in coronary heart disease. PMID: 11390424 [PubMed - indexed for MEDLINE] PR51: J Biol Chem. 2001 Aug 10;276(32):29805-14. Epub 2001 Jun 4. Requirement for high mobility group protein HMGI-C interaction with STAT3 inhibitor PIAS3 in repression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) transcription by Ras activation in salivary epithelial cells. Zentner MD, Lin HH, Deng HT, Kim KJ, Shih HM, Ann DK. Department of Molecular Pharmacology and Toxicology, Will Rogers Institute, Pulmonary Research Center, University of Southern California, Los Angeles, California 90033, USA. Previously, we have demonstrated that oxidative stress or Ras/ERK activation leads to the transcriptional repression of alpha-subunit of epithelial Na(+) channel (ENaC) in lung and salivary epithelial cells. Here, we further investigated the coordinated molecular mechanisms by which alpha-ENaC expression is regulated. Using both stable and transient transfection assays, we demonstrate that the overexpression of high mobility group protein I-C (HMGI-C), a Ras/ERK-inducible HMG-I family member, represses glucocorticoid receptor (GR)/dexamethasone (Dex)-stimulated alpha-ENaC/reporter activity in salivary epithelial cells. Northern analyses further confirm that the expression of endogenous alpha-ENaC gene in salivary Pa-4 cells is suppressed by an ectopic HMGI-C overexpression. Through yeast two-hybrid screening and co-immunoprecipitation assays from eukaryotic cells, we also discovered the interaction between HMGI-C and PIAS3 (protein inhibitor of activated STAT3 (signal transducer and activator of transcription 3)). A low level of ectopically expressed PIAS3 cooperatively inhibits GR/Dex-dependent alpha-ENaC transcription in the presence of HMGI-C. Reciprocally, HMGI-C expression also coordinately enhances PIAS3-mediated repression of STAT3-dependent transactivation. Moreover, overexpression of antisense HMGI-C construct is capable of reversing the repression mediated by Ras V12 on GR- and STAT3-dependent transcriptional activation. Together, our results demonstrate that Ras/ERK-mediated induction of HMGI-C is required to effectively repress GR/Dex-stimulated transcription of alpha-ENaC gene and STAT3-mediated transactivation. These findings delineate a network of inhibitory signaling pathways that converge on HMGI-C.PIAS3 complex, causally associating Ras/ERK activation with the repression of both GR and STAT3 signaling pathways in salivary epithelial cells. PMID: 11390395 [PubMed - indexed for MEDLINE] NR52: Arch Biochem Biophys. 2001 Jan 15;385(2):364-71. Low activity of mitochondrial HMG-CoA synthase in liver of starved piglets is due to low levels of protein despite high mRNA levels. Barrero MJ, Alho CS, Ortiz JA, Hegardt FG, Haro D, Marrero PF. Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Spain. The unusually low hepatic ketogenic capacity of piglets has been correlated with lack of expression of the mitochondrial HMG-CoA synthase gene. However, we have shown that starvation of 2-week-old piglets increased the mRNA levels of mitochondrial HMG-CoA synthase to a level similar to that observed in starved rats (S. H. Adams, C. S. Alho, G. Asins, F. G. Hegardt, and P. F. Marrero, 1997, Biochem. J. 324, 65-73). We now report that antibodies against pig mitochondrial HMG-CoA synthase detected the pig enzyme in mitochondria of 2-week-old starved piglets and that the pig mitochondrial HMG-CoA synthase cDNA encodes an active enzyme in the eukaryotic cell line Mev-1, with catalytic behavior similar to that of the rat enzyme when expressed in the same system. We also show that low activity of pig mitochondrial HMG-CoA synthase correlates with low expression of the pig enzyme. The discrepancy in mitochondrial HMG-CoA synthase gene expression between the high levels of mRNA and low levels of enzyme was not associated with differences in transcript maturation, which suggests that an attenuated translation of the pig mRNA is responsible for the diminished ketogenic capacity of pig mitochondria. PMID: 11368018 [PubMed - indexed for MEDLINE] PR53: J Biol Chem. 2001 Jul 13;276(28):25736-41. Epub 2001 May 8. Interaction of FACT, SSRP1, and the high mobility group (HMG) domain of SSRP1 with DNA damaged by the anticancer drug cisplatin. Yarnell AT, Oh S, Reinberg D, Lippard SJ. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. The structure-specific recognition protein SSRP1, initially isolated from expression screening of a human B-cell cDNA library for proteins that bind to cisplatin (cis-diamminedichloroplatinum(II))-modified DNA, contains a single DNA-binding high mobility group (HMG) domain. Human SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that alleviates the nucleosomal block to transcription elongation by RNAPII in vitro. The affinity and specificity of FACT, SSRP1, and the isolated HMG domain of SSRP1 for cisplatin-damaged DNA were investigated by gel mobility shift assays. FACT exhibits both affinity and specificity for DNA damaged globally with cisplatin compared with unmodified DNA or DNA damaged globally with the clinically ineffective trans-DDP isomer. FACT binds the major 1,2-d(GpG) intrastrand cisplatin adduct, but its isolated SSRP1 subunit fails to form discrete, high affinity complexes with cisplatin-modified DNA under similar conditions. These results suggest that Spt16 primes SSRP1 for cisplatin-damaged DNA recognition by unveiling its HMG domain. As expected, the isolated HMG domain of SSRP1 is sufficient for specific binding to cisplatin-damaged DNA and binds the major cisplatin 1,2-d(GpG) intrastrand cross-link. The affinity and specificity of FACT for cisplatin-modified DNA, as well as its importance for transcription of chromatin, suggests that the interaction of FACT and cisplatin-damaged DNA may be crucial to the anticancer mechanism of cisplatin. PMID: 11344167 [PubMed - indexed for MEDLINE] NR54: Insect Biochem Mol Biol. 2001 Mar 15;31(4-5):425-33. 3-Hydroxy-3-methylglutaryl coenzyme A synthase-1 of Blattella germanica has structural and functional features of an active retrogene. Casals N, Buesa C, Marrero PF, Belles X, Hegardt FG. Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, E-08028, Barcelona, Spain. ncasals@csc.unica.edu Blattella germanica has two cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase genes, HMG-CoA synthase-1 and -2. HMG-CoA synthase-1 gene shows several features of processed genes (retroposons): it contains no introns but has a short direct-repeat sequence (ATTATTATT) at both ends. An atypical feature is the presence at both ends of the gene of short inverse repeats flanked by direct repeats. There is neither a TATA box nor a CAAT box in the 5' region. Comparative analysis with other species suggests that the HMG-CoA synthase-1 gene derives from HMG-CoA synthase-2. Cultured embryonic B. germanica UM-BGE-1 cells express HMG-CoA synthase-1 but not HMG-CoA synthase-2, suggesting that the intron-less gene is functional. In addition, it can complement MEV-1 cell line, which is auxotrophic for mevalonate. We show that compactin and mevalonate do not significantly affect the mRNA levels of HMG-CoA synthase-1 in UM-BGE-1 cells. Compactin induces a 6.7-fold increase in HMG-CoA reductase activity, which is restored to normal levels by mevalonate. HMG-CoA synthase activity is not modified by either of these effectors, suggesting that the mevalonate pathway in this insect cell line is regulated by post-transcriptional mechanisms affecting HMG-CoA reductase but not HMG-CoA synthase. PMID: 11222952 [PubMed - indexed for MEDLINE] NR55: Mol Endocrinol. 2001 Mar;15(3):363-77. SRYand architectural gene regulation: the kinetic stability of a bent protein-DNA complex can regulate its transcriptional potency. Ukiyama E, Jancso-Radek A, Li B, Milos L, Zhang W, Phillips NB, Morikawa N, King CY, Chan G, Haqq CM, Radek JT, Poulat F, Donahoe PK, Weiss MA. Pediatric Surgical Research Laboratories, Massachusetts General Hospital Harvard Medical School Boston, Massachusetts 02114, USA. Protein-directed DNA bending is proposed to regulate assembly of higher-order DNA-multiprotein complexes (enhanceosomes and repressosomes). Because transcriptional initiation is a nonequilibrium process, gene expression may be modulated by the lifetime of such complexes. The human testis-determining factor SRY contains a specific DNA-bending motif, the high-mobility group (HMG) box, and is thus proposed to function as an architectural factor. Here, we test the hypothesis that the kinetic stability of a bent HMG box-DNA complex can in itself modulate transcriptional potency. Our studies employ a cotransfection assay in a mammalian gonadal cell line as a model for SRY-dependent transcriptional activation. Whereas sex-reversal mutations impair SRY-dependent gene expression, an activating substitution is identified that enhances SRY's potency by 4-fold. The substitution (I13F in the HMG box; fortuitously occurring in chimpanzees) affects the motif's cantilever side chain, which inserts between base pairs to disrupt base pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA complex to an extent similar to its enhanced function. By contrast, equilibrium properties (specific DNA affinity, specificity, and bending; thermodynamic stability and cellular expression) are essentially unchanged. This correlation between potency and lifetime suggests a mechanism of kinetic control. We propose that a locked DNA bend enables multiple additional rounds of transcriptional initiation per promoter. This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity and decreased lifetime. Aromatic DNA-intercalating agents exhibit analogous kinetic control of transcriptional elongation whereby chemotherapeutic potencies correlate with drug-DNA dissociation rates. PMID: 11222738 [PubMed - indexed for MEDLINE] NR56: Swiss Med Wkly. 2001 Jan 27;131(3-4):41-6. The HMG-CoA reductase inhibitor simvastatin inhibits IFN-gamma induced MHC class II expression in human vascular endothelial cells. Kwak B, Mulhaupt F, Veillard N, Pelli G, Mach F. Cardiology Division, Department of Medicine, University Hospital, Geneva Medical School, Foundation for Medical Research, Geneva, Switzerland. HMG-CoA reductase inhibitors, or statins, are lipid-lowering agents that have been shown to effectively decrease severe rejection periods and development of transplant coronary artery disease after heart transplantation. Precise regulation of major histocompatibility complex class II (MHC class II) gene expression plays a pivotal role in control of the immune response after transplantation. The aim of this study was to evaluate the potential role of HMG-CoA reductase inhibitors in regulating the immune response. We have examined the effects of simvastatin on MHC class II expression in primary human endothelial cells. Using RNAse protection assay and flow cytometry, we observed that simvastatin dose-dependently reduced interferon-gamma (IFN-gamma) induced MHC class II expression (mRNA and protein). In contrast, simvastatin did not affect the expression of MHC class I, pointing to specific actions in the MHC class II signalling cascade. The transcriptional coactivator CIITA is the general regulator of both constitutive and inducible MHC class II expression. After stimulation with IFN-gamma, the CIITA gene is selectively activated via one (promotor IV) of its four promoters. Interestingly, mRNA levels of CIITA were decreased after treatment with simvastatin. In addition, using transient transfections of promoter-reporter constructs we observed that the activity of CIITA promoter IV was decreased by simvastatin. In conclusion, simvastatin selectively decreases IFN-gamma-induced MHC class II expression in human primary endothelial cells through actions on the CIITA promoter IV. Thus, the beneficial effects of statins reported after heart transplantation may result from this immunosuppressive action, suggesting possible therapeutic use for other immunological disorders as well. PMID: 11219190 [PubMed - indexed for MEDLINE] DR57: J Cell Physiol. 2001 Mar;186(3):315-28. Coevolution of HMG domains and homeodomains and the generation of transcriptional regulation by Sox/POU complexes. Dailey L, Basilico C. Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA. dailel01@med.nyu.edu The highly conserved homeodomains and HMG domains are components of a large number of proteins that play a role in the transcriptional regulation of gene expression during embryogenesis. Both the HMG domain and the homeodomain serve as interfaces for factor interactions with DNA, as well as with other proteins, and it is likely that the high degree of structural and sequence conservation within these domains reflects the conservation of basic aspects of these interactions. Classical HMG domain proteins have an ancient origin, being found in all eukaryotes, and are thought to have given rise to the metazoan-specific class of HMG domain proteins called the Sox proteins. Similarly, the metazoan-specific POU domain proteins are thought to have arisen from genes encoding ancestral homeodomain proteins. In this review, we summarize several examples of different HMG-homeodomain interactions that illustrate not only the ancient origin of each of these protein families, but also their relationship to each other, and discuss how coevolution of HMG and homeodomains may have lead to creation of the specialized Sox/POU protein complexes. Using the FGF-4 gene as an example, we also speculate on how coevolution of regulatory Sox/POU target DNA sequences may have occurred, and how the summation of these changes may have lead to the emergence of new developmental pathways. Copyright 2001 Wiley-Liss, Inc. Publication Types: Review PMID: 11169970 [PubMed - indexed for MEDLINE] NR58: Nucleic Acids Res. 2001 Feb 1;29(3):799-808. Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites. Cohen BD, Sertil O, Abramova NE, Davies KJ, Lowry CV. Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA. The DAN/TIR mannoprotein genes of Saccharomyces cerevisiae (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3 and TIR4) are expressed in anaerobic cells while the predominant cell wall proteins Cwp1 and Cwp2 are down-regulated. Elements involved in activation and repression of the DAN/TIR genes were defined in this study, using the DAN1 promoter as a model. Nested deletions in a DAN1/lacZ reporter pinpointed regions carrying activation and repression elements. Inspection revealed two consensus sequences subsequently shown to be independent anaerobic response elements (AR1, consensus TCGTTYAG; AR2, consensus AAAAATTGTTGA). AR1 is found in all of the DAN/TIR promoters; AR2 is found in DAN1, DAN2 and DAN3. A 120 bp segment carrying two copies of AR1 preferentially activated transcription of lacZ under anaerobic conditions. A fusion of three synthetic copies of AR1 to MEL1 was also expressed anaerobically. Mutations in either AR1 site within the 120 bp segment caused a drastic loss of expression, indicating that both are necessary for activation and implying cooperativity between adjacent transcriptional activation complexes. A single AR2 site carried on a 46 bp fragment from the DAN1 promoter activated lacZ transcription under anaerobic conditions, as did a 26 bp synthetic AR2 fragment fused to MEL1. Nucleotide substitutions within the AR2 sequence eliminated the activity of the 46 bp segment. Ablation of the AR2 sequences in the full promoter caused a partial reduction of expression. The presence of the ATTGTT core (recognized by HMG proteins) in the AR2 sequence suggests that an HMG protein may activate through AR2. One region was implicated in aerobic repression of DAN1. It contains sites for the heme-induced Mot3 and Rox1 repressors. PMID: 11160904 [PubMed - indexed for MEDLINE] NR59: Int J Dev Biol. 2000 Oct;44(7):749-56. dTcf antagonises Wingless signalling during the development and patterning of the wing in Drosophila. Lawrence N, Dearden P, Hartley D, Roose J, Clevers H, Arias AM. Department of Genetics, University of Cambridge, UK. Members of the Tcf family of HMG box-containing transcriptional regulators mediate Wnt signalling in the nucleus. Current models suggest that in the absence of Wnt signalling, Tcf interacts with the repressor protein Groucho and suppresses the expression of Wnt targets. Wnt signalling leads to increases in the level of cytoplasmic beta catenin, which enters the nucleus, displaces Tcf from Groucho and leads to transcriptional activation. In order to test this model we have studied the effects of Drosophila Tcf (dTcf) on signalling by Wingless, a Drosophila member of the Wnt family. We show that overexpression of wild-type dTcf during the development and patterning of the wing antagonises Wingless signalling. Furthermore, increases in the concentration of Armadillo, the Drosophila homologue of beta catenin, do not appear to be sufficient to trigger the change from antagonism to activation. This leads us to suggest that the inactivation of the repressive activity of dTcf requires the activity of Wingless in a manner that is independent of Armadillo. We observe that a Groucho molecule devoid of the WD40 repeats can interact with dTcf and acts as a dominant repressor of Wingless signalling in vivo and in vitro. Coexpression of this molecule with dTcf however, does not lead to enhancement of the repressive effects of dTcf alone. This observation suggests that repression by dTcf might not simply be mediated by an interaction with Groucho but that dTcf may have an intrinsic repressive activity that has to be antagonised by Wingless signalling. PMID: 11128568 [PubMed - indexed for MEDLINE] PR60: EMBO J. 2000 Dec 15;19(24):6804-13. Chromatin-mediated transcriptional regulation by the yeast architectural factors NHP6A and NHP6B. Moreira JM, Holmberg S. Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. The Saccharomyces cerevisiae NHP6A and NHP6B proteins are chromatin architectural factors, functionally and structurally related to the mammalian high mobility group (HMG)-1 and -2 proteins, a family of non-sequence-specific DNA binding proteins. nhp6a nhp6b mutants have various morphological defects and are defective in the induced expression of several RNA polymerase II-transcribed genes. We found that NHP6A/B proteins are also required for full induction of the yeast CHA1 gene. Importantly, CHA1 basal level expression is increased 10-fold in an nhp6a nhp6b double deletion mutant. Micrococcal nuclease and DNase I analysis of the CHA1 gene in this strain showed an open promoter structure, characteristic of the activated state of this promoter, even under non-inducing conditions. To address the possible function of the NHP6A/B proteins in chromatin-mediated gene regulation, we performed whole-genome transcriptional profiling of a Deltanhp6a Deltanhp6b yeast strain. Our results suggest that NHP6A/B proteins play an important regulatory role, repressing as well as potentiating expression of genes involved in several cellular processes, and that NHP6A/B control is exerted at the level of the individual gene. PMID: 11118215 [PubMed - indexed for MEDLINE] PR61: Cancer Res. 2000 Nov 15;60(22):6303-6. The oncogenic potential of the high mobility group box protein Sox3. Xia Y, Papalopulu N, Vogt PK, Li J. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. Sox proteins belong to the superfamily of high mobility group (HMG) proteins. Sox3 is expressed predominantly in the immature neuroepithelium. Ectopic expression of Sox3 causes oncogenic transformation of chicken embryo fibroblasts (CEFs). The oncogenicity of Sox3 is correlated with nuclear localization and transcriptional regulatory activity; mutants containing deletions in the HMG box or the transactivation domain fail to induce foci of transformation. These observations suggest that Sox proteins can induce aberrant cell growth and strengthen the link of HMG proteins to oncogenesis. PMID: 11103788 [PubMed - indexed for MEDLINE] PR62: J Virol. 2000 Nov;74(22):10523-34. High-mobility-group protein I can modulate binding of transcription factors to the U5 region of the human immunodeficiency virus type 1 proviral promoter. Henderson A, Bunce M, Siddon N, Reeves R, Tremethick DJ. The John Curtin School of Medical Research, the Australian National University, Canberra, Australian Capital Territory 2601, Australia. HMG I/Y appears to be a multifunctional protein that relies on in its ability to interact with DNA in a structure-specific manner and with DNA, binding transcriptional activators via distinct protein-protein interaction surfaces. To investigate the hypothesis that HMG I/Y may have a role in human immunodeficiency virus type 1 (HIV-1) expression, we have analyzed whether HMG I/Y interacts with the 5' long terminal repeat and whether this interaction can modulate transcription factor binding. Using purified recombinant HMG I, we have identified several high-affinity binding sites which overlap important transcription factor binding sites. One of these HMG I binding sites coincides with an important binding site for AP-1 located downstream of the transcriptional start site, in the 5' untranslated region at the boundary of a positioned nucleosome. HMG I binding to this composite site inhibits the binding of recombinant AP-1. Consistent with this observation, using nuclear extracts prepared from Jurkat T cells, we show that HMG I (but not HMG Y) is strongly induced upon phorbol myristate acetate stimulation and this induced HMG I appears to both selectively inhibit the binding of basal DNA-binding proteins and enhance the binding of an inducible AP-1 transcription factor to this AP-1 binding site. We also report the novel finding that a component present in this inducible AP-1 complex is ATF-3. Taken together, these results argue that HMG I may play a fundamental role in HIV-1 expression by determining the nature of transcription factor-promoter interactions. PMID: 11044097 [PubMed - indexed for MEDLINE] DR63: J Biol Chem. 2001 Jan 19;276(3):1984-92. Epub 2000 Oct 16. Distinct organization of DNA complexes of various HMGI/Y family proteins and their modulation upon mitotic phosphorylation. Piekielko A, Drung A, Rogalla P, Schwanbeck R, Heyduk T, Gerharz M, Bullerdiek J, Wisniewski JR. III. Zoologisches Institut-Entwicklungsbiologie, Universitat Gottingen, D-37073 Gottingen, Germany. High mobility group (HMG) proteins HMGI, HMGY, HMGI-C, and Chironomus HMGI are DNA-binding proteins thought to modulate the assembly and the function of transcriptional complexes. Each of these proteins contains three DNA-binding domains (DBD), properties of which appear to be regulated by phosphorylation. High levels of these proteins are characteristic for rapidly dividing cells in embryonic tissues and tumors. On the basis of their occurrence, specific functions for each of these proteins have been postulated. In this study we demonstrate differences in the nature of contacts of these proteins with promoter region of the interferon-beta gene. We show that HMGI and HMGY interact with this DNA via three DBDs, whereas HMGI-C and Chironomus HMGI bind to this DNA using only two domains. Phosphorylation of HMGY protein by Cdc2 kinase leads to impairing of contacts between the N-terminally located DBD and a single promoter element. The perturbations in the architecture of the protein.DNA complexes involve changes in the degree of unbending of the intrinsically bent IFNbeta promoter. Our results provide first insights into the molecular basis of functional specificity of proteins of the HMGI/Y family and their regulation by phosphorylation. PMID: 11034995 [PubMed - indexed for MEDLINE] NR64: J Neurosci. 2000 Oct 1;20(19):7317-24. The architectural transcription factor high mobility group I(Y) participates in photoreceptor-specific gene expression. Chau KY, Munshi N, Keane-Myers A, Cheung-Chau KW, Tai AK, Manfioletti G, Dorey CK, Thanos D, Zack DJ, Ono SJ. The Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA. The nonhistone chromosomal proteins high mobility group I(Y) [HMG I(Y)] have been shown to function as architectural transcription factors facilitating enhanceosome formation on a variety of mammalian promoters. Specifically, they have been shown to act as a "molecular glue" mediating protein-protein and protein-DNA contacts within the enhanceosome complex. HMG I(Y) proteins are expressed at high levels in embryonic and transformed cells and have been implicated in transcriptional regulation in these cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, HMG I(Y). In contrast to these observations, we show here that adult mouse retinal photoreceptors, which are terminally differentiated cells, express high levels of these proteins. Using retinoblastoma cells as an approximate model, we further demonstrate in transiently transfected cells that inhibition of HMG I(Y) expression and mutation of HMG I(Y) binding sites significantly reduce rhodopsin promoter activity. DNase I footprint analysis indicates that HMG I protein interacts with a discrete site within the rhodopsin proximal promoter. This site overlaps with the binding site for Crx, a paired-like homeodomain transcription factor that is essential for photoreceptor functioning and that when mutated causes several forms of human photoreceptor degeneration. Both biochemical and functional experiments demonstrate that HMG I(Y) physically associate with Crx and that their interaction with DNA is required for high-level transcription of the rhodopsin gene. These data provide the first demonstration that HMG I(Y) can be important for gene activation in terminally differentiated cells. PMID: 11007889 [PubMed - indexed for MEDLINE] NR65: Biochem J. 2000 Sep 15;350 Pt 3:785-90. Contribution of steroidogenic factor 1 to the regulation of cholesterol synthesis. Mascaro C, Nadal A, Hegardt FG, Marrero PF, Haro D. Department of Biochemistry and Molecular Biology. School of Pharmacy, University of Barcelona, Avenida Diagonal 643, E 08028 Barcelona, Spain. Steroidogenic factor 1 (SF-1) is an orphan member of the nuclear receptor family expressed in steroidogenic tissues, where it has an essential role in the regulation of the steroid hormone biosynthesis, adrenal and gonadal development and endocrine responses fundamental for reproduction. Here we show that SF-1 regulates the transcription of cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which is essential for the endogenous synthesis of cholesterol. We have identified an element located 365 bp upstream of the gene for cytosolic HMG-CoA synthase; SF-1 binds as a monomer to this element and confers SF-1 responsiveness to homologous and heterologous promoters. It has been shown that in tissues with a high demand for cholesterol to be used in steroid synthesis, there is a lack of correlation between the cholesterol levels and the activity of the limiting enzymes of the mevalonate pathway. In accord with those results, we observed that cholesterol synthesis from acetate and either cytosolic HMG-CoA mRNA expression or transcriptional activity were not changed in response to 25-hydroxycholesterol in the SF-1-expressing steroidogenic Leydig tumour MA-10 cells. Moreover, the overexpression of SF-1 in non-steroidogenic CV-1 cells renders them less sensitive to the regulatory effects of cholesterol. This observation led to the hypothesis that in steroidogenic tissues the expression of SF-1 permits high levels of endogenous synthesis of cholesterol irrespective of the intracellular levels of this metabolite. PMID: 10970793 [PubMed - indexed for MEDLINE] DR66: Mol Cell Biol. 2000 Aug;20(15):5490-502. HMG-I/Y, a new c-Myc target gene and potential oncogene. Wood LJ, Mukherjee M, Dolde CE, Xu Y, Maher JF, Bunton TE, Williams JB, Resar LM. Hematology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. The HMG-I/Y gene encodes the HMG-I and HMG-Y proteins, which function as architectural chromatin binding proteins important in the transcriptional regulation of several genes. Although increased expression of the HMG-I/Y proteins is associated with cellular proliferation, neoplastic transformation, and several human cancers, the role of these proteins in the pathogenesis of malignancy remains unclear. To better understand the role of these proteins in cell growth and transformation, we have been studying the regulation and function of HMG-I/Y. The HMG-I/Y promoter was cloned, sequenced, and subjected to mutagenesis analysis. A c-Myc-Max consensus DNA binding site was identified as an element important in the serum stimulation of HMG-I/Y. The oncoprotein c-Myc and its protein partner Max bind to this site in vitro and activate transcription in transfection experiments. HMG-I/Y expression is stimulated by c-Myc in a Myc-estradiol receptor cell line in the presence of the protein synthesis inhibitor cycloheximide, indicating that HMG-I/Y is a direct c-Myc target gene. HMG-I/Y induction is decreased in Myc-deficient fibroblasts. HMG-I/Y protein expression is also increased in Burkitt's lymphoma cell lines, which are known to have increased c-Myc protein. Like Myc, increased expression of HMG-I protein leads to the neoplastic transformation of both Rat 1a fibroblasts and CB33 cells. In addition, Rat 1a cells overexpressing HMG-I protein form tumors in nude mice. Decreasing HMG-I/Y proteins using an antisense construct abrogates transformation in Burkitt's lymphoma cells. These findings indicate that HMG-I/Y is a c-Myc target gene involved in neoplastic transformation and a member of a new class of potential oncogenes. PMID: 10891489 [PubMed - indexed for MEDLINE] DR67: Nucleic Acids Res. 2000 Jul 1;28(13):2541-50. HMG I/Y regulates long-range enhancer-dependent transcription on DNA and chromatin by changes in DNA topology. Bagga R, Michalowski S, Sabnis R, Griffith JD, Emerson BM. Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA. The nature of nuclear structures that are required to confer transcriptional regulation by distal enhancers is unknown. We show that long-range enhancer-dependent beta-globin transcription is achieved in vitro upon addition of the DNA architectural protein HMG I/Y to affinity-enriched holo RNA polymerase II complexes. In this system, HMG I/Y represses promoter activity in the absence of an associated enhancer and strongly activates transcription in the presence of a distal enhancer. Importantly, nucleosome formation is neither necessary for long-range enhancer regulation in vitro nor sufficient without the addition of HMG I/Y. Thus, the modulation of DNA structure by HMG I/Y is a critical regulator of long-range enhancer function on both DNA and chromatin-assembled genes. Electron microscopic analysis reveals that HMG I/Y binds cooperatively to preferred DNA sites to generate distinct looped structures in the presence or absence of the beta-globin enhancer. The formation of DNA topologies that enable distal enhancers to strongly regulate gene expression is an intrinsic property of HMG I/Y and naked DNA. PMID: 10871404 [PubMed - indexed for MEDLINE] NR68: Biochem J. 2000 Jul 1;349(Pt 1):27-34. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase. Montalvetti A, Pena-Diaz J, Hurtado R, Ruiz-Perez LM, Gonzalez-Pacanowska D. Instituto de Parasitologia y Biomedicina Lopez-Neyra, Consejo Superior de Investigaciones Cientificas, C/Ventanilla 11, 18001-Granada, Spain. In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control. PMID: 10861207 [PubMed - indexed for MEDLINE] PR69: Mol Cell Biol. 2000 Jul;20(13):4666-79. Binding of HMG-I(Y) imparts architectural specificity to a positioned nucleosome on the promoter of the human interleukin-2 receptor alpha gene. Reeves R, Leonard WJ, Nissen MS. Biochemistry/Biophysics, School of Molecular Biosciences, Washington State University, Pullman 99164, USA. reevesr@mailwsu.edu Transcriptional induction of the interleukin-2 receptor alpha-chain (IL-2Ralpha) gene is a key event regulating T-cell-mediated immunity in mammals. In vivo, the T-cell-restricted protein Elf-1 and the general architectural transcription factor HMG-I(Y) cooperate in transcriptional regulation of the human IL-2Ralpha gene by binding to a specific positive regulatory region (PRRII) in its proximal promoter. Employing chromatin reconstitution analyses, we demonstrate that the binding sites for both HMG-I(Y) and Elf-1 in the PRRII element are incorporated into a strongly positioned nucleosome in vitro. A variety of analytical techniques was used to determine that a stable core particle is positioned over most of the PRRII element and that this nucleosome exhibits only a limited amount of lateral translational mobility. Regardless of its translational setting, the in vitro position of the nucleosome is such that DNA recognition sequences for both HMG-I(Y) and Elf-1 are located on the surface of the core particle. Restriction nuclease accessibility analyses indicate that a similarly positioned nucleosome also exists on the PRRII element in unstimulated lymphocytes when the IL-2Ralpha gene is silent and suggest that this core particle is remodeled following transcriptional activation of the gene in vivo. In vitro experiments employing the chemical cleavage reagent 1,10-phenanthroline copper (II) covalently attached to its C-terminal end demonstrate that HMG-I(Y) protein binds to the positioned PRRII nucleosome in a direction-specific manner, thus imparting a distinct architectural configuration to the core particle. Together, these findings suggest a role for the HMG-I(Y) protein in assisting the remodeling of a critically positioned nucleosome on the PRRII promoter element during IL-2Ralpha transcriptional activation in lymphocytes in vivo. PMID: 10848593 [PubMed - indexed for MEDLINE] NR70: J Biol Chem. 2000 Apr 28;275(17):12497-502. Transcriptional regulation of the ATP citrate-lyase gene by sterol regulatory element-binding proteins. Sato R, Okamoto A, Inoue J, Miyamoto W, Sakai Y, Emoto N, Shimano H, Maeda M. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. aroysato@mail.ecc.u-tokyo.ac.jp In an attempt to identify unknown target genes for SREBP-1, total RNA from a stable Chinese hamster ovary cell line (CHO-487) expressing a mature form of human SREBP-1a (amino acids 1-487) with a LacSwitch Inducible Mammalian Expression System was subjected to a polymerase chain reaction subtraction method. One of the fragments was found to have 90 and 86% homology with rat and human ATP citrate-lyase (ACL) cDNA, respectively. When Hep G2 cells are cultured under either sterol-loaded or -depleted conditions, expression of the gene is induced approximately 2-3-fold by sterol depletion. To investigate the direct effect of SREBP-1a on transcription, luciferase assays using the promoter of the human ACL gene were performed. These deletion studies indicated that a minimum 160-base pair segment contains the information required for the transcriptional regulation brought about by enforced expression of SREBP-1a. Luciferase assays using mutant reporter genes revealed that SREBP-dependent transcriptional regulation is mediated by two nearby motifs, the SREBP-binding site (a TCAGGCTAG sequence) and the NF-Y-binding site (a CCAAT box). It was confirmed by gel mobility shift assays that recombinant SREBP-1a binds to the sequence. Data from studies with transgenic mice and reporter assays show that the ACL gene promoter is activated by SREBP-1a more strongly than SREBP-2 in contrast to the HMG CoA synthase and LDL receptor gene promoters, which exhibit the same preference for the two factors. Therefore, SREBPs transcriptionally regulates ACL enzyme activity, which generates the cytosolic acetyl CoA required for both cholesterol and fatty acid synthesis. PMID: 10777536 [PubMed - indexed for MEDLINE] NR71: J Pharmacol Exp Ther. 2000 May;293(2):315-20. New molecular targets for cholesterol-lowering therapy. Izzat NN, Deshazer ME, Loose-Mitchell DS. Department of Integrative Biology and Pharmacology, University of Texas Health Sciences Center, Houston, Texas 77225, USA. The use of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) in randomized clinical trials has established that cholesterol-lowering treatment reduces the risk of both cardiovascular and total mortality. This reduction in risk occurs in patients with or without existing cardiovascular disease and in patients with high or average plasma cholesterol concentrations. Aggressive treatment to lower plasma cholesterol has been shown to slow progression of atherosclerosis and in some instances may be as successful as angioplasty in reducing ischemic events. These studies suggest that reduction of plasma cholesterol to levels even below 100 mg/dl might be desirable. New targets for cholesterol-lowering therapy with mechanisms of action different from the statins have been identified. One of these targets is the Na(+)-dependent bile acid transporter that is expressed in the terminal ileum. This protein is responsible for recycling bile acids from the intestine to the liver. Several compounds that demonstrate the ability to decrease transporter activity and to lower plasma cholesterol have been investigated. Absorption of cholesterol from the small intestine is another potential target. Compounds that inhibit cholesterol absorption may act by interacting stoichiometrically with cholesterol within the intestinal lumen or substoichiometrically, presumably within the enterocyte. Finally, the transcriptional regulation of cholesterol 7alpha-hydroxylase by members of the nuclear receptor superfamily provides at least two other molecular targets for cholesterol-lowering drugs. Publication Types: Review Review, Tutorial PMID: 10772997 [PubMed - indexed for MEDLINE] NR72: J Cell Sci. 2000 May;113 ( Pt 10):1759-70. A beta-catenin/engrailed chimera selectively suppresses Wnt signaling. Montross WT, Ji H, McCrea PD. Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. beta-catenin plays an integral role in cell-cell adhesion by linking the cadherin complex of the adherens junction to the underlying actin cytoskeleton. In addition, beta-catenin transduces intracellular signals within the Wnt developmental pathway that are crucial to the proper establishment of embryonic axes and pattern formation of early mesoderm and ectoderm. For example, in the context of a defined dorsal 'organizer' region of early Xenopus embryos, beta-catenin enters the nucleus and associates with transcription factors of the HMG (High Mobility Group) Lef/Tcf protein family. Consequently, genes such as siamois, a homeobox gene contributing to the specification of the dorsoanterior axis, are activated. To further examine the role that beta-catenin plays in Wnt signaling, we generated a chimeric protein, beta-Engrailed (beta-Eng), in which the C-terminal trans-activation domain of beta-catenin is replaced with the transcriptional repression domain of Drosophila Engrailed. Dorsal overexpression of this mRNA in early Xenopus embryos leads to suppression of organizer-specific molecular markers such as siamois, Xnr-3 and goosecoid, corresponding with the dramatic morphological ventralization of embryos. Ventralized embryos further exhibit reduced activity of the Wnt pathway, as indicated by the loss of the notochord/organizer marker, chordin. Importantly, beta-Eng associates and functions normally with the known components of the cadherin complex, providing the experimental opportunity to repress beta-catenin's signaling function apart from its role in cadherin-mediated cell-cell adhesion. PMID: 10769207 [PubMed - indexed for MEDLINE] DR73: Endocr Res. 2000 Feb;26(1):1-21. The regulatory effects of thyroid hormone on the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Choi JW, Choi HS. Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea. jwchoiphar@yumc.yonsei.ac.kr The effects of 3, 3', 5-triiodothyronine (T3) on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were evaluated in the C100 baby hamster kidney cell line. Cells cultured in Minimal Essential Medium (MEM) were supplemented with 10% thyroid hormone-depleted fetal bovine serum (THDS-MEM) and had a 70.1% lower level of HMG-CoA reductase activity than the cells grown in a medium supplemented with fetal bovine serum (FBS). When T3 was added to THDS-MEM, the reduction of the reductase activity was blocked in a dose-dependent manner. In the cells grown in THDS-MEM for 48 hours, T3 (10(-6) M) treatment rapidly increased HMG-CoA reductase activity, achieving the control level six hours after treatment. Such effects of T3 were blocked by actinomycin D (5 microg/ml) or cycloheximide (10 microg/ml). The transcriptional rate of the HMG-CoA reductase gene did not change significantly regardless of the presence of T3, while T3 inhibited the 25-hydroxycholesterol-mediated decay of the reductase mRNA significantly. Our results show that T3-dependent regulation of HMG-CoA reductase activity, via the de novo synthesis of the reductase enzyme, seems to be mediated at least partially by the stabilization of HMG-CoA reductase mRNA. PMID: 10711719 [PubMed - indexed for MEDLINE] NR74: J Biol Chem. 2000 Mar 3;275(9):6368-74. NBP-45, a novel nucleosomal binding protein with a tissue-specific and developmentally regulated expression. Shirakawa H, Landsman D, Postnikov YV, Bustin M. Institute of Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20892, USA. Here we characterize a novel murine nuclear protein, which we named NBP-45, that is related to the ubiquitous nuclear proteins HMG-14/-17, binds specifically to nucleosome core particles, and can function as a transcriptional activator. NBP-45 mRNA is expressed at low levels and in variable amounts in all mouse tissues tested but is especially abundant in RNA extracted from 7-day-old mouse embryos, suggesting that it functions in early embryonic development. NBP-45 is composed of 406 amino acids and is encoded by a single size transcript. The region spanning the N-terminal 85 amino acids contains three segments that are highly homologous to functionally important domains in the HMG-14/-17 protein family: the nuclear localization signal, the nucleosome binding domain, and the chromatin unfolding domain. The protein region spanning the C-terminal 321 amino acids has a 42% content of negatively charged residues. The first 23 amino acids contain a region necessary for nuclear entry of the protein, the region spanning residues 12-40 is the main nucleosomal binding domain of the protein, and the negatively charged, C-terminal domain is necessary for transcription activation. The functional domains of NBP-45 are indicative of a nuclear protein that binds to nucleosomes, thereby creating a chromatin region of high local negative charge. Our studies establish the nucleosomal binding domain as a protein motif that is present in other than just the ubiquitous HMG-14/-17 proteins. We suggest that the nucleosomal binding domain motif is a protein module that facilitates binding to nucleosomes in chromatin. PMID: 10692437 [PubMed - indexed for MEDLINE] PR75: Arch Biochem Biophys. 2000 Feb 15;374(2):286-92. Sterol regulatory element binding protein-mediated effect of fluvastatin on cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase transcription. Mascaro C, Ortiz JA, Ramos MM, Haro D, Hegardt FG. Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, E-08028, Spain. The effects of acute treatment with fluvastatin, a hypocholesteremic drug, on the mRNA levels of several regulatory enzymes of cholesterogenesis and of the LDL receptor were determined in rat liver. Fluvastatin increased the hepatic mRNA levels for HMG-CoA reductase up to 12-fold in 5 weeks of treatment at a daily dose of 6. 3 mg/kg. The effect was less marked in cytosolic HMG-CoA synthase, farnesyl-PP synthase, squalene synthetase, and LDL receptor. SREBP-2 mRNA levels were also increased, but SREBP-1 were not. De novo synthesis of cholesterol in several cultured cells was reduced by increasing concentrations of fluvastatin, and the IC(50) values of fluvastatin in HepG2, CV-1, and CHO cells were respectively 0.01, 0. 05, and 0.1 microM. When CHO cells stably transfected with a chimeric gene composed of the promoter of cytosolic HMG-CoA synthase and the CAT gene as a reporter were incubated with fluvastatin, the CAT gene was overexpressed, an effect which was similar to the cotransfection with the processed form of SREBP-1a. Both ALLN and fluvastatin increased the transcriptional activity of cytosolic HMG-CoA synthase. Mutation in either SRE or NF-Y boxes abolished the increase in transcriptional rate caused by fluvastatin in the promoter of cytosolic HMG-CoA synthase. These results indicate that the increase in transcriptional activity in the HMG-CoA synthase gene attributable to fluvastatin is a consequence of the activation of the proteolytic cleavage of SREBPs by reduced levels of intracellular cholesterol. Copyright 2000 Academic Press. PMID: 10666309 [PubMed - indexed for MEDLINE] NR76: Glia. 2000 Jan 15;29(2):118-23. Transcriptional control in myelinating glia: the basic recipe. Wegner M. Zentrum fur Molekulare Neurobiologie, Universitat Hamburg, Hamburg, Germany. Development of myelinating glia and the myelination process both require dramatic changes in the pattern of gene expression. During these processes, cells of the oligodendrocyte and Schwann cell lineages have to alter the transcription rates of numerous genes in a highly coordinated manner. This is made possible by the combined action of a set of transcription factors with preferential expression in these cells. Recent years have seen the identification of such glial transcription factors, including several homeodomain proteins, zinc finger proteins and HMG-domain proteins. Understanding their mode of action will help to unravel the molecular basis of myelination and will provide insights into the causes of myelinopathies and related diseases. Copyright 2000 Wiley-Liss, Inc. Publication Types: Review Review, Tutorial PMID: 10625329 [PubMed - indexed for MEDLINE] NR77: J Lipid Res. 1999 Nov;40(11):2099-110. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation. Raspe E, Madsen L, Lefebvre AM, Leitersdorf I, Gelman L, Peinado-Onsurbe J, Dallongeville J, Fruchart JC, Berge R, Staels B. U325, INSERM, Departement d'Atherosclerose/U325 INSERM, Institut Pasteur de Lille, 59019 Lille, France. 3-Thia fatty acids are modified fatty acids that promote hepatic peroxisome proliferation and decrease serum triacylglycerol, cholesterol and free fatty acid levels in rats. In vivo administration of tetradecylthioacetic acid (TTA) to rats led to a significant decrease in liver apolipoproteins apoA-I, A-II, A-IV, and C-III mRNA levels, and to an increase of liver acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-II, and 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA synthase) mRNA levels and activities. By contrast, no significant changes of lipoprotein lipase (LPL) mRNA levels were detected in rat epididymal adipose tissue. Liver carnitine palmitoyltransferase-I, apoB, apoE, and LDL receptor mRNA levels were not significantly affected. When tested in vitro, TTA increased rat ACO and carnitine palmitoyltransferase-I mRNA levels in primary rat hepatocytes and also LPL mRNA levels in 3T3-L1 preadipocytes. TTA also enhanced the transcriptional activity of chimeras containing the DNA binding domain of the yeast transcription factor Gal4 fused to the ligand binding domain of either human PPARalpha or human PPARgamma. The effect depended on the concentration tested and the cell type.In conclusion, our data suggest that in vitro, TTA activates both PPARalpha and PPARgamma, but the latter with much lower affinity. TTA affects serum lipid levels in vivo in rats by acting mainly on the liver via PPARalpha where it decreases the liver expression of genes involved in vascular lipid transport and increases the expression of genes involved in intracellular fatty acid metabolism. -Raspe, E., L. Madsen, A-M. Lefebvre, I. Leitersdorf, L. Gelman, J. Peinado-Onsurbe, J. Dallongeville, J-C. Fruchart, R. Berge, and B. Staels. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation. PMID: 10553013 [PubMed - indexed for MEDLINE] NR78: J Intern Med. 1999 Oct;246(4):399-407. Deoxycholic acid treatment in patients with cholesterol gallstones: failure to detect a suppression of cholesterol 7alpha-hydroxylase activity. Hillebrant C, Nyberg B, Angelin B, Axelson M, Bjorkhem I, Rudling M, Einarsson C. Department of Gastroenterology, Huddinge University Hospital, Sweden. Hillebrant C-G, Nyberg B, Angelin B, Axelson M, Bjorkhem I, Rudling M, Einarsson C (Huddinge University Hospital and Karolinska Hospital, Karolinska Institute, Stockholm, Sweden). Deoxycholic acid treatment in patients with cholesterol gallstones: failure to detect a suppression of cholesterol 7alpha-hydroxylase activity. J Intern Med 1999; 246: 399-407. OBJECTIVES: Based on animal studies, hydrophobic bile acids have been postulated to be particularly strong inhibitors of bile acid synthesis. The present study was undertaken to characterize in humans the effects of one of the most hydrophobic of the common bile acids, deoxycholic acid (DCA), on the transcriptional regulation and activity of the cholesterol 7alpha-hydroxylase, on hepatic cholesterol metabolism and on biliary lipid metabolism and plasma lipids. DESIGN, SUBJECTS AND SETTINGS: Thirteen patients with cholesterol gallstone disease were treated with DCA (750 mg day-1) for 3 weeks prior to cholecystectomy. Blood samples were collected before and during treatment. At operation, a liver biopsy and gallbladder bile were obtained. Twenty-eight untreated gallstone patients undergoing cholecystectomy served as controls. The study was carried out at a university hospital. RESULTS: Deoxycholic acid comprised 72 +/- 6% (mean +/- SEM) of total biliary bile acids in DCA-treated patients (n = 8), and 21 +/- 2% in the controls (n = 16; P < 0.001). Cholesterol saturation of gallbladder bile averaged 102% in both treated (n = 7) and untreated (n = 16) patients. Cholesterol 7alpha-hydroxylase and HMG CoA reductase activities and mRNA levels were not different between DCA-treated and untreated gallstone patients. The LDL receptor mRNA levels were similar in both groups of patients. Plasma levels of total cholesterol were lowered by 10% upon DCA treatment (P < 0.05). CONCLUSIONS: Treatment with DCA did not significantly affect mRNA levels and activity of hepatic cholesterol 7alpha-hydroxylase or HMG CoA reductase in patients with cholesterol gallstones. There was no effect on the saturation of gallbladder bile, Thus, the present study could not verify that the hydrophobicity of the bile acid pool is a major factor regulating human hepatic cholesterol 7alpha-hydroxylase activity. Publication Types: Clinical Trial Randomized Controlled Trial PMID: 10583711 [PubMed - indexed for MEDLINE] PR79: Adv Cancer Res. 2000;77:1-24. The Yin-Yang of TCF/beta-catenin signaling. Barker N, Morin PJ, Clevers H. Department of Immunology, University Hospital, Utrecht, The Netherlands. Wingless/Wnt signaling directs cell-fate choices during embryonic development. In Drosophila, Wingless signaling mediates endoderm induction and the establishment of segment polarity in the developing embryo. The fly Wingless cascade is strikingly similar to the vertebrate Wnt signaling pathway, which controls a number of key developmental decisions such as dorsal-ventral patterning in Xenopus. Factors of the TCF/LEF HMG domain family (Tcfs) have recently been established as the downstream effectors of the Wingless/Wnt signal transduction pathways. Upon Wingless/Wnt signaling, a cascade is initiated that results in the accumulation of cytoplasmic beta-catenin (or its fly homolog, Armadillo). There is also a concomitant translocation of beta-catenin/Armadillo to the nucleus, where it interacts with a specific sequence motif at the N terminus of Tcfs to generate a transcriptionally active complex. This bipartite transcription factor is targeted to the upstream regulatory regions of Tcf target genes including Siamois and Nodal related gene-3 in Xenopus, engrailed and Ultrabithorax in Drosophila via the sequence-specific HMG box, and mediates their transcriptional activation by virtue of transactivation domains contributed by beta-catenin/Armadillo. In the absence of Wingless/Wnt signals, a key negative regulator of the pathway, GSK3 beta, is activated, which mediates the downregulation of cytoplasmic beta-catenin/Armadillo via the ubiquitin-proteasome pathway. In the absence of nuclear beta-catenin, the Tcfs recruit the corepressor protein Groucho to the target gene enhancers and actively repress their transcription. An additional corepressor protein, CREB-binding protein (CBP), may also be involved in this repression of Tcf target gene activity. Several other proteins, including adenomatous polyposis coli (APC), GSK3 beta, and Axin/Conductin, are instrumental in the regulation of beta-catenin/Armadillo. In APC-deficient colon carcinoma cell lines, beta-catenin accumulates and is constitutively complexed with nuclear Tcf-4. A proportion of APC wild-type colon carcinomas and melanomas also contains constitutive nuclear Tcf-4/beta-catenin complexes as a result of dominant mutations in the N terminus of beta-catenin that render it insensitive to downregulation by APC, GSK3 beta, and Axin/Conductin. This results in the unregulated expression of Tcf-4 target genes such as c-myc. Based on the established role for Tcf-4 in maintaining intestinal stem cells it is likely that deregulation of c-myc expression as a result of constitutive Tcf-4/beta-catenin activity promotes uncontrolled intestinal cell proliferation. This would readily explain the formation of intestinal polyps during colon carcinogenesis. Similar mechanisms leading to deregulation of Tcf target gene activity are likely to be involved in melanoma and other forms of cancer. Publication Types: Review PMID: 10549354 [PubMed - indexed for MEDLINE] NR80: Mech Dev. 1999 Jun;84(1-2):103-20. Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: their expression during embryonic organogenesis of the chicken. Uchikawa M, Kamachi Y, Kondoh H. Institute for Molecular and Cellular Biology, Osaka University, Japan. Group B Sox genes, Sox1, -2 and -3 are known to activate crystallin genes and to be involved in differentiation of lens and neural tissues. Screening of chicken genomic sequences for more Group B Sox genes identified two additional genes, Sox14 and Sox21. Proteins encoded by Sox14 and Sox21 genes are similar to each other but distinct from those coded by Sox1-3 (subgroup B1) except for the HMG domain and Group B homology immediately C-proximal of the HMG domain. C-terminal domains of SOX21 and SOX14 proteins function as strong and weak repression domains, respectively, when linked to the GAL4 DNA binding domain. These SOX proteins strongly (SOX21) or moderately (SOX14) inhibited activation of delta1-crystallin DC5 enhancer by SOX1 or SOX2, establishing that Sox14 and Sox21 are repressing subgroup (B2) of Group B Sox genes. This provides the first evidence for the occurrence of repressor SOX proteins. Activating (B1) and repressing (B2) subgroups of Group B Sox genes display interesting overlaps of expression domains in developing tissues (e.g. optic tectum, spinal cord, inner ear, alimentary tract, branchial arches). Within each subgroup, most expression domains of Sox1 and -3 are included in those of Sox2 (e.g. CNS, PNS, inner ear), while co-expression of Sox14 and Sox21 occurs in highly restricted sites of the CNS, with the likely temporal order of Sox21 preceding Sox14 (e.g. interneurons of the spinal cord). These expression patterns suggest that target genes of Group B SOX proteins are finely regulated by the counterbalance of activating and repressing SOX proteins. PMID: 10473124 [PubMed - indexed for MEDLINE] NR81: Proc Soc Exp Biol Med. 1999 Sep;221(4):294-311. Isoprenoid-mediated inhibition of mevalonate synthesis: potential application to cancer. Elson CE, Peffley DM, Hentosh P, Mo H. Department of Nutritional Sciences, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. elson@nutrisci.wisc.edu Pure and mixed isoprenoid end products of plant mevalonate metabolism trigger actions that suppress 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity. These actions modulate HMG CoA reductase mRNA translation and the proteolytic degradation of HMG CoA reductase. Such post-transcriptional events, we propose, are activated directly by acyclic isoprenoids and indirectly by cyclic isoprenoids. Isoprenoids, acting secondarily to the dominant transcriptional effector of sterologenesis, modestly lower cholesterol levels, if and only if, sterologenesis is not repressed by a saturating imput of dietary cholesterol. An anomaly associated with tumor growth-a sterol feedback-resistant HMG CoA reductase activity-ensures a pool of sterologenic pathway intermediates. Such intermediates provide lipophilic anchors essential for membrane attachment and biological activity of growth hormone receptors, nuclear lamins A and B, and oncogenic ras. Tumor HMG CoA reductase retains high sensitivity to the isoprenoid-mediated secondary regulation. Repression of mevalonate synthesis by plant-derived isoprenoids reduces ras and lamin B processing, arrests cells in G1, and initiates cellular apoptosis. This unique tumor cell-specific sensitivity allows isoprenoids to be used for tumor therapy, an application emulating that of the statins, but one free of adverse effects. When evaluated at levels provided by a typical diet, isoprenoids individually have no impact on cholesterol synthesis and tumor growth. Nonetheless, isoprenoid-mediated activities are additive, and, sometimes synergistic. Therefore, the combined actions of the estimated 23,000 isoprenoid constituents of plant materials, acting in concert with other chemopreventive phytochemicals, may explain the lowered cancer risk associated with a diet rich in plant products. In contrast, that lowering of cancer risk does not correspond to supplemental intake of other dietary factors associated with fruits, vegetables, and cereal grains, namely fiber, beta-carotene, vitamin C, and vitamin E, and only weakly to supplemental folate. Publication Types: Review Review, Tutorial PMID: 10460692 [PubMed - indexed for MEDLINE] PR82: Development. 1999 Jun;126(14):3159-70. XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development. Brannon M, Brown JD, Bates R, Kimelman D, Moon RT. Department of Biochemistry, Howard Hughes Medical Institute, Center for Developmental Biology, University of Washington School of Medicine, Seattle, Washington 98195-7350, USA. kimelman@u.washington.edu. XTcf-3 is an HMG box transcription factor that mediates Xenopus dorsal-ventral axis formation. As a Wnt pathway effector, XTcf-3 interacts with beta-catenin and activates the expression of the dorsal organizing gene siamois, while in the absence of beta-catenin, XTcf-3 functions as a transcriptional repressor. We show that XTcf-3 contains amino- and carboxy-terminal repressor domains and have identified a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) as the C-terminal co-repressor. We show that two XCtBP binding sites near the XTcf-3 carboxy-terminus are required for the interaction of XTcf-3 and XCtBP and for the transcriptional repression mediated by the XTcf-3 carboxy-terminal domain. By fusing the GAL4 activation domain to XCtBP we have generated an antimorphic protein, XCtBP/G4A, that activates siamois transcription through an interaction with endogenous XTcf-3. Ectopic expression of XCtBP/G4A demonstrates that XCtBP functions in the regulation of head and notochord development. Our data support a role for XCtBP as a co-repressor throughout Xenopus development and indicate that XCtBP/G4A will be a useful tool in determining how XCtBP functions in various developmental processes. PMID: 10375506 [PubMed - indexed for MEDLINE] PR83: Annu Rev Immunol. 1999;17:149-87. Transcriptional regulation of T lymphocyte development and function. Kuo CT, Leiden JM. Department of Medicine, University of Chicago, Illinois 60637, USA. The development and function of T lymphocytes are regulated tightly by signal transduction pathways that include specific cell-surface receptors, intracellular signaling molecules, and nuclear transcription factors. Since 1988, several families of functionally important T cell transcription factors have been identified. These include the Ikaros, LKLF, and GATA3 zinc-finger proteins; the Ets, CREB/ATF, and NF-kappa B/Rel/NFAT transcription factors; the Stat proteins; and HMG box transcription factors such as LEF1, TCF1, and Sox4. In this review, we summarize our current understanding of the transcriptional regulation of T cell development and function with particular emphasis on the results of recent gene targeting and transgenic experiments. In addition to increasing our understanding of the molecular pathways that regulate T cell development and function, these results have suggested novel targets for genetic and pharmacological manipulation of T cell immunity. Publication Types: Review PMID: 10358756 [PubMed - indexed for MEDLINE] DR84: EMBO J. 1999 Jun 1;18(11):3074-89. The role of HMG I(Y) in the assembly and function of the IFN-beta enhanceosome. Yie J, Merika M, Munshi N, Chen G, Thanos D. Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, NY 10032, USA. Transcriptional activation of the virus inducible enhancer of the human interferon-beta (IFN-beta) gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappaB, ATF-2/c-Jun, IRFs and the architectural protein of the mammalian high mobility group I(Y) [HMG I(Y)]. Here, we demonstrate that the first step in enhanceosome assembly, i.e. HMG I(Y)-dependent recruitment of NF-kappaB and ATF-2/c-Jun to the enhancer, is facilitated by discrete regions of HMG I and is mediated by allosteric changes induced in the DNA by HMG I(Y) and not by protein-protein interactions between HMG I(Y) and these proteins. However, we show that completion of the enhanceosome assembly process requires protein-protein interactions between HMG I(Y) and the activators. Finally, we demonstrate that once assembled, the IFN-beta enhanceosome is an unusually stable nucleoprotein structure that can activate transcription at high levels by promoting multiple rounds of reinitiation of transcription. PMID: 10357819 [PubMed - indexed for MEDLINE] NR85: J Mol Endocrinol. 1999 Jun;22(3):295-304. Regulation of the expression and activity by progestins of a member of the SOX gene family of transcriptional modulators. Graham JD, Hunt SM, Tran N, Clarke CL. Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, New South Wales 2145, Australia. The mammalian testis-determining gene Sry and the related Sox genes define a family of transcriptional regulators widely expressed during embryogenesis. Tightly controlled temporal profiles of expression are a feature of the Sox gene family and may be required for initiation of a cascade of gene expression, yet the molecular mechanisms that control Sox gene expression are unknown. We now show that human SOX4 is expressed in the normal breast and in breast cancer cells. In these cells SOX4 is a progesterone-regulated gene, the expression of which is increased by progestins, leading to a marked increase in SOX-mediated transcriptional activity. Treatment of T-47D breast cancer cells with the synthetic progestin ORG 2058 directly increased SOX4 transcription, resulting in a 4-fold increase in SOX4 mRNA levels within 4 h of treatment. No effect of ORG 2058 was noted on other SOX genes measured, nor were other hormone-regulated HMG box proteins detected in this system, suggesting that the observed ability of progestin to increase SOX mRNA expression was confined to SOX4. The increase in SOX4 transcription was reflected in increased SOX4 protein expression, as progestin treatment of T-47D cells transfected with a SOX-responsive reporter resulted in a marked increase in reporter gene expression. Progesterone is essential for normal development and differentiation of the female reproductive system, plays an essential role in regulating growth and differentiation of the mammary gland and is required for opposing the proliferative effects of estrogen in specific cell types. The detection of SOX4 expression in the normal and malignant breast and the demonstration that SOX4 expression is under progesterone control suggests that changes in SOX4 gene expression may play a role in commitment to the differentiated phenotype in the normal and malignant mammary gland. PMID: 10343288 [PubMed - indexed for MEDLINE] NR86: J Biol Chem. 1999 May 14;274(20):14508-13. YY1 is a negative regulator of transcription of three sterol regulatory element-binding protein-responsive genes. Ericsson J, Usheva A, Edwards PA. Departments of Biological Chemistry and Medicine, UCLA, Los Angeles, California 90095, USA. Ying Yang 1 (YY1) is shown to bind to the proximal promoters of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, farnesyl diphosphate (FPP) synthase, and the low density lipoprotein (LDL) receptor. To investigate the potential effect of YY1 on the expression of SREBP-responsive genes, HepG2 cells were transiently transfected with luciferase reporter constructs under the control of promoters derived from either HMG-CoA synthase, FPP synthase, or the LDL receptor genes. The luciferase activity of each construct increased when HepG2 cells were incubated in lipid-depleted media or when the cells were cotransfected with a plasmid encoding mature sterol regulatory element-binding protein (SREBP)-1a. In each case, the increase in luciferase activity was attenuated by coexpression of wild-type YY1 but not by coexpression of mutant YY1 proteins that are known to be defective in either DNA binding or in modulating transcription of other known YY1-responsive genes. In contrast, incubation of cells in lipid-depleted media resulted in induction of an HMG-CoA reductase promoter-luciferase construct by a process that was unaffected by coexpression of wild-type YY1. Electromobility shift assays were used to demonstrate that the proximal promoters of the HMG-CoA synthase, FPP synthase, and the LDL receptor contain YY1 binding sites and that YY1 displaced nuclear factor Y from the promoter of the HMG-CoA synthase gene. We conclude that YY1 inhibits the transcription of specific SREBP-dependent genes and that, in the case of the HMG-CoA synthase gene, this involves displacement of nuclear factor Y from the promoter. We hypothesize that YY1 plays a regulatory role in the transcriptional regulation of specific SREBP-responsive genes. PMID: 10318878 [PubMed - indexed for MEDLINE] NR87: Gene. 1999 Apr 1;230(1):1-5. Plant chromosomal HMGI/Y proteins and histone H1 exhibit a protein domain of common origin. Krech AB, Wulff D, Grasser KD, Feix G. Institut fur Biologie III, Albert-Ludwigs-Universitat Freiburg, Schanzlestr. 1, D-79104, Freiburg, Germany. The chromosomal high-mobility-group (HMG) proteins of the HMGI/Y family interact with A/T-rich stretches in duplex DNA, and are considered assistant factors in transcriptional regulation. A cDNA encoding an HMGI/Y protein of 190 amino acid residues was isolated from maize and characterized. Like other plant HMGI/Y proteins, the maize HMGI/Y protein contains four copies of the AT-hook DNA-binding motif and an amino-terminal 'histone H1-like region' with a similarity to the globular domain of H1. The maize hmgi/y gene that was isolated from a genomic DNA library contains a single intron that is localized in the region of sequence similarity to histone H1. Interestingly, the genes encoding plant H1 contain an intron at exactly the same relative position, indicating an evolutionary relationship of the plant genes encoding HMGI/Y and H1 proteins. PMID: 10196467 [PubMed - indexed for MEDLINE] NR88: J Surg Res. 1999 Apr;82(2):194-200. Transcriptional arrest of the human E-selectin gene. Boyle EM Jr, Sato TT, Noel RF Jr, Verrier ED, Pohlman TH. Department of Surgery, The University of Washington, Seattle, Washington, 98104-9796, USA. BACKGROUND. E-selectin transcription requires binding of transcription factors, NF-kappaB, ATF-2, and HMG-I(Y). Here we characterize the mechanism responsible for the transcriptional downregulation of E-selectin expression. MATERIALS AND METHODS. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. HUVEC E-selectin expression was measured by enzyme-linked immunosorbent assay, Northern blotting, and nuclear run-on assays, and NF-kappa B was assessed by electrophoretic gel mobility shift assays (EMSAs). RESULTS. (1) E-selectin surface expression peaked at 4 h and then diminished over the next 20 h. (2) Transcription of E-selectin began within 1 h of TNF-alpha exposure and ceased by 8 h, despite continuous stimulation of HUVECs with TNF-alpha. (3) EMSAs revealed persistent binding activity of NF-kappa B proteins to two NF-kappa B-binding sites during 24 h of continuous stimulation with TNF-alpha. However, binding activity of proteins that recognize a third NF-kappa B element, -126 to -116 bp from the transcription start site, was lost after 4 h during 24 h of continuous stimulation with TNF-alpha; ATF-2 binding was unchanged over 24 h stimulation with TNF-alpha. CONCLUSION. The termination of E-selectin expression is controlled at the level of transcription, with loss of protein-DNA interactions at only one of three NF-kappa B-binding sites in the E-selectin promoter. Copyright 1999 Academic Press. PMID: 10090829 [PubMed - indexed for MEDLINE] NR89: Mol Cell Biol. 1999 Apr;19(4):2872-9. Recruitment of TATA-binding protein-TAFI complex SL1 to the human ribosomal DNA promoter is mediated by the carboxy-terminal activation domain of upstream binding factor (UBF) and is regulated by UBF phosphorylation. Tuan JC, Zhai W, Comai L. Department of Molecular Microbiology and Immunology and Norris Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles, California 90033, USA. Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any sequence-specific DNA binding activity, and its recruitment to the promoter is mediated by specific protein interactions with UBF. Once on the promoter, the SL1 complex makes direct contact with the DNA promoter and directs promoter-specific initiation of transcription. To investigate the mechanism of UBF-dependent transcriptional activation, we first performed protein-protein interaction assays between SL1 and a series of UBF deletion mutants. This analysis indicated that the carboxy-terminal domain of UBF, which is necessary for transcriptional activation, makes direct contact with the TBP-TAFI complex SL1. Since this region of UBF can be phosphorylated, we then tested whether this modification plays a functional role in the interaction with SL1. Alkaline phosphatase treatment of UBF completely abolished the ability of UBF to interact with SL1; moreover, incubation of the dephosphorylated UBF with nuclear extracts from exponentially growing cells was able to restore the UBF-SL1 interaction. In addition, DNase I footprinting analysis and in vitro-reconstituted transcription assays with phosphatase-treated UBF provided further evidence that UBF phosphorylation plays a critical role in the regulation of the recruitment of SL1 to the ribosomal DNA promoter and stimulation of UBF-dependent transcription. PMID: 10082553 [PubMed - indexed for MEDLINE] NR90: Mol Cell Biol. 1999 Apr;19(4):2613-23. Multiple layers of cooperativity regulate enhanceosome-responsive RNA polymerase II transcription complex assembly. Ellwood K, Huang W, Johnson R, Carey M. Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737, USA. Two coordinate forms of transcriptional synergy mediate eukaryotic gene regulation: the greater-than-additive transcriptional response to multiple promoter-bound activators, and the sigmoidal response to increasing activator concentration. The mechanism underlying the sigmoidal response has not been elucidated but is almost certainly founded on the cooperative binding of activators and the general machinery to DNA. Here we explore that mechanism by using highly purified transcription factor preparations and a strong Epstein-Barr virus promoter, BHLF-1, regulated by the virally encoded activator ZEBRA. We demonstrate that two layers of cooperative binding govern transcription complex assembly. First, the architectural proteins HMG-1 and -2 mediate cooperative formation of an enhanceosome containing ZEBRA and cellular Sp1. This enhanceosome then recruits transcription factor IIA (TFIIA) and TFIID to the promoter to form the DA complex. The DA complex, however, stimulates assembly of the enhanceosome itself such that the entire reaction can occur in a highly concerted manner. The data reveal the importance of reciprocal cooperative interactions among activators and the general machinery in eukaryotic gene regulation. PMID: 10082527 [PubMed - indexed for MEDLINE] PR91: Biochem J. 1999 Mar 15;338 ( Pt 3):569-82. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis. Hegardt FG. Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Av/ Diagonal 643, 08028 Barcelona, Spain. hegardt@farmacia.far.ub.es Cytosolic and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases were first recognized as different chemical entities in 1975, when they were purified and characterized by Lane's group. Since then, the two enzymes have been studied extensively, one as a control site of the cholesterol biosynthetic pathway and the other as an important control site of ketogenesis. This review describes some key developments over the last 25 years that have led to our current understanding of the physiology of mitochondrial HMG-CoA synthase in the HMG-CoA pathway and in ketogenesis in the liver and small intestine of suckling animals. The enzyme is regulated by two systems: succinylation and desuccinylation in the short term, and transcriptional regulation in the long term. Both control mechanisms are influenced by nutritional and hormonal factors, which explains the incidence of ketogenesis in diabetes and starvation, during intense lipolysis, and in the foetal-neonatal and suckling-weaning transitions. The DNA-binding properties of the peroxisome-proliferator-activated receptor and other transcription factors on the nuclear-receptor-responsive element of the mitochondrial HMG-CoA synthase promoter have revealed how ketogenesis can be regulated by fatty acids. Finally, the expression of mitochondrial HMG-CoA synthase in the gonads and the correction of auxotrophy for mevalonate in cells deficient in cytosolic HMG-CoA synthase suggest that the mitochondrial enzyme may play a role in cholesterogenesis in gonadal and other tissues. Publication Types: Review PMID: 10051425 [PubMed - indexed for MEDLINE] NR92: Cytogenet Cell Genet. 1998;83(1-2):139-46. Characterisation and mapping of the human SOX14 gene. Arsic N, Rajic T, Stanojcic S, Goodfellow PN, Stevanovic M. Institute of Molecular Genetics and Genetic Engineering, Belgrade (Yugoslavia). SOX genes comprise a family of genes that are related to the mammalian sex determining gene SRY in the region that encodes the HMG-box domain responsible for the sequence-specific DNA-binding activity. SOX genes encode putative transcriptional regulators implicated in the decision of cell fates during development and the control of diverse developmental processes. We have cloned and characterised SOX14, a novel member of the human SOX gene family. Based on the HMG-box sequence, human SOX14 is a member of the B subfamily. SOX14 is expressed in human foetal brain, spinal cord and thymus, and like other members of the B subfamily, it might have a role in regulation of nervous system development. While other members of the B subfamily show similarity outside the HMG-box, the regions flanking the HMG box of the human SOX14 gene are unique. SOX14 has been mapped to human chromosome 3q22--> q23, close to the marker D3S1549. This location places SOX14 within a chromosome interval associated with two distinct syndromes that affect craniofacial development: Blepharophimosis-ptosis-epicantus inversus syndrome and Mobius syndrome. PMID: 9925951 [PubMed - indexed for MEDLINE] DR93: Biochimie. 1998 Oct;80(10):803-6. Transcriptional regulation of mitochondrial HMG-CoA synthase in the control of ketogenesis. Hegardt FG. Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Spain. Mitochondrial and cytosolic HMG-CoA synthases are encoded by two different genes. Control of ketogenesis is exerted by transcriptional regulation of mitochondrial HMG-CoA synthase. Fasting, cAMP, and fatty acids increase its transcriptional rate, while refeeding and insulin repress it. Fatty acids increase transcription through peroxisomal proliferator regulatory element (PPRE), to which peroxisome proliferator activated receptor (PPAR) can bind. Other transcription factors such as chicken ovalbumin upstream promoter transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF-4) compete for the PPRE site, modulating the response of PPAR. Publication Types: Review Review, Tutorial PMID: 9893938 [PubMed - indexed for MEDLINE] NR94: Dev Genet. 1998;23(4):324-34. DSP1 gene of Drosophila melanogaster encodes an HMG-domain protein that plays multiple roles in development. Mosrin-Huaman C, Canaple L, Locker D, Decoville M. Centre de Biophysique Moleculaire, CNRS, Orleans, France. DSP1 is an HMG-box containing protein of Drosophila melanogaster which was first identified as a co-repressor of the Dorsal protein. Recently, the analysis of the structure of the gene has led us to propose that DSP1 is the Drosophila equivalent of the ubiquitous vertebrate HMG 1/2 proteins. In the present paper, the patterns of expression of DSP1 protein and RNA in adult flies and during development are reported. In the adults DSP1 protein is located in nurse cells of ovaries and in brain. During eggs development uniform expression of DSP1 protein persists until the end of germband retraction. At later stages, expression is restricted to the ventral nerve chord and brain. Using P-element mutagenesis, we have isolated a mutant deficient in DSP1 functions. Genetic studies of this mutant show that DSP1 protein is essential for the growth and the development of Drosophila. In addition to be a co-repressor of the transcriptional activator Dorsal our results provide compelling evidence that DSP1 is a regulator involved in several pathways necessary for the development of the fly. PMID: 9883584 [PubMed - indexed for MEDLINE] DR95: J Biol Chem. 1999 Jan 15;274(3):1525-32. Induction of high mobility group-I(Y) protein by endotoxin and interleukin-1beta in vascular smooth muscle cells. Role in activation of inducible nitric oxide synthase. Pellacani A, Chin MT, Wiesel P, Ibanez M, Patel A, Yet SF, Hsieh CM, Paulauskis JD, Reeves R, Lee ME, Perrella MA. Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA. Nonhistone chromosomal proteins of the high mobility group (HMG) affect the transcriptional regulation of certain mammalian genes. For example, HMG-I(Y) controls cytokine-mediated promoters that require transcription factors, such as nuclear factor-kappaB, for maximal expression. Even though a great deal is known about how HMG-I(Y) facilitates expression of other genes, less is known about the regulation of HMG-I(Y) itself, especially in cells in primary culture. Therefore we investigated the effect of endotoxin and the cytokine interleukin-1beta on HMG-I(Y) expression in vascular smooth muscle cells. Induction of HMG-I(Y) peaked after 48 h of interleukin-1beta stimulation (6.2-fold) in cells in primary culture, and this increase in mRNA corresponded to an increase in HMG-I(Y) protein. Moreover, immunohistochemical staining revealed a dramatic increase in HMG-I(Y) protein expression in vascular smooth muscle cells after endotoxin stimulation in vivo. This increase in HMG-I(Y) expression (both in vitro and in vivo) mirrored an up-regulation of inducible nitric oxide synthase, a cytokine-responsive gene. The functional significance of this coinduction is underscored by our finding that HMG-I(Y) potentiated the response of inducible nitric oxide synthase to nuclear factor-kappaB transactivation. Taken together, these studies suggest that induction of HMG-I(Y), and subsequent transactivation of iNOS, may contribute to a reduction in vascular tone during endotoxemia and other systemic inflammatory processes. PMID: 9880529 [PubMed - indexed for MEDLINE] NR96: Genetics. 1999 Jan;151(1):45-55. The MSN1 and NHP6A genes suppress SWI6 defects in Saccharomyces cerevisiae. Sidorova J, Breeden L. Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. Ankyrin (ANK) repeats were first found in the Swi6 transcription factor of Saccharomyces cerevisiae and since then were identified in many proteins of eukaryotes and prokaryotes. These repeats are thought to serve as protein association domains. In Swi6, ANK repeats affect DNA binding of both the Swi4/Swi6 and Mbp1/Swi6 complexes. We have previously described generation of random mutations within the ANK repeats of Swi6 that render the protein temperature sensitive in its ability to activate HO transcription. Two of these SWI6 mutants were used in a screen for high copy suppressors of this phenotype. We found that MSN1, which encodes a transcriptional activator, and NHP6A, which encodes an HMG-like protein, are able to suppress defective Swi6 function. Both of these gene products are involved in HO transcription, and Nhp6A may also be involved in CLN1 transcription. Moreover, because overexpression of NHP6A can suppress caffeine sensitivity of one of the SWI6 ANK mutants, swi6-405, other SWI6-dependent genes may also be affected by Nhp6A. We hypothesize that Nhp6A and Msn1 modulate Swi6-dependent gene transcription indirectly, through effects on chromatin structure or other transcription factors, because we have not been able to demonstrate that either Msn1 or Nhp6A interact with the Swi4/Swi6 complex. PMID: 9872947 [PubMed - indexed for MEDLINE] NR97: J Lipid Res. 1999 Jan;40(1):24-38. HMG-CoA reductase regulation: use of structurally diverse first half-reaction squalene synthetase inhibitors to characterize the site of mevalonate-derived nonsterol regulator production in cultured IM-9 cells. Petras SF, Lindsey S, Harwood HJ Jr. Department of Metabolic Diseases, Pfizer Central Research, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA. The activity of HMG-CoA reductase (HMGR) is tightly regulated, in part through post-transcriptional mechanisms that are mediated by nonsterol products of mevalonate metabolism. Previous reports have suggested that these mediators are derived from farnesyl pyrophosphate (FPP). Recent studies have implicated FPP hydrolysis products (e.g., farnesol), the squalene synthetase (SQS) reaction products presqualene pyrophosphate (PSQPP) and squalene, or their metabolites. To distinguish among these possible mediators, we evaluated the ability of HMGR and SQS inhibitors to induce compensatory increases in HMGR activity in cultured IM-9 cells. Mevinolin (HMGR inhibitor) produced predicted increases in HMGR activity that were related to the degree of cholesterolgenesis inhibition (e.g., 4-fold, 9-fold, and 17-fold increases relative to 50%, 76%, and 90% inhibition, respectively). By contrast, a variety of structurally distinct reversible, competitive, first half-reaction SQS inhibitors all reduced cholesterolgenesis by up to 90% with no appreciable increases in HMGR activity.These observations strongly suggest that nonsterol-mediated post-transcriptional mechanisms regulating HMGR activity remain intact after SQS first half-reaction inhibition, indicating that nonsterol regulator production is independent of SQS action and ruling out PSQPP, squalene and their metabolites as possible mediators. Unexpectedly, the SQS mechanism-based irreversible inactivator, zaragozic acid A (ZGA) exhibited the greatest degree of HMGR modulation, producing 5-fold, 11-fold, and 40-fold increases in HMGR activity at concentrations that produced 25%, 50%, and 75% cholesterolgenesis inhibition, respectively. The markedly greater magnitude of HMGR stimulation by ZGA versus mevinolin at similar levels of cholesterolgenesis inhibition suggests that ZGA may directly interfere with the production or action of the nonsterol regulator. PMID: 9869647 [PubMed - indexed for MEDLINE] NR98: Cell Struct Funct. 1998 Aug;23(4):187-92. Characterization of two Chinese hamster ovary cell lines expressing the COOH-terminal domains of sterol regulatory element-binding protein (SREBP)-1. Kawabe Y, Imanaka T, Kodama T, Takano T, Sato R. Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan. Sterol regulatory element-binding proteins (SREBPs) regulate transcription of genes encoding enzymes in the cholesterol biosynthetic pathway and the LDL receptor. These proteins are synthesized as membrane-bound precursors and processed to generate the NH2-terminal domains, mature transcription factors. We established two Chinese hamster ovary (CHO) cell lines, CHO-421 expressing the truncated hamster SREBP-1 (amino acids 421-1133) with two transmenbrane domains and CHO-557 expressing another truncated SREBP-1 (amino acids 557-1133) without any transmembrane domains, to investigate the fate of the COOH terminus after cleavage of the NH2-terminal mature SREBP. The cell fractionation experiments revealed that the two proteins, regardless of the absence of transmembrane domains in the SREBP (557-1133), similarly localized in the nuclear envelope and the microsomal membrane fractions, suggesting that these proteins appear to be tightly bound to a membrane protein(s) localizing on the nuclear and endoplasmic reticulum (ER) membranes. Although we predicted that overexpression of the COOH-terminal domains, which were thought to be involved in the regulation of SREBP processing, would result in disruption of the SREBP-dependent transcriptional regulation of several genes, the mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase in these two cell lines were regulated in a sterol-dependent manner. Pulse-chase experiments revealed that the SREBP (421-1133) was relatively rapidly degraded (t1/2 = 4-6 hr) and that the intracellular cholesterol level did not affect the half-life time. The degradation of the SREBP (421-1133) was not suppressed by the treatment of a calpain inhibitor, N-acetyl-leucyl-leucyl-norleucynal (ALLN), which blocks the proteolysis of some proteins within or near the ER. In CHO-557 cells the SREBP (557-1133) was much more rapidly degraded (t1/2 = 1-2 hr), suggesting that the cytosolic COOH-terminal domain is accessible to the enzymatic attacks from the cytoplasm. Taken together, overexpression of the COOH-terminal domains does not affect the regulation of SREBP processing and the domains are rapidly turned over by the cytosolic proteolytic process distinct from the ALLN-sensitive ER degradative pathway. PMID: 9855111 [PubMed - indexed for MEDLINE] NR99: EMBO J. 1998 Dec 1;17(23):6972-8. A mitochondrial ketogenic enzyme regulates its gene expression by association with the nuclear hormone receptor PPARalpha. Meertens LM, Miyata KS, Cechetto JD, Rachubinski RA, Capone JP. Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMG-CoAS) is a key enzyme in ketogenesis, catalyzing the condensation of acetyl-CoA and acetoacetyl-CoA to generate HMG-CoA, which is eventually converted to ketone bodies. Transcription of the nuclear-encoded gene for mHMG-CoAS is stimulated by peroxisome proliferator-activated receptor (PPAR) alpha, a fatty acid-activated nuclear hormone receptor. Here we show that the mHMG-CoAS protein physically interacts with PPARalpha in vitro, and potentiates PPARalpha-dependent transcriptional activation via the cognate PPAR response element of the mHMG-CoAS gene in vivo. Immunofluorescence of transiently transfected cells demonstrated that in the presence of PPARalpha, mHMG-CoAS is translocated into the nucleus. Binding to PPARalpha, stimulation of PPARalpha activity and nuclear penetration require the integrity of the sequence LXXLL in mHMG-CoAS, a motif known to mediate the interaction between nuclear hormone receptors and coactivators. These findings reveal a novel mechanism of gene regulation whereby the product of a PPARalpha-responsive gene, normally resident in the mitochondria, directly interacts with this nuclear hormone receptor to autoregulate its own nuclear transcription. PMID: 9843503 [PubMed - indexed for MEDLINE] NR100: Mol Cell Biol. 1998 Dec;18(12):7317-26. The mating-type proteins of fission yeast induce meiosis by directly activating mei3 transcription. Van Heeckeren WJ, Dorris DR, Struhl K. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. Cell type control of meiotic gene regulation in the budding yeast Saccharomyces cerevisiae is mediated by a cascade of transcriptional repressors, a1-alpha2 and Rme1. Here, we investigate the analogous regulatory pathway in the fission yeast Schizosaccharomyces pombe by analyzing the promoter of mei3, the single gene whose expression is sufficient to trigger meiosis. The mei3 promoter does not appear to contain a negative regulatory element that represses transcription in haploid cells. Instead, correct regulation of mei3 transcription depends on a complex promoter that contains at least five positive elements upstream of the TATA sequence. These elements synergistically activate mei3 transcription, thereby constituting an on-off switch for the meiosis pathway. Element C is a large region containing multiple sequences that resemble binding sites for Mc, an HMG domain protein encoded by the mating-type locus. The function of element C is extremely sensitive to spacing changes but not to linker-scanning mutations, suggesting the possibility that Mc functions as an architectural transcription factor. Altered-specificity experiments indicate that element D interacts with Pm, a homeodomain protein encoded by the mating-type locus. This indicates that Pm functions as a direct activator of the meiosis pathway, whereas the homologous mating-type protein in S. cerevisiae (alpha2) functions as a repressor. Thus, despite the strong similarities between the mating-type loci of S. cerevisiae and S. pombe, the regulatory logic that governs the tight control of the key meiosis-inducing genes in these organisms is completely different. PMID: 9819418 [PubMed - indexed for MEDLINE] PR101: Mol Cell. 1998 Oct;2(4):457-67. Acetylation of HMG I(Y) by CBP turns off IFN beta expression by disrupting the enhanceosome. Munshi N, Merika M, Yie J, Senger K, Chen G, Thanos D. Department of Biochemistry, and Molecular Biophysics, Columbia University, New York, New York 10032, USA. The transcriptional coactivators CBP and P/CAF are required for activation of transcription from the IFN beta enhanceosome. We show that CBP and P/CAF acetylate HMG I(Y), the essential architectural component required for enhanceosome assembly, at distinct lysine residues, causing distinct effects on transcription. Thus, in the context of the enhanceosome, acetylation of HMG I by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly. We demonstrate that acetylation of HMG I(Y) by CBP is essential for turning off IFN beta gene expression. Finally, we show that the acetyltransferase activities of CBP and P/CAF modulate both the strength of the transcriptional response and the kinetics of virus-dependent activation of the IFN beta gene. PMID: 9809067 [PubMed - indexed for MEDLINE] NR102: Dev Dyn. 1998 Oct;213(2):159-69. Alx-4, a transcriptional activator whose expression is restricted to sites of epithelial-mesenchymal interactions. Hudson R, Taniguchi-Sidle A, Boras K, Wiggan O, Hamel PA. Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada. We have recently demonstrated that the retinoblastoma family of negative cell cycle regulators can form complexes with a class of developmental factors which contain paired-like (PL) homeodomains (Wiggan et al. [1998] Oncogene 16:227-236). Our screens led to the isolation of a novel PL-homeodomain protein which had been isolated independently by another group and called Alx-4 (Qu et al. [1997] Development 124:3999-4008). Mice homozygous for a targeted null mutation of Alx-4 have several abnormalities, including preaxial polydactyly, suggesting that Alx-4 plays a role in pattern formation in limb buds. In data that we present here, we show that Alx-4 is expressed in mesenchymal condensations of a diverse group of tissues whose development is dependent on epithelial-mesenchymal interactions, many of which are additionally dependent on expression of the HMG-box-containing protein, LEF-1. Alx-4-expressing tissues include osteoblast precursors of most bones, the dermal papilla of hair and whisker follicles, the dental papilla of teeth, and a subset of mesenchymal cells in pubescent mammary glands. We show further that Alx-4 strongly activates transcription from a promoter containing the homeodomain binding site, P2. Optimal activation requires specific sequences in the N-terminal portion of Alx-4 as well as a proline-rich region downstream of the PL-homeodomain, but not the paired-tail at the C terminus. Taken together, our results demonstrate that Alx-4 is a potent transcriptional activator that is expressed at sites of epithelial-mesenchymal interactions during murine embryonic development. PMID: 9786416 [PubMed - indexed for MEDLINE] PR103: Mol Reprod Dev. 1998 Oct;51(2):218-24. Effects of differentiation on the transcriptional regulation of the FGF-4 gene: critical roles played by a distal enhancer. Lamb KA, Rizzino A. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA. Embryonal carcinoma (EC) cells are used widely as a model system for studying the expression of developmentally regulated genes, in particular genes that are regulated at the transcriptional level when EC cells differentiate. This review focuses on the molecular mechanisms that govern the transcription of the fibroblast growth factor-4 (FGF-4) gene, which appears to be the first FGF expressed during mammalian development. Interest in this gene has increased considerably with the finding that FGF-4 is essential for mammalian embryogenesis. The FGF-4 gene has also generated considerable interest because it is inhibited at the transcriptional level when EC cells undergo differentiation and because this gene is regulated by a powerful distal enhancer located 3 kb downstream of the transcription start site in the last exon of the gene. Hence, study of the FGF-4 gene is likely to shed light on the molecular mechanisms by which distal enhancers regulate gene expression. In addition to being regulated by the downstream enhancer, the expression of this gene is influenced by a regulatory region located just upstream of the transcription start site, which contains two Sp1 motifs and a CCAAT box motif. Examination of the downstream enhancer has identified three functional cis-regulatory elements: a high mobility group (HMG) protein binding motif, an octamer binding motif, and an Sp1 motif, which are likely to bind Sox-2, Oct-3, and Sp1/Sp3, respectively, in vivo. Interestingly, Sox-2 and Oct-3 expression, like FGF-4 expression, decreases when EC cells differentiate, which suggests that the loss of these transcription factors is responsible, at least in part, for the transcriptional turn-off of the FGF-4 gene. In view of these and other findings, we present a model for the differential expression of the FGF-4 gene that includes not only the contributions of specific transcription factors, but also the contribution of chromatin structure before and after differentiation. Publication Types: Review Review, Tutorial PMID: 9740330 [PubMed - indexed for MEDLINE] NR104: J Biol Chem. 1998 Sep 11;273(37):24266-71. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. Laufs U, Liao JK. Cardiovascular Division, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity. PMID: 9727051 [PubMed - indexed for MEDLINE] NR105: FEBS Lett. 1998 Jul 17;431(2):180-4. The 5'-AMP-activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response complex. Leclerc I, Kahn A, Doiron B. Institut Cochin de Genetique Moleculaire, INSERM, Unite 129, Paris, France. 5-Amino-4-imidazolecarboxamide riboside (AICAR) is known to stimulate rat liver 5'-AMP-activated protein kinase (AMPK). AMPK is the mammalian homologue of Snf1p in yeast, involved in derepression of glucose-repressed genes. We used AICAR to test if AMPK could also play a role in the regulation of glucose-dependent genes in mammalian cells. At a concentration which induces phosphorylation-dependent inactivation of HMG-CoA reductase, AICAR blocked glucose activation of three glucose responsive genes, namely L-type pyruvate kinase (L-PK), Spot 14 and fatty acid synthase genes in primary cultured hepatocytes, but was without any action on glucose phosphorylation to glucose 6-phosphate and on expression of PEPCK, albumin and beta-actin genes. AICAR was also found to inhibit activation of the L-PK gene promoter by glucose in transiently transfected hepatoma cells. Therefore our results suggest that AMPK is probably involved in the glucose signal pathway regulating gene expression in the liver. PMID: 9708898 [PubMed - indexed for MEDLINE] NR106: Mol Cell. 1998 Jan;1(2):277-87. Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Merika M, Williams AJ, Chen G, Collins T, Thanos D. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA. Transcriptional activation of the IFN beta gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappa B, IRF1, ATF2/c-Jun, and the architectural protein HMG I(Y). The level of transcription generated by all of these activators is greater than the sum of the levels generated by individual factors, a phenomenon designated transcriptional synergy. We demonstrate that this synergy, in the context of the enhanceosome, requires a new protein-protein interaction domain in the p65 subunit of NF-kappa B. Transcriptional synergy requires recruitment of the CBP/p300 coactivator to the enhanceosome, via a new activating surface assembled from the novel p65 domain and the activation domains of all of the activators. Deletion, substitution, or rearrangement of any one of the activation domains in the context of the enhanceosome decreases both recruitment of CBP and transcriptional synergy. PMID: 9659924 [PubMed - indexed for MEDLINE] NR107: Mol Cell. 1997 Dec;1(1):119-29. The mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome. Kim TK, Maniatis T. Harvard University, Department of Molecular and Cellular Biology, Cambridge, Massachusetts 02138, USA. A functional interferon-beta gene enhanceosome was assembled in vitro using the purified recombinant transcriptional activator proteins ATF2/c-JUN, IRF1, and p50/p65 of NF-kappa B. Maximal levels of transcriptional synergy between these activators required the specific interactions with the architectural protein HMG I(Y) and the correct helical phasing of the binding sites of these proteins on the DNA helix. Analyses of the in vitro assembled enhanceosome revealed that the transcriptional synergy is due, at least in part, to the cooperative assembly and stability of the complex. Reconstitution experiments showed that the formation of a stable enhanceosome-dependent preinitiation complex require cooperative interactions between the enhanceosome; the general transcription factors TFID, TFIIA, and TFIIB; and the cofactor USA. These studies provide a direct biochemical demonstration of the importance of the structure and function of natural multicomponent transcriptional enhancer complexes in gene regulation. PMID: 9659909 [PubMed - indexed for MEDLINE] NR108: J Biol Chem. 1998 Jul 10;273(28):17865-70. CBP is required for sterol-regulated and sterol regulatory element-binding protein-regulated transcription. Ericsson J, Edwards PA. Departments of Biological Chemistry and Medicine, UCLA, Los Angeles, California 90095, USA. Cells were transfected with luciferase reporter genes, under the control of promoters derived from either the farnesyl diphosphate (FPP) synthase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, or low density lipoprotein receptor genes. The increase in luciferase activity that occurred when cells were either incubated in sterol-depleted medium or cotransfected with a cDNA encoding sterol regulatory element-binding protein (SREBP)-1a was prevented by coexpression of wild-type E1A or a Gal4-CBP (1-451) fusion protein. The inhibitory effect of E1A was overcome by coexpression of CBP. The increase in reporter gene activity noted above was not affected when the cells were cotransfected with cDNAs that encoded either a mutant E1A that is unable to interact with the transcriptional activator CBP or Gal4-CBP fusion proteins encoding separate fragments of CBP, which span the remainder of the CBP molecule. A preformed SREBP-1a:[32P]DNA complex bound specifically to membrane-immobilized GST-CBP fusion proteins that contained amino-terminal portions of CBP. In order to investigate the role of CBP in the regulation of endogenous genes, we isolated stable transformants that express Gal4-CBP(1-451) in response to added doxycycline. Induction of endogenous FPP synthase and HMG-CoA synthase mRNAs, in response to cellular cholesterol depletion, was prevented when cells expressed Gal4-CBP(1-451). We conclude that when cells are incubated in the absence of sterols, the transcriptional activation of the HMG-CoA synthase, HMG-CoA reductase, FPP synthase, and low density lipoprotein receptor genes is dependent on a specific interaction between SREBP, which is bound to the promoter DNA, and the amino-terminal domain (amino acids 1-451) of CBP. PMID: 9651391 [PubMed - indexed for MEDLINE] NR109: Gene. 1998 May 12;211(2):251-7. Rainbow trout Sox24, a novel member of the Sox family, is a transcriptional regulator during oogenesis. Kanda H, Kojima M, Miyamoto N, Ito M, Takamatsu N, Yamashita S, Shiba T. Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan. We isolated a cDNA clone encoding a novel SRY-type HMG box (Sox) protein, designated Sox24, from a rainbow trout ovary cDNA library. On the basis of the HMG box amino acid sequence, Sox24 can be categorized into the same subgroup of Sox proteins as SOX4, SOX11, and SOX22. The proteins in this group also share a highly conserved sequence at the C-terminus. The Sox24 mRNA is expressed at high levels in the ovary, and in-situ hybridization localized its expression to oocytes. The recombinant protein containing the Sox24 HMG box region bound to an AACAAT sequence strongly in a gel retardation assay. Upon co-transfection into CHO cells, the full-length Sox24 transactivated transcription from a reporter plasmid through the AACAAT binding motif. We used GAL4/Sox24 chimeras with the DNA binding domain of yeast GAL4 at the N-terminus to map the transactivation function to the C-terminal region, which included the conserved sequence. These results suggest that Sox24 plays a role as a transcriptional regulator during oogenesis. PMID: 9602142 [PubMed - indexed for MEDLINE] NR110: Mech Dev. 1998 May;73(2):169-82. Gene regulatory functions of Drosophila fish-hook, a high mobility group domain Sox protein. Ma Y, Niemitz EL, Nambu PA, Shan X, Sackerson C, Fujioka M, Goto T, Nambu JR. Neuroscience and Behavior Program, University of Massachusetts, Amherst, MA 01003, USA. In this study we investigate the gene regulatory functions of Drosophila Fish-hook (Fish), a high mobility group (HMG) Sox protein that is essential for embryonic segmentation. We show that the Fish HMG domain binds to the vertebrate Sox protein consensus DNA binding sites, AACAAT and AACAAAG, and that this binding induces an 85 degrees DNA bend. In addition, we use a heterologous yeast system to show that the NH2-terminal portion of Fish protein can function as a transcriptional activator. Fish directly regulates the expression of the pair rule gene, even-skipped (eve), by binding to multiple sites located in downstream regulatory regions that direct formation of eve stripes 1, 4, 5, and 6. Fish may function along with the Drosophila POU domain proteins Pdm-1 and Pdm-2 to regulate eve transcription, as genetic interactions were detected between fish and pdm mutants. Finally, we determined that Fish protein is expressed in a dynamic pattern throughout embryogenesis, and is present in nuclear and cytoplasmic compartments. Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved. PMID: 9622621 [PubMed - indexed for MEDLINE] DR111: J Biochem (Tokyo). 1998 Jun;123(6):1191-8. Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase. Inoue J, Sato R, Maeda M. Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan. The expression of the human SREBP-2 gene is transcriptionally regulated in a cooperative manner by sterol regulatory element-binding proteins (SREBPs) and the general transcription factor NF-Y [Sato, R., Inoue, J., Kawabe, Y., Kodama, T., Takano, T., and Maeda, M. (1996) J. Biol. Chem. 271, 26461-26464]. To understand the sterol-dependent transcriptional regulation by these factors in detail, we have examined the regulation of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and squalene synthase genes, whose promoters have multiple potential sterol regulatory elements (SRE, SREBP binding site) and NF-Y binding sites. The promoter of the human HMG CoA synthase gene was cloned, sequenced, and functionally characterized by means of reporter gene assays. The results indicate that an inverted CCAAT box, two SRE motifs and two Sp1 sites localized in a 90-bp region coordinately regulate the transcription. In the case of the human squalene synthase promoter, two SRE motifs and an inverted CCAAT box between the motifs localized in a 51-bp region are responsible for the sterol-regulated transcription of the gene. Gel mobility shift assay reveals that these two inverted CCAAT boxes are recognized by NF-Y. The involvement of multiple responsive elements in the transcription of HMG CoA synthase and squalene synthase seems to induce a higher level of sterol-dependent regulation (3.5 to 5. 8-fold) compared with that of the SREBP-2 promoter, which contains a single pair of SRE motif and CCAAT box (1.8 to 2.6-fold). Reporter gene assays using constructs containing various nucleotide spacing lengths between the SRE motif and the CCAAT box demonstrate that the 16 to 20-bp spacing range is required for maximal transcriptional regulation. These results agree with the findings that the distances between the two motifs in the known sterol responsive elements in several genes, including the human HMG CoA synthase and squalene synthase genes, are in this range. PMID: 9604010 [PubMed - indexed for MEDLINE] PR112: J Biol Chem. 1998 Apr 24;273(17):10618-23. Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21(WAF1/CIP1) in human prostate carcinoma cells. Lee SJ, Ha MJ, Lee J, Nguyen P, Choi YH, Pirnia F, Kang WK, Wang XF, Kim SJ, Trepel JB. Medicine Branch, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. Progression through the cell cycle is controlled by the induction of cyclins and the activation of cognate cyclin-dependent kinases. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor lovastatin induces growth arrest and cell death in certain cancer cell types. We have pursued the mechanism of growth arrest in PC-3-M cells, a p53-null human prostate carcinoma cell line. Lovastatin treatment increased protein and mRNA levels of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1), increased binding of p21 with Cdk2, markedly inhibited cyclin E- and Cdk2-associated phosphorylation of histone H1 or GST-retinoblastoma protein, enhanced binding of the retinoblastoma protein to the transcription factor E2F-1 in vivo, and induced the activation of a p21 promoter reporter construct. By using p21 promoter deletion constructs, the lovastatin-responsive element was mapped to a region between -93 and -64 relative to the transcription start site. Promoter mutation analysis indicated that the lovastatin-responsive site coincided with the previously identified transforming growth factor-beta-responsive element. These data indicate that in human prostate carcinoma cells an inhibitor of the HMG-CoA reductase pathway can circumvent the loss of wild-type p53 function and induce critical downstream regulatory events leading to transcriptional activation of p21. PMID: 9553123 [PubMed - indexed for MEDLINE] NR113: Gene. 1998 Mar 27;210(1):109-16. Molecular approaches for production of pravastatin, a HMG-CoA reductase inhibitor: transcriptional regulation of the cytochrome p450sca gene from Streptomyces carbophilus by ML-236B sodium salt and phenobarbital. Watanabe I, Serizawa N. Biomedical Research Laboratories, Sankyo Co. Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140, Japan. We have characterized the transcriptional regulation of ML-236B.Na and phenobarbital-inducible cytochrome P450sca-2 (CytP450sca-2) from Streptomyces carbophilus, an industrial pravastatin-producing strain. ML-236B.Na and phenobarbital enhanced the expression of the cytP450sca-2 gene in S. carbophilus. The cytP450sca-2 gene was also ML-236B.Na-inductive in S. lividans. Analysis of various deletion and mutation of the 5'-flanking region of the cytP450sca-2 gene revealed that the 1-kb region was required for ML-236B.Na-dependent CytP450sca-2 induction. We have found a putative ORF in the 5'-flanking region that encodes a protein of 174 amino acid residues containing a helix-turn-helix DNA-binding motif. A gel mobility shift assay showed that the protein was bound by an imperfect palindromic sequence between -46bp and -24bp in the 5'-flanking region, and ML-236B.Na was found to inhibit its binding. These findings suggest that induction of cytP450sca-2 is negatively regulated at the transcriptional level and that the protein encoded by the putative ORF is possibly functional as a repressor of the cytP450sca-2 gene. PMID: 9524240 [PubMed - indexed for MEDLINE] NR114: J Biochem (Tokyo). 1998 Apr;123(4):596-601. Hypocholesterolemic effects of the LDL receptor gene transcriptional upregulator CP-230821. Shimokawa T, Goto S, Ida M, Goto M, Nishijima S, Kodama T. Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305. simokawa@yamanouchi.co.jp. CP-230821 is a novel, potent LDL receptor gene transcriptional upregulator which decreases total plasma cholesterol level. Interestingly, this plasma LDL decrease does not alter hepatic lipid contents. A series of experiments was undertaken to study the molecular biology of this phenomenon. Twelve hours after CP-230821 treatment, the transcriptional activity and mRNA level of the LDL receptor gene in HepG2 cells were increased by 264% and 426%, respectively. Although treatment with the HMG-CoA reductase inhibitor compactin also increased LDL receptor gene transcription and mRNA, CP-230821 did not increase the level of HMG-CoA reductase gene transcription or mRNA. These results indicate that LDL receptor gene activity may play an important role in the decrease of plasma LDL level. These results further suggest that the LDL receptor gene and the HMG-CoA reductase gene are not strictly coordinately controlled. PMID: 9538248 [PubMed - indexed for MEDLINE] NR115: Mol Cell Biol. 1998 May;18(5):2617-28. The HMG domain protein SSRP1/PREIIBF is involved in activation of the human embryonic beta-like globin gene. Dyer MA, Hayes PJ, Baron MH. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. The human embryonic beta-like globin (epsilon-globin) gene is expressed in primitive erythroid cells of the yolk sac during the first few weeks of development. We have previously shown that developmental stage-specific expression of the epsilon-globin gene is mediated by multiple positive and negative regulatory elements upstream of the start of transcription. Of particular interest is one positive regulatory element, PRE II, that works together with other elements (PRE I and PRE V) to confer developmental stage- and/or tissue-specific expression on a minimal promoter. An approximately 85- to 90-kDa PRE II binding factor (PREIIBF) was identified in the nuclei of erythroid cells and shown to bind specifically to a novel 19-bp region within PRE II; binding of this protein to PRE II resulted in bending of the target DNA and was required for promoter activation. In this report, we present the cDNA expression cloning of PREIIBF. The cDNA encodes a previously identified member of the HMG domain family of DNA binding proteins termed SSRP1. By a number of biochemical and immunological criteria, recombinant SSRP1 appears to be identical to the PREII binding factor from erythroid nuclei. A hallmark of HMG domain proteins is their ability to bend their target DNAs; therefore, as we speculated previously, DNA bending by SSRP1/PREIIBF may contribute to the mechanism by which PRE II synergizes with other regulatory elements located upstream and downstream. In contrast with reports from other investigators, we demonstrate that SSRP1 binds DNA with clear sequence specificity. Moreover, we show that SSRP1/PREIIBF lacks a classical activation domain but that binding by this protein to PRE II is required for activation of a minimal promoter in stable erythroid cell lines. These studies provide the first evidence that SSRP1 plays a role in transcriptional regulation. SSRP1/PREIIBF may serve an architectural function by helping to coordinate the assembly of a multiprotein complex required for stage-specific regulation of the human epsilon-globin gene. PMID: 9566881 [PubMed - indexed for MEDLINE] PR116: J Lipid Res. 1998 Apr;39(4):901-12. Effect of a coffee lipid (cafestol) on cholesterol metabolism in human skin fibroblasts. Halvorsen B, Ranheim T, Nenseter MS, Huggett AC, Drevon CA. Institute for Nutrition Research, Faculty of Medicine, University of Oslo, Norway. Consumption of boiled coffee promotes an elevation of plasma cholesterol concentration in humans. The active compounds found in the lipid fraction of the coffee have been identified as the diterpenes cafestol and kahweol. We have studied the effects of pure cafestol on cholesterol metabolism in human skin fibroblasts (HSF). The uptake of [125I]-labeled tyramine cellobiose-labeled low density lipoprotein ([125I]TC-LDL) was decreased by about 50% (P< 0.05) after 18 h preincubation time with cafestol (20 microg/ml), as compared to the control cells. The specific binding of radiolabeled LDL was reduced by 54% (P < 0.05) after preincubation for 18 h with cafestol. A reduced amount of LDL receptors was demonstrated by a protein-normalized Scatchard plot analysis (20% decrease in Bmax) as well as by immunoblotting (25%) after cafestol incubation. No significant effect was observed on the level of mRNA for the LDL receptor after 11 and 23 h incubation with cafestol. Furthermore, we transfected HSF cells with a promoter region for the LDL receptor gene linked to a reporter gene, chloramphenicol acetyl transferase (CAT). No change was seen in the CAT activity after incubation with cafestol (20 microg/ml). Moreover, cafestol caused a 2.3-fold (P < 0.05) higher incorporation of radiolabeled [14C]oleic acid into cholesteryl esters after 24 h incubation, as compared to control cells, suggesting an increased acyl-CoA:cholesterol acyl transferase (ACAT) activity. Incorporation of [14C]acetate into cholesterol was reduced by approximately 40% (P < 0.05) with cafestol (20 microg/ml), as compared to control after 24 h preincubation, indicating a decreased 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity. Our results suggest that intake of cafestol may cause increased concentration of plasma cholesterol via the down-regulation of low density lipoprotein receptors by post-transcriptional mechanisms. PMID: 9555953 [PubMed - indexed for MEDLINE] PR117: Arch Biochem Biophys. 1998 Mar 15;351(2):159-66. Compensatory responses to inhibition of hepatic squalene synthase. Lopez D, Chambers CM, Keller RK, Ness GC. College of Medicine and the Institute for Biomolecular Science, University of South Florida, Tampa, Florida, 33612-4799, USA. The mechanism by which depletion of hepatic cholesterol levels, achieved by inhibition of squalene synthase, alters hepatic LDL receptor, HMG-CoA reductase, and cholesterol 7alpha-hydroxylase gene expression was investigated by measuring transcription rates, mRNA stability, rates of translation, translational efficiency, and levels of sterol response element binding proteins. It was found that the transcription of both hepatic LDL receptor and HMG-CoA reductase were increased about twofold. The increase in LDL receptor transcription occurred within 2 h after giving 2 mg/kg zaragozic acid A, a potent inhibitor of squalene synthase. This preceded the increase in transcription of HMG-CoA reductase that occurred at 4 h. Increases in the stability of both of these mRNAs were also observed. These changes account for the increases in LDL receptor and HMG-CoA reductase mRNA levels previously observed. The rate of transcription of hepatic cholesterol 7alpha-hydroxylase was decreased to about 25% of control within 3 h after administration of zaragozic acid A, which correlates with the decrease in this mRNA. The rates of translation, as determined by pulse labeling, of both hepatic HMG-CoA reductase and LDL receptor were increased two- to threefold. The translational efficiency of these two mRNAs was also increased as judged by polysome profile analysis. There was an increase in mRNA associated with the heaviest polysome fraction and a decrease in that associated with monosomes. No significant change was observed in the levels of sterol response element binding protein 2, the form that mediates induced transcription, in response to zaragozic acid A treatment, indicating that this protein might not be involved in mediating the observed transcriptional changes. An increase in sterol response element binding protein -1 was observed 30 min after giving zaragozic acid A. The results suggest that compensatory responses to depletion of squalene-derived products involve alterations in the rates of transcription, mRNA stability, and translational of key proteins involved in cholesterol homeostasis. Copyright 1998 Academic Press. PMID: 9514656 [PubMed - indexed for MEDLINE] NR118: Protein Sci. 1998 Jan;7(1):178-84. Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon. Rosenthal RS, Rodwell VW. Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA. The mvaAB operon of Pseudomonas mevalonii encodes HMG-CoA reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the mvaAB cis-regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA-binding activity, comigrated with P15 and P16 during DNA-affinity chromatography, size-exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31-kDa polypeptide complex that contained N-terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size-exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N-termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa. PMID: 9514272 [PubMed - indexed for MEDLINE] NR119: Biochem Biophys Res Commun. 1998 Jan 26;242(3):692-6. The hepatocyte nuclear factor 4 (HNF-4) represses the mitochondrial HMG-CoA synthase gene. Rodriguez JC, Ortiz JA, Hegardt FG, Haro D. Unit of Biochemistry, School of Pharmacy, University of Barcelona, Spain. We have recently shown that the gene for the mitochondrial HMG-CoA synthase is a target for PPAR and that this receptor mediates the induction of this gene by fatty acids. With the aim of gaining further insight into the function and regulation of this gene we examined the effect of other members of the nuclear hormone receptor superfamily on its expression. We previously identified a regulatory element in the mitochondrial HMG-CoA synthase gene promoter that confers transcriptional regulation by PPAR, RXR and the orphan nuclear receptor COUP-TF. In this study we demonstrate a trans-repressing regulatory function for HNF-4 at this same nuclear receptor response element (NRRE). HNF-4 binds to the mitochondrial HMG-CoA synthase NRRE, and, in cotransfection assays in HepG2 cells, it represses PPAR-dependent activation of reporter gene linked to the mitochondrial HMG-CoA synthase gene promoter. These results suggest that the mitochondrial HMG-CoA synthase gene is subject to differential regulation by the interplay of multiple members of the nuclear hormone receptor superfamily. PMID: 9464279 [PubMed - indexed for MEDLINE] NR120: J Biol Chem. 1998 Jan 16;273(3):1349-56. Sterol regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene through a direct interaction between sterol regulatory element binding protein and the trimeric CCAAT-binding factor/nuclear factor Y. Dooley KA, Millinder S, Osborne TF. Department of Molecular Biology and Biochemistry, University of California, Irvine 92697-3900, USA. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, a key regulatory enzyme in the pathway for endogenous cholesterol synthesis, is a target for negative feedback regulation by cholesterol. The promoter for HMG-CoA synthase contains two binding sites for the sterol regulatory element-binding proteins (SREBPs). When cellular sterol levels are low, the SREBPs are released from the endoplasmic reticulum membrane, allowing them to translocate to the nucleus and activate SREBP target genes. In all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are required. In the HMG-CoA synthase promoter there are several potential co-regulatory transcription factor binding sites, including an inverted CCAAT box. A similar element has been shown to function with SREBP to mediate sterol regulation of another gene involved in cholesterol metabolism, farnesyl diphosphate synthase. Here, we show that CCAAT binding factor/nuclear factor Y (CBF/NF-Y) binding to the CCAAT box is required for sterol-regulated transcription of HMG-CoA synthase. The SREBP sites and the inverted CCAAT box are normally separated by 17 base pairs, and we show that increasing this distance results in a decrease in the level of transcriptional regulation by sterols. Furthermore, we provide evidence that there is a direct interaction between CBF/NF-Y and the basic helix-loop-helix-zipper region of SREBP. Interestingly, this interaction does not occur efficiently with any of the isolated subunits and appears to require all three nonidentical CBF/NF-Y subunits in a preassembled complex. Since CBF/NF-Y only binds to DNA when all three subunits are in a complex, this would prevent SREBP from forming nonproductive associations with the individual subunits. PMID: 9430668 [PubMed - indexed for MEDLINE] NR121: Trends Genet. 1997 Dec;13(12):485-9. TCF/LEF factor earn their wings. Clevers H, van de Wetering M. Department of Immunology, University Hospital Utrecht, The Netherlands. h.clevers@lab.azu.nl Factors of the TCF/LEF HMG domain family (TCFs) exist in vertebrates, Drosophila melanogaster and Caenorhabditis elegans. It has very recently become evident that TCFs interact with the vertebrate WNT effector beta-catenin to mediate axis formation in Xenopus. Likewise, Armadillo (the Drosophila ortholog of beta-catenin) is genetically upstream of a Drosophila TCF in the Wingless pathway. Upon Wingless/Wnt signaling, Armadillo/beta-catenin associate with nuclear TCFs and contribute a trans-activation domain to the resulting bipartite transcription factor. The cytoplasmic tumor-suppressor protein APC binds to beta-catenin causing its destruction. In APC-deficient colon carcinoma cells, beta-catenin accumulates and is constitutively complexed with TCF factors. In APC-positive colon carcinomas and melanomas, dominant mutations in beta-catenin render it indestructable, providing an alternative mechanism to activate transcription of TCF target genes inappropriately. So, transcriptional activation of TCF target genes by beta-catenin appears to be a central event in development and cellular transformation. Publication Types: Review Review, Tutorial PMID: 9433138 [PubMed - indexed for MEDLINE] NR122: Mol Cell Biol. 1997 Nov;17(11):6321-9. Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites. Ambrosetti DC, Basilico C, Dailey L. Department of Microbiology and Kaplan Cancer Center, New York University School of Medicine, New York 10016, USA. Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. We have previously shown that fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. We now demonstrate that increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer "spacing mutants" which failed to activate transcription were also compromised in their ability to form the Oct* complexes even though they could still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes. PMID: 9343393 [PubMed - indexed for MEDLINE] DR123: Dev Dyn. 1997 Oct;210(2):79-86. Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction. Hargrave M, Wright E, Kun J, Emery J, Cooper L, Koopman P. Centre for Molecular and Cellular Biology, The University of Queensland, Brisbane, Australia. Sry, the mammalian Y-linked testis determining gene, is a member of a family of genes known as Sox genes, which encode transcription factors related by a DNA-binding motif termed the HMG box. Sox genes are known to have diverse roles in vertebrate differentiation and development. We report here the cloning and characterisation of one of these genes, Sox11, in mice. In addition to an N-terminal HMG box domain, the deduced SOX11 protein contains a number of highly conserved C-terminal motifs, which may function in transcriptional regulation. Expression of Sox11 in mouse embryos was prominent in the periventricular cells of the central nervous system, suggesting a role in neuronal maturation. Expression was also observed in a wide range of tissues involved in epithelial-mesenchymal interactions, suggesting an additional role in tissue modelling during development. PMID: 9337129 [PubMed - indexed for MEDLINE] PR124: Genes Dev. 1997 Sep 15;11(18):2359-70. A beta-catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus. Brannon M, Gomperts M, Sumoy L, Moon RT, Kimelman D. Department of Biochemistry, University of Washington School of Medicine, Seattle 98195-7350, USA. The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of beta-catenin and HMG box transcription factors of the Lef/Tcf family. We show that the promoter of the dorsalizing homeo box gene siamois is a direct target for the beta-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, we show that a 0.8-kb fragment of the siamois promoter is strongly activated by beta-catenin. The proximal 0.5 kb, which is also activated by beta-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the beta-catenin-mediated activation of siamois and show that siamois is regulated by the beta-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of beta-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Furthermore, elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of beta-catenin. Finally, we find that the endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. We propose that the dorsal activation of siamois by the beta-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos. PMID: 9308964 [PubMed - indexed for MEDLINE] NR125: Biochem J. 1997 Sep 1;326 ( Pt 2):587-92. Chicken ovalbumin upstream-promoter transcription factor (COUP-TF) could act as a transcriptional activator or repressor of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene. Rodriguez JC, Ortiz JA, Hegardt FG, Haro D. Unitat de Bioquimica, Facultat de Farmacia, Barcelona, Spain. The chicken ovalbumin upstream-promoter transcription factor (COUP-TF) has a dual effect on the regulation of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene. COUP-TF could act as a transcriptional activator or repressor of this gene through different DNA sequences. COUP-TF induces expression of a reporter gene linked to the mitochondrial HMG-CoA synthase gene promoter in human hepatoma HepG2 cells, but represses it in a Leydig tumour cell line (R2C); in both these cell lines the expression of the mitochondrial HMG-CoA synthase gene mimics that of liver and testis. The activation is promoted by a fragment of the gene from coordinates -62 to +28, which contains a GC box and a TATA box, and where no COUP-TF binding site was observed by in vitro DNA binding studies. On the other hand, the COUP-TF inhibitory effect is mainly due to repression of peroxisome-proliferator-activated receptor-dependent activation of the gene, interacting with the region from -104 to -92. To our knowledge this work represents the second example of a target gene for COUP-TF I that could be either activated or repressed by the action of this receptor through different DNA sequences of the same gene. PMID: 9291136 [PubMed - indexed for MEDLINE] PR126: Mol Cell Biol. 1997 Aug;17(8):4230-7. Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein. Voit R, Schafer K, Grummt I. German Cancer Research Center, Division of Molecular Biology of the Cell II, Heidelberg. The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis. PMID: 9234680 [PubMed - indexed for MEDLINE] DR127: Arch Biochem Biophys. 1997 Jul 1;343(1):118-22. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors unmask cryptic regulatory mechanisms. Lopez D, Chambers CM, Ness GC. Department of Biochemistry and Molecular Biology, College of Medicine, and Institute for Biomolecular Science, University of South Florida, Tampa 33612, USA. The possibility that potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may alter the mechanisms by which dietary cholesterol and farnesol regulate this gene was investigated by comparing the regulatory responses of rats maintained on diets with or without 0.04% Lovastatin supplementation to dietary cholesterol. It was found that the rate of hepatic HMG-CoA reductase transcription was significantly decreased by dietary cholesterol in animals fed Lovastatin-supplemented diets, whereas animals maintained on a normal chow diet showed no decrease in the rate of transcription. The levels of reductase mRNA were decreased to about 10% of controls in Lovastatin-supplemented animals in response to dietary cholesterol but not affected in nonsupplemented animals. Administration of farnesol, reputed to be the nonsterol regulator of reductase, to rats maintained on a diet containing Lovastatin decreased hepatic HMG-CoA reductase protein by 30% and the half-life of reductase immunoreactive protein to 4.0 h, which is close to that observed in chow-fed animals. In contrast, farnesol treatment does not affect the turnover rate of reductase protein in rats fed a normal chow diet. These results suggest that potent inhibitors of HMG-CoA reductase may unmask transcriptional regulation by dietary cholesterol and accelerated degradation of the reductase by the putative nonsterol regulator farnesol. PMID: 9210653 [PubMed - indexed for MEDLINE] DR128: Mol Cell Biol. 1997 Jul;17(7):3649-62. Intra- and intermolecular cooperative binding of high-mobility-group protein I(Y) to the beta-interferon promoter. Yie J, Liang S, Merika M, Thanos D. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA. The mammalian high-mobility-group protein I(Y) [HMG I(Y)], while not a typical transcriptional activator, is required for the expression of many eukaryotic genes. HMG I(Y) appears to recruit and stabilize complexes of transcriptional activators through protein-DNA and protein-protein interactions. The protein binds to the minor groove of DNA via three short basic repeats, preferring tracts of adenines and thymines arranged on the same face of the DNA helix. However, the mode by which these three basic repeats function together to recognize HMG I(Y) binding sites has remained unclear. Here, using deletion mutants of HMG I(Y), DNase I footprinting, methylation interference, and in vivo transcriptional assays, we have characterized the binding of HMG I(Y) to the model beta-interferon enhancer. We show that two molecules of HMG I(Y) bind to the enhancer in a highly cooperative fashion, each molecule using a distinct pair of basic repeats to recognize the tandem AT-rich regions of the binding sites. We have also characterized the function of each basic repeat, showing that only the central repeat accounts for specific DNA binding and that the presence of a second repeat bound to an adjacent AT-rich region results in intramolecular cooperativity in binding. Surprisingly, the carboxyl-terminal acidic tail of HMG I(Y) is also important for specific binding in the context of the full-length protein. Our results present a detailed examination of HMG I(Y) binding in an important biological context, which can be extended not only to HMG I(Y) binding in other systems but also to the binding mode of many other proteins containing homologous basic repeats, which have been conserved from bacteria to humans. PMID: 9199299 [PubMed - indexed for MEDLINE] NR129: Biochem J. 1997 May 15;324 ( Pt 1):65-73. Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet. Adams SH, Alho CS, Asins G, Hegardt FG, Marrero PF. Unit of Biochemistry, School of Pharmacy, University of Barcelona, Avda. Diagonal, 643, 08028 Barcelona, Spain. The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting. PMID: 9164842 [PubMed - indexed for MEDLINE] NR130: Oncogene. 1997 Apr 3;14(13):1617-22. ESX: a structurally unique Ets overexpressed early during human breast tumorigenesis. Chang CH, Scott GK, Kuo WL, Xiong X, Suzdaltseva Y, Park JW, Sayre P, Erny K, Collins C, Gray JW, Benz CC. Cancer Research Institute and Division of Oncology-Hematology, University of California at San Francisco, 94143, USA. The >30 known members of the Ets multigene family of transcriptional regulators are increasingly being recognized for their involvement in early embryonic development and late tissue maturation, directing stage-specific and tissue-restricted programs of target gene expression. Identifiable primarily by their 85 amino acid ETS DNA-binding domain and dispersed across all metazoan lineages into distinct subfamilies, Ets genes also produce malignancies in humans and other vertebrates when overexpressed or rearranged into chimeras retaining the ETS domain, suggesting that their oncogenic potential is determined by the program of target genes they regulate. Searching for Ets factors that regulate expression of the HER2/neu (c-erbB2) oncogene in human breast cancer, we identified a new epithelium-restricted Ets encoding an ETS domain homologous to the Drosophila E74/human Elf-1 subfamily, an amino-terminal region (A-region or Pointed domain) homologous to the distantly related Ets-1 subfamily, and a serine-rich box homologous to the transactivating domain of the lymphocyte-restricted High Mobility Group (HMG) protein, SOX4. Recombinant protein encoded by ESX (for epithelial-restricted with serine box) exhibits Ets-like DNA binding specificity in electrophoretic mobility shift assays and, in transient transfection assays, transactivates Ets-responsive promoter elements including that found in the HER2/neu oncogene. ESX is located at chromosome 1q32 in a region known to be amplified in 50% of early breast cancers, is heregulin-inducible and overexpressed in HER2/neu activated breast cancer cells. Tissue hybridization suggests that ESX becomes overexpressed at an early stage of human breast cancer development known as ductal carcinoma in situ (DCIS). PMID: 9129154 [PubMed - indexed for MEDLINE] NR131: Genes Dev. 1997 Mar 1;11(5):640-53. ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function. Bruhn L, Munnerlyn A, Grosschedl R. Department of Microbiology, University of California, San Francisco 94143-0414, USA. LEF-1 is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCR alpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins. With the aim of gaining insight into the function of context-dependent activation domains, we cloned ALY, a novel LEF-1-interacting protein. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1 (CBF alpha2, PEBP2 alpha(B), which is another protein component of the TCR alpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCR alpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCR alpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCR alpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. We propose that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCR alpha enhancer complex. PMID: 9119228 [PubMed - indexed for MEDLINE] DR132: Nucleic Acids Res. 1997 Jan 15;25(2):339-46. Distamycin prolongs E-selectin expression by interacting with a specific NF-kappaB-HMG-I(Y) binding site in the promoter. Ghersa P, Whelan J, Cambet Y, DeLamarter JF, Hooft van Huijsduijnen R. Geneva Biomedical Research Institute, 14, Chemin des Aulx, Case Postale 674, 1228 Plan-les-Ouates, Geneva, Switzerland. pfg8091@ggr.co.uk The E-selectin cell adhesion protein plays a critical role in mediating adherence of leukocytes to endothelium at sites of inflammation. Cytokine-induced E-selectin expression on the surface of endothelial cells is transient; mRNA expression peaks at 3-4 h after induction and returns to basal levels within 24 h. The mechanism for this transcriptional down-modulation is not known. Promoter binding factors responsible for induced gene expression include NF-kappaB, which binds at three sites within the E-selectin promoter, and HMG-I(Y), which binds to the A/T-rich core found at the centre of these binding sites. Distamycin is an antibiotic that also binds A/T-rich DNA and inhibits HMG-I(Y) DNA binding. To study the role of HMG-I(Y) in E-selectin expression, we have examined the effect of distamycin on the cytokine-induced E-selectin expression cycle. We found that distamycin prolonged E-selectin expression, both by sustaining mRNA transcription and by extending the transcript's half-life. The distamycin effect on transcription was mediated through one of the three NF-kappaB-HMG-I(Y) binding sites (NF-kappaBII) within the promoter. This suggests that the NF-kappaB-HMG-I(Y) complex interacting at the NF-kappaBII site plays a role not only in cytokine induction of E-selectin expression, but also in its down-modulation. PMID: 9016563 [PubMed - indexed for MEDLINE] DR133: J Cancer Res Clin Oncol. 1997;123(3):133-40. High-mobility-group proteins and cancer--an emerging link. Wunderlich V, Bottger M. In the last few years, considerable interest has been generated in the role of high-mobility-group (HMG) proteins, and HMG box proteins generally, in cancer development and therapy. These proteins were discovered in the early 1970s (Goodwin et al. 1973) as a group of nonhistone proteins. Some members of the HMG protein family (i) constitute a class of important architectural proteins involved in transcriptional regulation of genes, (ii) are frequently expressed in transformed cells at levels that correlate with the degree of neoplastic cell transformation, (iii) participate in gene rearrangements, which are linked to the emergence of benign solid tumors, (iv) confer the ability to recognize DNA-cisplatin adducts selectively, and (v) provide a new delivery system for efficient gene transfer. It should be considered that some HMG proteins, acting as architectural proteins that bring many of the transcription factors into precise three-dimensional shapes, may have a similar critical role in neoplastic transformation to that of some transcription factors themselves. Publication Types: Editorial PMID: 9119878 [PubMed - indexed for MEDLINE] NR134: J Immunol. 1996 Dec 15;157(12):5411-21. Cloning of the human platelet endothelial cell adhesion molecule-1 promoter and its tissue-specific expression. Structural and functional characterization. Almendro N, Bellon T, Rius C, Lastres P, Langa C, Corbi A, Bernabeu C. Department of Immunology, Center of Biological Investigations, High Council of Scientific Investigations, Madrid, Spain. Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a cell adhesion molecule involved in transendothelial migration and expressed by hemopoietic and endothelial cells. To understand the mechanisms underlying its regulated expression, a genomic clone containing 1555 bp of the 5'-flanking region and the first exon of the human PECAM-1 gene has been isolated. The 5'-flanking region of the PECAM-1 gene lacks a consensus TATA box, but contains consensus motifs for Sp1, EGR1, ets, helix-loop-helix (HLH) box, GATA, AP-2, C/EBP, YY1, CCACC, LyF-1, imperfect octamer, heptamer, high mobility group proteins (HMG) box, and nuclear factor-kappaB, as well as shear stress-, retinoic acid-, glucocorticoid-, and acute phase-responsive elements, and an Alu sequence. Successive 5' to 3' or 3' to 5' deletions revealed tissue-specific promoter activity within the two contiguous 0.22-kb NheI/BglII and 0.44-kb BglII/PstI fragments. The transcriptional activity displayed by the 0.22-kb NheI/BglII fragment was specific for the myeloid lineage, whereas the promoter activity of the 0.44-kb BglII/PstI fragment was apparently restricted to endothelial cells. The transcriptional activity of the 0.22-kb NheI/BglII fragment was confirmed by 5' RACE (rapid amplification of 5' cDNA ends) and S1 nuclease protection experiments that revealed previously unidentified transcription start sites. The 0.22-kb NheI/BglII promoter exhibited PMA inducibility in myeloid cells and contained a PMA-responsive element recognized by Sp1 and EGR-1 transcription factors. Isolation and characterization of the human PECAM-1 promoter represent an initial step in elucidating the controlled expression of the PECAM-1 gene. PMID: 8955189 [PubMed - indexed for MEDLINE] DR135: Jpn J Hum Genet. 1996 Dec;41(4):363-79. Molecular basis governing primary sex in mammals. Nagai K. Department of Biochemistry, Tokyo Medical College, Japan. The function of Sry for inducing a male gonad was identified due to a development of a transgenic XX male mouse with testes by introducing a single gene into an embryo. The intronless Sry encodes a putative transcriptional protein harboring an HMG motif. The sequence similarity within the HMG motif has been highly conserved despite less conservation in other domains. Hence, the HMG motif must play a critical role in the transcriptional regulation, leading to the development of a male gonad. However, a non HMG box C terminal domain of Sry protein may also be indispensable for inducing normal testicular development. Further, several autosomal genes, such as SF1, WT1, SOX and MIS, as well as a unique X chromosomal DAX1 were suggested to be associated with the development of gonadal sex in mammals. Therefore, the significance on the involvement of these genes in the molecular mechanism of mammalian sex determination should be also considered. Publication Types: Review Review, Tutorial PMID: 9088107 [PubMed - indexed for MEDLINE] NR136: Mol Cell Biol. 1996 Dec;16(12):6829-40. Erratum in: Mol Cell Biol. 1997 Apr;17(4):2351. Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene. Lecine P, Algarte M, Rameil P, Beadling C, Bucher P, Nabholz M, Imbert J. INSERM U119, Marseille, France. The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor. PMID: 8943338 [PubMed - indexed for MEDLINE] PR137: Biochim Biophys Acta. 1996 Oct 18;1303(3):199-206. Lovastatin inhibits gene expression of type-I scavenger receptor in THP-1 human macrophages. Umetani N, Kanayama Y, Okamura M, Negoro N, Takeda T. First Department of Internal Medicine, Osaka City University Medical School, Japan. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits the synthesis of mevalonic acid and is widely used as an anti-atherosclerotic drug. The macrophage scavenger receptor (SCR), a trimeric membrane glycoprotein, is postulated to play a key role in atheroma macrophage foam cell formation. HMG-CoA reductase is involved in the control of the synthesis of glycoproteins and farnesylated proteins, including ras proteins, which are involved in the transcriptional regulation of SCR gene expression. Accordingly, we examined whether lovastatin alters the gene expression of SCRs in THP-1 cell derived human macrophages. Lovastatin (5-15 microM) caused a significant dose-related reduction in steady state levels of type-I SCR mRNA in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells. The addition of exogenous mevalonate (1 mM) completely restored the lovastatin-induced decrease of type-I SCR mRNA levels. While the addition of the isoprenoid end-product, isopentenyl adenine (50 microM), had little effect on the type-I SCR mRNA levels in lovastatin treated cells, the addition of isoprenoid farnesol (5 microM) largely restored the lovastatin-induced decrease of type-I SCR mRNA levels. Actinomycin D treatment showed that degradation rates of type-I SCR mRNA did not differ between the THP-1 derived cells with and without lovastatin treatment. Nuclear run-on assays showed that lovastatin markedly decreased the transcription of SCR gene in the cells. These results suggest that lovastatin inhibits the transcription of type-I SCR gene by affecting mevalonate metabolism, possibly through the farnesyl-pyrophosphate related end-product(s) in the THP-1-derived macrophages. PMID: 8908154 [PubMed - indexed for MEDLINE] DR138: EMBO J. 1996 Sep 16;15(18):4981-91. HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation. Zappavigna V, Falciola L, Helmer-Citterich M, Mavilio F, Bianchi ME. DIBIT, Istituto Scientifico San Raffaele, Milano, Italy. High mobility group protein 1 (HMG1) is a non-histone, chromatin-associated nuclear protein with a proposed role in the regulation of eukaryotic gene expression. We show that HMG1 interacts with proteins encoded by the HOX gene family by establishing protein-protein contacts between the HMG box domains and the HOX homeodomain. The functional role of these interactions was studied using the transcriptional activity of the human HOXD9 protein as a model. HMG1 enhances, in a dose-dependent fashion, the sequence-specific DNA binding activity in vitro, and the transcriptional activation in a co-transfection assay in vivo, of the HOXD9 protein. Functional interaction between HMG1 and HOXD9 is dependent on the DNA binding activity of the homeodomain, and requires the HOXD9 transcriptional activation domain. HMG1 enhances activation by HOXD9, but not by HOXD8, of the HOXD9-controlled element. Specific target recognition and functional interaction with HMG1 can be transferred to HOXD8 by homeodomain swapping. We propose that HMG1-like proteins might be general co-factors in HOX-mediated transcriptional activation, which facilitate access of HOX proteins to specific DNA targets, and/or introduce architectural constraints in the assembly of HOX-containing transcriptional complexes. PMID: 8890171 [PubMed - indexed for MEDLINE] NR139: J Interferon Cytokine Res. 1996 Aug;16(8):595-600. Repression of interleukin-2 and interleukin-4 promoters by tumor suppressor protein p53. Pesch J, Brehm U, Staib C, Grummt F. Institut fur Biochemie, Universitat Wurzburg, Germany. Interleukin 2 (IL-2) and interleukin 4 (IL-4) secreted by activated but not by resting mature T cells are pleiotropic cytokines affecting growth and differentiation of diverse cell types, such as T cells, B cells, and mast cells. There is little information about the molecular basis for the constitutive repression of IL-2 and IL-4 gene expression in unstimulated T cells. We investigated the possibility that wild-type (wt) p53, a nuclear tumor suppressor protein, might serve to repress IL-2 and IL-4 gene expression in murine E14 T lymphoma and in human Jurkat cells. We transiently cotransfected these cells with constitutive simian virus 40 (SV 40) early promoter expression plasmids overproducing wt or mutant murine p53 and with appropriate luciferase (luc) reporter plasmids containing the promoter elements of murine IL-2 and IL-4 genes to evaluate the effect of various p53 species on these promoters. Murine wt p53 derived from pSG5p53cD strongly repressed the IL-2 and IL-4 promoters in both cell lines induced by the phorbol ester TPA and the Ca2+ ionophore ionomycin but not, however, in uninduced cells. In similar transient transfection experiments with lymphoma cells, overexpression of deletion mutant species of murine p53 revealed that the N-terminal and C-terminal domains are crucial for inhibition of both IL-2 and IL-4 gene expression. These parts of p53 comprise the transactivation domain at the amino terminal side, which has previously also been shown to interact with the TATA-box binding-protein TBP and the carboxy-terminal oligomerization domain. Additionally, it was shown that a previously described inhibitory protein, the high-mobility-group protein HMG-I/Y, does not functionally interact with p53. Cotransfection of expression plasmids for both p53 and HMG-I/Y did not alter the extent of inhibition by the individual proteins. These data suggest that p53 can downmodulate both IL-2 and IL-4 gene expression and that both the transactivation and oligomerization domains of the tumor suppressor protein are essential for this transcriptional repression. PMID: 8877730 [PubMed - indexed for MEDLINE] NR140: Mol Reprod Dev. 1996 Aug;44(4):460-71. Binding of transcription factors to widely-separated cis-regulatory elements of the murine FGF-4 gene. Lamb K, Rosfjord E, Brigman K, Rizzino A. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA. Embryonal carcinoma (EC) cells and their embryo-derived counterparts, embryonic stem (ES) cells, have been used extensively to study the transcriptional regulation of the fibroblast growth factor-4 (FGF-4) gene. The FGF-4 gene is expressed in EC cells and ES cells, but it is repressed in their retinoic acid (RA)-induced differentiated counterparts. Previous studies have shown that the transcription of the FGF-4 gene is controlled by cis-regulatory elements located in the 5' flanking region of the gene, and by a powerful enhancer located approximately 3 kb downstream from the transcription start site. In the current study, gel mobility shift analysis was used to examine the binding of nuclear proteins to cis-regulatory elements involved in the transcription of the FGF-4 gene. We demonstrate that the transcription factors Sp1 and Sp3 in nuclear extracts prepared from EC cells bind to three Sp1 motifs, one located in the downstream enhancer, and two located in the 5' flanking region of the gene. We also show that Sp1 and Sp3 bind to each of the Sp1 motifs when nuclear extracts prepared from EC-derived differentiated cells are used. In contrast, differentiation of EC cells and ES cells drastically reduces the ability of nuclear factors to bind to an octamer motif and an adjacent High Mobility Group (HMG) motif, which have been shown previously to play essential roles in the functioning of the FGF-4 enhancer. Together, these findings provide a mechanistic explanation of how the distant FGF-4 enhancer promotes transcription of this gene in EC cells and ES cells, and how differentiation of these cells represses transcription of the FGF-4 gene. PMID: 8844688 [PubMed - indexed for MEDLINE] DR141: Oncogene. 1996 Jun 20;12(12):2515-25. Analysis of the role of v-rel in transcriptional regulation of high mobility group 14. Walker AK, Enrietto PJ. Department of Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA. The oncogene, v-rel is a member of the rel/NF-kappaB family of transcription factors. It causes a rapidly fatal lymphoma in young chicks and is capable of transforming both fibroblasts and primitive hematopoietic cells in culture. To understand the role of v-rel in transformation we constructed an inducible form of v-rel and used it to identify potential cellular target genes for v-rel regulation. In this paper we show that High Mobility Group Protein 14 (HMG 14) is expressed in a wide variety of v-rel transformed cell types. In addition we show that v-rel participates in the transcriptional regulation of HMG 14 and that extracts from v-relER cells interact with the HMG 14 promoter. These experiments suggest a role for v-rel in the regulation of a unique gene whose protein product may influence gene transcription in a global fashion. PMID: 8700510 [PubMed - indexed for MEDLINE] PR142: J Biol Chem. 1996 Jun 7;271(23):13731-8. Mechanisms of murine RANTES chemokine gene induction by Newcastle disease virus. Lokuta MA, Maher J, Noe KH, Pitha PM, Shin ML, Shin HS. Department of Pathology, University of Maryland, School of Medicine, Baltimore, Maryland 21201, USA. We have previously defined the lipopolysaccharide (LPS)-responsive element (LRE) in the promoters of murine RANTES (regulated on activation normal T-cell expressed) (MuRantes) and murine IP-10/crg-2, chemokines which have potent chemotactic properties for inflammatory cells including monocytes and T lymphocytes. In the present work, we studied the transcriptional mechanism of MuRantes gene induction by virus and compared it with that of LPS in an effort to understand the host responses to virus and bacterial toxins at the molecular level. MuRantes mRNA expression is induced by Newcastle disease virus (NDV) and LPS in the RAW 264.7 macrophage cell line and peritoneal macrophages of LPS-responsive C3HeB/FeJ mice. In LPS-hyporesponsive C3H/HeJ mice, only NDV induces this chemokine gene, indicating that the pathways of transcriptional activation by NDV and LPS are not identical. Using a transient transfection assay, the minimal virus-responsive element (VRE) was localized between nt -175 and -116. The VRE contains previously defined LRE motif 1 (TCAYRCTT) and motif 3 ((T/A)GRTTTCA(G/C)TTT), which were shown to also be important for initiation of transcription by virus. NDV-stimulated nuclear extracts were tested for trans-activating factors able to bind the VRE. The chromosomal protein HMG-I(C) was shown to bind the 3'-A.T-rich domains of the VRE, and the presence of HMG-I(C) was demonstrated in the VRE-protein complex formed with nuclear extracts from NDV-stimulated, but not unstimulated cells. These findings demonstrate the role of HMG-I(C) in activation of MuRantes promoter by NDV. PMID: 8662857 [PubMed - indexed for MEDLINE] NR143: J Cell Biol. 1996 May;133(3):667-81. Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. Kanai Y, Kanai-Azuma M, Noce T, Saido TC, Shiroishi T, Hayashi Y, Yazaki K. Department of Ultrastructural Research, Tokyo Metropolitan Institute of Medical Science, Japan. The different mRNA isoforms of the mouse Sox17 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Sry-related protein of 419 amino acids containing a single high mobility group box near the NH2-terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Sox17 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Sox17 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Sox17 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Sox17 products is lower in comparison to the high accumulation of t-Sox17 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Sox17 and t-Sox17 proteins, Sox17 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Sox17 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Sox17 could stimulate transcription through its binding site, but t-Sox17 had little effect on reporter gene expression. Thus, these findings suggest that Sox17 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Sox17 may lead to the loss of its function in the postmeiotic germ cells. PMID: 8636240 [PubMed - indexed for MEDLINE] NR144: Mol Gen Genet. 1996 Apr 10;250(6):767-74. Transcriptional analysis of the mtA idiomorph of Neurospora crassa identifies two genes in addition to mtA-1. Ferreira AV, Saupe S, Glass NL. Department of Botany, University of British Columbia, Vancouver, Canada. In Neurospora crassa, mating and heterokaryon formation between opposite mating-types is controlled by a single locus with two alternate forms termed mt A and mt a. Previously, an open reading frame (mt A-1) that confers mating identity and heterokaryon incompatibility was characterized in the 5.3 kb mt A idiomorph. In this study, we describe the structural and transcriptional characterization of two additional genes in the mt A idiomorph, Mt A-2 and mt A-3. The 373 amino acid mt A-2 ORF has 23% identity to the SMR1 ORF of Podospora anserina. DNA sequence analysis of a mutation affecting ascospore to 129 amino acids. The 324 amino acids mt A-3 ORF has an HMG domain and shows 22% amino acid identity to SMR2 of P. anserina. Transcripts from mt A-2 and mt A-3 are constitutively expressed during both vegetative and sexual reproduction. The presence of upstream ORFs in the mt A-2 and mt A-3 transcripts suggests the possibility of post-transcriptional regulation of the expression mt A-2 and mt A-3 polypeptides. PMID: 8628238 [PubMed - indexed for MEDLINE] NR145: J Biol Chem. 1996 Apr 5;271(14):8053-6. Transcriptional regulation of squalene epoxidase by sterols and inhibitors in HeLa cells. Nakamura Y, Sakakibara J, Izumi T, Shibata A, Ono T. Department of Biochemistry, Niigata University School of Medicine, Niigata 951, Japan. Regulation of squalene epoxidase (SE) gene expression was studied in comparison with those of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and low density lipoprotein (LDL) receptor. An increased expression of SE mRNA and protein content in mouse L929 cells grown in 10% lipoprotein-deficient fetal bovine serum (LPDS) for 48 h was found by performing immunoblot and Northern blot analyses when compared with the culture in the presence of fetal bovine serum (FBS). The same results in mRNA levels were seen using human cell lines HepG2, HeLa, and Chang liver cells. The increase of SE mRNA in HeLa cells grown in LPDS was preventable in a dose-dependent manner by feeding cells with 25-hydroxycholesterol or cholesterol. When an SE inhibitor, NB-598, was fed to HeLa cells grown in LPDS, it caused further increases in mRNA levels of SE, HMG-CoA reductase, and LDL receptor. In contrast, NB-598 had no effect on the message levels of these genes when fed to HeLa cells grown in FBS. These results suggest that sterol produced endogenously can also regulate SE expression at the level of transcription. PMID: 8626488 [PubMed - indexed for MEDLINE] PR146: Mol Endocrinol. 1996 Apr;10(4):393-407. Progesterone receptor-induced bending of its target DNA: distinct effects of the A and B receptor forms. Prendergast P, Pan Z, Edwards DP. Department of Pathology, University of Colorado Health Sciences Center Denver 80262, USA. We have used circular permutation and phasing electrophoretic mobility shift assays to determine the ability of the A and B forms of human progesterone receptor (PR) to bend target DNA. Studies were done with baculovirus-expressed full-length receptors purified to apparent homogeneity. By circular permutation analysis, both forms of PR induced substantial distortions in the structure of target DNA with calculated distortion angles (alpha D) of 57 degrees for PR-A and 84 degrees for PR-B. The apparent bend centers for both forms of PR were similarly located a few base pairs (-4 to -2 bp) from the middle of the progesterone response element. No differences were detected in the magnitude of distortion or apparent bend centers when PR was bound to hormone agonist (R5020) or the antagonist RU486. Phasing analysis, which can determine the orientation of a DNA bend, revealed that both forms of PR mediated directional bends toward the major groove of the DNA helix. Calculated directed bend angles (alpha B) were 40 degrees for PR-B and 31 degrees for PR-A. The chromatin high mobility group protein HMG-1, which acts as an accessory factor to enhance the binding affinity of purified PR for progesterone response elements, had minimal influence on PR-mediated DNA bending. This result, taken together with the fact that HMG-1 can form a ternary complex with PR and DNA, is consistent with the conclusion that HMG-1 facilitates PR binding by stabilizing a receptor-induced DNA conformation that is required for assembly of a high affinity PR-DNA complex. The results of this study also suggest that DNA bending may be coupled to transcriptional regulation since PR-B is generally a stronger transcriptional activator than PR-A and also mediates a larger bend in target DNA than PR-A. PMID: 8721984 [PubMed - indexed for MEDLINE] PR147: Hypertension. 1996 Apr;27(4):1025-9. Mechanisms of transcriptional synergism of eukaryotic genes. The interferon-beta paradigm. Thanos D. Department of Biochemistry and Molecular Biophysics, Columbia University, New York 10032, USA. The virus-inducible enhancer of the human interferon-beta gene has served as an excellent example for the mechanisms controlling the activation and repression of transcription. This enhancer is activated by three different transcription factors that, with the help of the high mobility group protein HMG I(Y), assemble in a unique nucleoprotein complex that interacts as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcription factors may provide the specificity required for regulation of complex patterns of gene expression in higher eukaryotes. Publication Types: Review Review, Tutorial PMID: 8613258 [PubMed - indexed for MEDLINE] NR148: Plant Physiol. 1996 Feb;110(2):645-55. Light suppresses 3-Hydroxy-3-methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana. Learned RM. Section of Molecular and Cellular Biology, University of California, Davis 95616, USA. rmlearned@ucdavis.edu 3-Hydroxy-3-methylglutaryl (HMG) coenzyme A reductase mRNA accumulates preferentially in dark-grown Arabidopsis plants. As one step toward understanding the role that light plays in the regulation of the mevalonate pathway in plants, we characterized the suppression of HMG1 gene expression in response to illumination wavelength, duration, and fluence rate. The accumulation of HMG1 mRNA by dark treatment is suppressed by continuous exposure to white light and is dependent on the amount of light perceived during the period of illumination. By using promoter/reporter gene fusions we also demonstrate that this reaction is mediated by cis-acting elements that reside in the Arabidopsis HMG1 promoter and, therefore, is likely to be controlled at the transcriptional level. HMG1 expression is differentially responsive to continuous blue and red light but not to far-red light. In contrast, changes in HMG1 mRNA levels were not observed in response to brief light pulses of any spectrum, suggesting that continuous illumination is required for sustained and maximal suppression of HMG coenzyme A reductase expression. Taken together, these data indicate that light-mediated control of the HMG1 gene is mediated by a regulatory circuit that monitors aspects of both spectral quality and fluence and involves either multiple photoreceptors or a single photoreceptor that is differentially sensitive to both blue and red light. PMID: 8742338 [PubMed - indexed for MEDLINE] DR149: Cell. 1995 Dec 29;83(7):1091-100. Virus induction of human IFN beta gene expression requires the assembly of an enhanceosome. Thanos D, Maniatis T. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. We present evidence that transcriptional activation of the human interferon-beta (IFN beta) gene requires the assembly of a higher order transcription enhancer complex (enhanceosome). This multicomponent complex includes at least three distinct transcription factors and the high mobility group protein HMG I(Y). Both the in vitro assembly and in vivo transcriptional activity of this complex require a precise helical relationship between individual transcription factor-binding sites. In addition, HMG I(Y), which binds specifically to three sites within the enhancer, promotes cooperative binding of transcriptional factors in vitro and is required for transcriptional synergy between these factors in vivo. Thus, HMG I(Y) plays an essential role in the assembly and function of the IFN beta gene enhanceosome. PMID: 8548797 [PubMed - indexed for MEDLINE] DR150: Cell Struct Funct. 1995 Dec;20(6):421-7. Regulation of intracellular cholesterol metabolism. Sato R, Takano T. Department of Microbiology and Molecular Pathology, Teikyo University, Kanagawa, Japan. Animal cells synthesize cholesterol from acetyl CoA through a series of more than 20 enzymatic reactions. In addition, cells obtain cholesterol from plasma in the form of low-density lipoprotein (LDL), which is internalized via the LDL receptor and hydrolyzed to free cholesterol in lysosomes. Each cell must balance these internal and external sources while avoiding sterol shortage or overaccumulation. Both the biosynthetic and uptake pathways are well-regulated through feedback control. When cells are cultured in the presence of LDL, the activity of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase decline by more than 90% and the number of LDL receptors also decreases (1). In the absence of LDL, the cells maintain high activities of these two enzymes, which are rate-limiting enzymes of the biosynthetic pathway, and also maintain a large number of LDL receptors on their surface. In this review we assess recent progress in understanding the mechanisms involved in transcriptional and posttranscriptional regulation of intracellular cholesterol metabolism. Publication Types: Review Review, Tutorial PMID: 8825062 [PubMed - indexed for MEDLINE] NR151: Cell. 1995 Nov 17;83(4):599-609. pop-1 encodes an HMG box protein required for the specification of a mesoderm precursor in early C. elegans embryos. Lin R, Thompson S, Priess JR. Howard Hughes Medical Institute, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. In C. elegans embryogenesis, the MS blastomere produces predominantly mesodermal cell types, while its sister E generates only endodermal tissue. We show that a maternal gene, pop-1, is essential for the specification of MS fate and that a mutation in pop-1 results in MS adopting an E fate. Previous studies have shown that the maternal gene skn-1 is required for both MS and E development and that skn-1 encodes a transcription factor. We show here that the pop-1 gene encodes a protein with an HMG box similar to the HMG boxes in the vertebrate lymphoid-specific transcriptional regulators TCF-1 and LEF-1. We propose that POP-1 and SKN-1 function together in the early embryo to allow MS-specific differentiation. PMID: 7585963 [PubMed - indexed for MEDLINE] NR152: Arch Biochem Biophys. 1995 Oct 1;322(2):475-85. The length of 5'-untranslated leader sequences influences distribution of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in polysomes: effects of lovastatin, oxysterols, and mevalonate. Gayen AK, Peffley DM. Department of Pharmacology and Molecular Biology, University of Health Sciences, Chicago Medical School, Illinois 60064, USA. Transcripts for hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are heterogeneous in length. This heterogeneity is due to variations in the length of 5'-untranslated leader (UTL) sequences, which are generated by both alternate splicing within the first exon as well as alternate transcription start sites. Because mRNA 5'-UTL sequences have a role in regulating translational efficiency, the level and distribution of HMG-CoA reductase transcripts were measured in both total cellular RNA and polysomes from the Syrian hamster cell line C100. Cells were treated with either lovastatin alone, lovastatin and 25-hydroxycholesterol (25-OH C), or lovastatin, 25-OH C, and mevalonate, three treatment regimens used in an earlier study to demonstrate nonsterol-mediated translational control of HMG-CoA reductase synthesis [D. M. Peffley (1992) Somat. Cell Mol. Genet. 18, 19-32]. When reductase mRNA was measured by 5'-extension analysis under the same conditions, levels of transcripts with 5'-UTL regions ranging from 41 to 81 bases were reduced approximately four- to eightfold. In contrast, transcripts with 5'-UTL regions 93 to 100 bases in length were not reduced, and transcripts with 5'-UTL regions approximately 300-400 bases in length increased twofold. The addition of 25-OH C alone or both 25-OH C and mevalonate to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels fivefold in total cellular RNA as determined by RNase protection assay. No comparable change was observed with control ribosomal protein S17 mRNA. Postmitochondrial supernatants representing both translationally inactive monosomes and translationally active polysomes were prepared by sucrose gradient fractionation from cells incubated with the standard three treatments. Because 5'-UTL sequences of many mRNAs have a role in regulating translational efficiency we isolated RNA from each fraction and measured levels of reductase transcripts by 5'-extension analysis. Under all three conditions, transcripts with 5'-UTL sequences 41-103 bases in length were primarily associated with dense sucrose fractions that contain polysomes. In contrast, reductase transcripts with leader sequences 300 to 400 bases were almost exclusively associated with the less dense sucrose fractions containing monosomes. These results indicate that both the level and polysome distribution of individual reductase transcripts are influenced by the length of 5'-UTL sequences. PMID: 7574724 [PubMed - indexed for MEDLINE] NR153: Biochem Pharmacol. 1995 Aug 8;50(4):529-44. Azalanstat (RS-21607), a lanosterol 14 alpha-demethylase inhibitor with cholesterol-lowering activity. Burton PM, Swinney DC, Heller R, Dunlap B, Chiou M, Malonzo E, Haller J, Walker KA, Salari A, Murakami S, et al. Institute of Biochemistry and Cell Biology, Syntex Research, Palo Alto, CA 94304, USA. Agents that inhibit hepatic cholesterol biosynthesis reduce circulating cholesterol levels in experimental animals and humans, and may be of pharmacological importance in the prevention of atherosclerosis. Azalanstat (RS-21607), a synthetic imidazole, has been shown to inhibit cholesterol synthesis in HepG2 cells, human fibroblasts, hamster hepatocytes and hamster liver, by inhibiting the cytochrome P450 enzyme lanosterol 14 alpha-demethylase. When administered orally to hamsters fed regular chow, RS-21607 (50 mg/kg/day) lowered serum cholesterol in a dose-dependent manner (ED50 = 62 mg/kg) in a period of 1 week. It preferentially lowered low density lipoprotein (LDL) cholesterol and apo B relative to high density lipoprotein (HDL) cholesterol and apo A-1. It also lowered plasma cholesterol levels in hamsters fed a high saturated fat and cholesterol diet. RS-21607 inhibited hepatic microsomal hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity in hamsters in a dose-dependent manner (ED50 = 31 mg/kg), and this was highly correlated with serum cholesterol lowering (r = 0.97). Cholesterol lowering by azalanstat and cholestyramine was additive, and the increase in HMG-CoA reductase brought about by cholestyramine was attenuated significantly by azalanstat. In vitro studies with HepG2 cells indicated that this modulation of reductase activity was indirect, occurring at a post-transcriptional step, and it is proposed that a regulatory oxysterol derived from dihydrolanosterol (or lanosterol) may be responsible for this regulation. Azalanstat does not appear to lower circulating cholesterol in the hamster by up-regulation of the hepatic LDL receptor, suggesting that other mechanisms are involved. Orally administered azalanstat (50-75 mg/kg) stimulated hepatic microsomal cholesterol 7 alpha-hydroxylase activity by 50-400% in hamsters, and it is postulated that this may result from modified cholesterol absorption and bile acid synthesis. PMID: 7646560 [PubMed - indexed for MEDLINE] NR154: Mol Cell Biol. 1995 Jul;15(7):3738-47. Functional interaction between the POU domain protein Tst-1/Oct-6 and the high-mobility-group protein HMG-I/Y. Leger H, Sock E, Renner K, Grummt F, Wegner M. Zentrum fur Molekulare Neurobiologie, Universitat Hamburg, Germany. The POU domain protein Tst-1/Oct-6 is a transcriptional activator of human papovavirus JC virus in transient transfections. Because of its endogenous expression in myelinating glia, Tst-1/Oct-6 might also be an important determinant for the glia specificity of JC virus in vivo. Activation of viral early and late genes depends on the ability of Tst-1/Oct-6 to interact with an AT-rich element within the viral regulatory region. Here, we show that this element not only is bound by Tst-1/Oct-6 but, in addition, serves as a binding site for the high-mobility-group protein HMG-I/Y. In the presence of HMG-I/Y, Tst-1/Oct-6 exhibited an increased affinity for this AT-rich element. The specificity of this effect was evident from the fact that no stimulation of Tst-1/Oct-6 binding was observed on a site that did not allow binding of HMG-I/Y. In addition, both proteins interacted with each other in solution. Direct contacts were identified between the POU domain of Tst-1/Oct-6 and a short stretch of 10 amino acids in the central portion of HMG-I/Y. These results point to an accessory role for HMG-I/Y in the activation of JC viral gene expression by the POU domain protein Tst-1/Oct-6. In agreement with such a role, HMG-Y synergistically supported the function of Tst-1/Oct-6 in transient transfections, measured on the early promoter of JC virus or on an artificial promoter consisting of only a TATA box and the common binding element for Tst-1 and HMG-I/Y. PMID: 7791781 [PubMed - indexed for MEDLINE] NR155: Arterioscler Thromb Vasc Biol. 1995 Jul;15(7):872-8. In vivo LDL receptor and HMG-CoA reductase regulation in human lymphocytes and its alterations during aging. Stulnig TM, Klocker H, Harwood HJ Jr, Jurgens G, Schonitzer D, Jarosch E, Huber LA, Amberger A, Wick G. Institute for General and Experimental Pathology, University of Innsbruck, Austria. The LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play primary roles in the regulation of cellular cholesterol metabolism. To investigate the transcriptional regulation of lipid metabolism under physiological conditions ex vivo and its alterations during aging, we analyzed both the activity and mRNA concentration of the LDL receptor and HMG-CoA reductase in freshly isolated lymphocytes from healthy young and elderly donors. Data from fluorescent reverse transcriptase-polymerase chain reaction indicated that not only plasma LDL but also plasma HDL downregulates lymphocyte LDL receptor mRNA. Downregulation by HDL was three times more effective than that by LDL and presumably involved specific HDL binding sites. There was coordinate regulation of HMG-CoA reductase mRNA with LDL receptor mRNA that was independent of plasma lipoprotein concentrations. Despite elevated plasma concentrations of LDL, lymphocytes from elderly donors paradoxically expressed increased levels of the LDL receptor (P = .030) and HMG-CoA reductase mRNA (P = .062). The age-related dysregulation of the LDL receptor was predominantly due to impaired downregulation by plasma LDL rather than by HDL. Thus, not only LDL but also HDL and age significantly influences the transcriptional regulation of the LDL receptor in extrahepatic cells in vivo. PMID: 7541292 [PubMed - indexed for MEDLINE] DR156: J Biol Chem. 1995 Jun 9;270(23):14235-42. Differential regulation of a multipromoter gene. Selective 12-O-tetradecanoylphorbol-13-acetate induction of a single transcription start site in the HMG-I/Y gene. Ogram SA, Reeves R. Department of Biochemistry/Biophysics, Washington State University, Pullman 99164-4660, USA. The human HMG-I/Y gene, encoding the non-histone "high mobility group" proteins HMG-I and HMG-Y, is transcriptionally activated in human K562 erythroleukemia cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induces differentiation of K562 cells within 2-4 days after treatment. In this report, we show that transcriptional activation of the HMG-I/Y gene is dependent on protein synthesis and is an early event (2 h after induction) in the TPA-mediated differentiation process. Of the four functional transcription start sites present in the gene, only one (start site 2) is preferentially induced upon TPA treatment. This is the first report, to our knowledge, of the preferential utilization of a specific transcription start site in response to a particular stimulus in a gene that contains multiple promoters. This indicates that each start site in the gene has the potential to be independently regulated instead of being coordinately controlled as shown in a number of other genes. In addition, sequences upstream of the inducible start site, which contains a TPA-responsive element, mediates TPA inducibility through AP1 (or an AP1-like) transcription factor. The HMG-I/Y proteins function as key regulators of gene expression and play a significant role in chromatin structural changes as well. The cloning and sequence analyses previously reported indicated the structure of the HMG-I/Y gene to be highly complex and predicted its expression to be tightly regulated. The results presented here confirm and extend these earlier findings. PMID: 7775485 [PubMed - indexed for MEDLINE] NR157: J Lipid Res. 1995 Jun;36(6):1168-77. Regulation of cholesterol 7 alpha-hydroxylase expression by sterols in primary rat hepatocyte cultures. Doerner KC, Gurley EC, Vlahcevic ZR, Hylemon PB. Department of Medicine, Medical College of Virginia--Virginia Commonwealth, Richmond 23298, USA. The importance of cholesterol and "oxysterols" in the regulation of cholesterol 7 alpha-hydroxylase is not clear. Previous in vivo studies suggest that cholesterol may up-regulate cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, but these studies are open to question as they were carried out in whole animals. Therefore, we used primary rat hepatocytes, cultured in serum-free medium, to determine the effects of cholesterol on the regulation of cholesterol 7 alpha-hydroxylase. Squalestatin, a specific squalene synthase inhibitor, was used to block sterol but not isoprenoid biosynthesis in this system. Squalestatin (1 microM) decreased cholesterol 7 alpha-hydroxylase specific activity to undetectable levels and decreased steady-state mRNA and transcriptional activity to 13% and 47% of controls, respectively. Mevalonolactone (2 mM) failed to restore cholesterol 7 alpha-hydroxylase specific activity or steady-state mRNA levels in squalestatin-treated cells. Addition of cholesterol, delivered in beta-cyclodextrin, to squalestatin-treated cells restored cholesterol 7 alpha-hydroxylase specific activity and steady-state mRNA to control levels in a concentration (25 microM to 200 microM) -dependent manner. In contrast, the individual addition of selected "oxysterols" (5-cholesten-3 beta, 7 alpha-diol; 5 alpha-cholestan-3 beta, 6 alpha-diol; cholestan-3 beta, 5 alpha,6 beta-triol; 5-(25R)-cholesten-3 beta,26-diol, all at 50 microM) failed to restore cholesterol 7 alpha-hydroxylase mRNA levels in squalestatin-treated cells. These experiments provide evidence that cholesterol rather than "oxysterols" regulate cholesterol 7 alpha-hydroxylase gene expression. Squalestatin (1 microM) treatment increased HMG-CoA reductase specific activity by 229% of controls. Addition of cholesterol (200 microM), but not mevalonolactone (2 mM), to squalestatin-treated cells decreased HMG-CoA reductase specific activity to 19% of control. The primary rat hepatocyte culture system in conjunction with a specific squalene synthetase inhibitor should be a useful model for elucidating the mechanism of regulation of cholesterol 7 alpha-hydroxylase gene expression by sterols. PMID: 7665995 [PubMed - indexed for MEDLINE] NR158: J Biol Chem. 1995 Apr 7;270(14):8023-31. Brefeldin A renders Chinese hamster ovary cells insensitive to transcriptional suppression by 25-hydroxycholesterol. Ridgway ND, Lagace TA. Department of Pediatrics and Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada. The effect of disruption of the Golgi apparatus on 25-hydroxycholesterol-mediated transcriptional suppression and activation of acyl-CoA:cholesterol acyltransferase was examined. In Chinese hamster ovary (CHO) cells, brefeldin A (BFA) caused dose-dependent inhibition of 25-hydroxycholesterol-mediated suppression of mRNAs for four sterol-regulated genes: 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, HMG-CoA synthase, farnesyl-diphosphate synthase, and the low density lipoprotein receptor. BFA prevented suppression whether added prior to or following a 4-h pretreatment with 25-hydroxycholesterol. In the presence of BFA (1 microgram/ml), 25-hydroxycholesterol-mediated suppression of mRNAs for HMG-CoA reductase, the low density lipoprotein receptor, and farnesyl-diphosphate synthase was almost completely blocked. HMG-CoA synthase mRNA was 80-90% suppressed by 25-hydroxycholesterol compared with 50-60% suppression in the presence of BFA. These effects of BFA were not due to alterations in mRNA stability. Disruption of the Golgi apparatus, as assessed by staining with a fluorescent lectin, correlated with concentrations of BFA that reversed mRNA suppression. Monensin was also found to block the effects of 25-hydroxycholesterol on suppression of HMG-CoA reductase. However, this ionophore decreased the other three sterol-regulated mRNAs to a similar degree as 25-hydroxycholesterol. In contrast to CHO cells, BFA-resistant PtK1 cells displayed normal down-regulation of HMG-CoA reductase and an intact Golgi apparatus in the presence of BFA and 25-hydroxycholesterol. Cholesterol esterification in CHO cells was stimulated to a similar extent by BFA (1 microgram/ml) and 25-hydroxycholesterol, and simultaneous treatment of CHO cells with both compounds was 60-70% additive. These results suggest that an intact Golgi apparatus is required for 25-hydroxycholesterol-mediated suppression of mRNA. PMID: 7713903 [PubMed - indexed for MEDLINE] DR159: Biochem J. 1995 Apr 1;307 ( Pt 1):233-8. 3'-untranslated sequences mediate post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA by 25-hydroxycholesterol. Choi JW, Peffley DM. Department of Pharmacology and Molecular Biology, University of Health Sciences, Chicago Medical School, IL 60064, USA. In an earlier study [Choi, Lundquist and Peffley (1993) Biochem. J. 296, 859-866], we determined that 25-hydroxycholesterol regulates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA through a post-transcriptional mechanism that requires protein synthesis. To investigate whether 3'-untranslated sequences play a role in 25-hydroxycholesterol-mediated post-transcriptional control, we ligated approx. 1400 bp of the 3'-untranslated region of HMG-CoA reductase cDNA to the coding region of human beta-globin DNA. beta-Globin-3'-untranslated reductase fusion constructs were then transiently expressed in Chinese hamster ovary fibroblasts under conditions known to regulate reductase mRNA. There were no differences in beta-globin RNA levels in transfected cells incubated with or without lovastatin, a competitive inhibitor of reductase. However, in the presence of lovastatin and an oxysterol, 25-hydroxycholesterol, beta-globin RNA levels were decreased approx. 2-fold. Inhibition of protein synthesis with cycloheximide blocked the effects of 25-hydroxycholesterol on beta-globin RNA. Moreover, replacing the 3'-untranslated sequences with 1367 bp of the simian virus 40 enhancer region eliminated the regulatory effect of 25-hydroxycholesterol. Because the fusion construct has no sterol regulatory elements necessary for transcription, our results indicate that the change in beta-globin RNA occurred at a post-transcriptional level. In addition, we have shown that the 3'-untranslated region of HMG-CoA reductase cDNA imparted oxysterol-mediated post-transcriptional regulation to beta-globin RNA, an effect that required protein synthesis. PMID: 7717981 [PubMed - indexed for MEDLINE] NR160: J Biol Chem. 1995 Mar 31;270(13):7515-22. The high mobility group transcription factor, SOX4, transactivates the human CD2 enhancer. Wotton D, Lake RA, Farr CJ, Owen MJ. Imperial Cancer Research Fund, London, United Kingdom. A strong T cell-specific enhancer is located 3' to the human CD2 gene. Six sequences within this enhancer are bound by proteins present in T cell nuclear extracts. These sequences share homology with sequences bound by several transcription factors involved in T cell- and lymphoid-specific transcription. The results presented here demonstrate that the human T cell-specific transcription factor, SOX4, is able to bind to one of these regions; further, SOX4 transactivates transcription of a reporter gene via three tandem copies of this sequence. The binding of SOX4 to this site is not via a canonical HMG protein binding sequence, identifying a novel class of binding site for this protein. A second sequence within the CD2 enhancer closely resembles the IL-2 NF-AT site. We show that it is bound by the ets-related factor, Elf1. However, unlike the IL-2 NF-AT sequence, the CD2 NF-AT-like sequence is unable to confer transcriptional inducibility on a reporter gene. Consistent with this result, we show that the observed increase in expression of CD2 protein on the cell surface following T cell activation is a post-transcriptional event. PMID: 7706298 [PubMed - indexed for MEDLINE] DR161: EMBO J. 1995 Mar 15;14(6):1198-208. High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors. Zwilling S, Konig H, Wirth T. Zentrum fur Molekulare Biologie Heidelberg, Germany. The octamer transcription factors Oct1 and Oct2 are involved in the transcriptional regulation of both lymphoid-specific and ubiquitously expressed genes. Their activity depends critically on their interaction with distinct cellular cofactors. Therefore, we have isolated cDNAs encoding proteins that physically interact with Oct2. Here we describe the analysis of one such clone, representing the murine homologue of high mobility group (HMG) protein 2. We have mapped the interaction domains for both proteins and have shown that HMG2 and Oct2 interact via their HMG domains and POU homeodomains, respectively. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 was fused directly to the HMG domains of HMG2, stimulated the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators. PMID: 7720710 [PubMed - indexed for MEDLINE] NR162: Arch Biochem Biophys. 1995 Feb 20;317(1):235-43. Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevalonate. Straka MS, Panini SR. Eleanor Roosevelt Institute, Denver, Colorado 80206. We have examined the mechanisms of sterol-independent regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase by mevalonate in Chinese hamster ovary (CHO) cells. Serum lipoproteins, 25-hydroxycholesterol, or mevalonate each repress HMG-CoA reductase activity by fivefold or more, and mevalonate lowers the rate of reductase synthesis by twofold. However, while the expression of the HMG-CoA reductase promoter construct, T42 delta CAT, in stable transfectants is also repressed by serum lipoproteins and 25-hydroxycholesterol, mevalonate is without effect. In addition, while 25-hydroxycholesterol reduces the steady-state level of endogenous HMG-CoA reductase mRNA by more than threefold, mevalonate again has no effect. Mevalonate does partially regulate the expression of both the artificial promoter construct pTK-Kx3-CAT, containing three copies of the sterol regulatory element, SRE-1, and the full-length LDL receptor promoter construct, pLDLRCAT-6500 as well as the expression of functional LDL receptors. This transcriptional regulation appears to be mediated by sterol end products generated from added mevalonate. In CHO cells starved for mevalonate due to a mutation in the biosynthetic pathway, addition of 20 mM mevalonate accelerates the rate of degradation of HMG-CoA reductase by threefold whether new sterol biosynthesis is blocked or not. In such cells, addition of 25-hydroxycholesterol, by itself, also decreases the half-life of reductase from 11.6 to 2.3 h. In contrast, in cells acutely treated with a reductase inhibitor, sterol-accelerated degradation of reductase is only observed in the presence of submillimolar level of mevalonate. We conclude that large concentrations of exogenous mevalonate fail to generate a transcriptional regulator of HMG-CoA reductase in CHO cells but do lead to the formation of translational regulator(s) of reductase synthesis. In contrast, sterol regulators derived from exogenous mevalonate appear to be capable of downregulating the LDL receptor promoter. We further conclude that in the absence of pretreatment with a reductase inhibitor, the regulatory signals generated by sterols and nonsterols for accelerated degradation of HMG-CoA reductase are mutually independent. However, the enzyme synthesized in the presence of reductase inhibitors appears to exhibit an obligatory corequirement for low-dose mevalonate for sterol-accelerated degradation. PMID: 7872789 [PubMed - indexed for MEDLINE] NR163: Gastroenterology. 1995 Feb;108(2):533-44. Failure of intravenous infusion of taurocholate to down-regulate cholesterol 7 alpha-hydroxylase in rats with biliary fistulas. Pandak WM, Heuman DM, Hylemon PB, Chiang JY, Vlahcevic ZR. Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond. BACKGROUND/AIMS: The decrease in cholesterol 7 alpha-hydroxylase induced by intraduodenal infusion of taurocholate in bile fistula rats may be indirect, i.e., mediated through release or absorption of an intestinal factor in response to the presence of bile salts in the intestine. The aim of this study was to determine if negative feedback regulation of cholesterol 7 alpha-hydroxylase can be shown when equimolar concentrations of taurocholate are administered intravenously, thus bypassing the intestine. METHODS: After 96 hours of biliary diversion, taurocholate (36 mumol.h-1.100 g, rat-1) was infused into the rats either intravenously or intraduodenally for the final 24 hours. Livers were then harvested for analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase specific activity, cholesterol 7 alpha-hydroxylase specific activity, messenger RNA levels, and transcriptional activity. RESULTS: Intraduodenally administered taurocholate significantly decreased HMG-CoA reductase and cholesterol 7 alpha-hydroxylase specific activity by more than 50% and cholesterol 7 alpha-hydroxylase steady-state messenger RNA levels and transcriptional activity by 50%-75%. In contrast, intravenous administration of taurocholate failed to down-regulate either cholesterol 7 alpha-hydroxylase or HMG-CoA reductase. CONCLUSIONS: Passage of taurocholate through the intestine strongly potentiates negative feedback regulation of cholesterol 7 alpha-hydroxylase. A putative intestinal factor, released or absorbed in the presence of bile acids in the intestinal lumen, may play a role in the regulation of bile acid synthesis. PMID: 7835596 [PubMed - indexed for MEDLINE] NR164: Biochem Biophys Res Commun. 1995 Jan 26;206(3):916-26. Identification of novel cellular genes transcriptionally suppressed by v-src. Frankfort BJ, Gelman IH. Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029. Our aim is to identify cellular genes whose transcriptional suppression by the v-src oncogene contributes directly and specifically to the transformed and tumorigenic phenotype. We used a modified PCR-based subtractive hybridization technique to isolate 9 cDNAs whose abundance in NIH3T3 fibroblasts is decreased 3-15-fold following transformation by the activated oncogene, v-src. Sequence analysis reveals that 3 cDNAs are unlike those in GenBank. The remaining 6 cDNAs are indentical or highly similar to rat helix-destabilizing protein gene (hnRNP A1), mouse CTLA-2 alpha cysteine protease, rat cytochrome c oxidase (COX) VIc subunit, mouse Type I collagen, human gravin and a partial human cDNA (clone A7C09) isolated by random automated sequencing. Northern blot analysis indicates that the basal level of transcripts in untransformed NIH3T3 of all the genes except mouse Type I collagen was at least 10-fold lower than that of HMG Co-A Reductase, which is abundantly transcribed. These data suggest that the down-regulation of some or all of these genes contributes to v-src-induced changes in mitogenic control or cell morphology. PMID: 7832805 [PubMed - indexed for MEDLINE] DR165: Biochemistry. 1994 Dec 13;33(49):14690-5. Stimulation of transcription in cultured cells by high mobility group protein 1: essential role of the acidic carboxyl-terminal region. Aizawa S, Nishino H, Saito K, Kimura K, Shirakawa H, Yoshida M. Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan. Several in vitro studies have suggested that high mobility group (HMG) protein 1 has a role in gene regulation as a trans activator or quasi-transcription factor. However, data on the molecular functions of HMG1 protein in these reactions are contradictory or obscure. In order to assess whether HMG1 protein does, in fact, have transcriptional activation potential, two assay systems in cultured cells were employed. HMG1 protein introduced into COS-1 cells as a complex with a reporter plasmid carrying the lacZ gene enhanced the level of the gene expression. Cotransfection of an expression plasmid carrying HMG1 cDNA into the cells with the reporter plasmid enhanced the activity of beta-galactosidase 2-3-fold in comparison with that of the control effector plasmid. The enhancement was proved to be dependent not on the replication but on the transcription of the reporter plasmid. In the cotransfection experiments, an expression plasmid the HMG1 molecule lacking the acidic carboxyl terminus repressed the expression of the reporter gene. The binding of an HMG1 protein variant lacking the acidic carboxyl terminus to DNA gave an extremely large shift of gel retardation in comparison with the complete HMG1 molecule. Together, these results indicate that HMG1 protein can enhance expression in cells in culture at the step of gene transcription and that the DNA binding domains comprising two-thirds of the HMG1 protein molecule are responsible for the inhibition property. Also, the acidic terminus of the HMG1 molecule is essential for the enhancement of gene expression in addition to elimination of the repression caused by the DNA binding. (ABSTRACT TRUNCATED AT 250 WORDS) PMID: 7993897 [PubMed - indexed for MEDLINE] NR166: Mol Cell Biol. 1994 Dec;14(12):7758-69. Interaction between a novel F9-specific factor and octamer-binding proteins is required for cell-type-restricted activity of the fibroblast growth factor 4 enhancer. Dailey L, Yuan H, Basilico C. Rockefeller University, New York, New York 10021. Understanding how diverse transcription patterns are achieved through common factor binding elements is a fundamental question that underlies much of developmental and cellular biology. One example is provided by the fibroblast growth factor 4 (FGF-4) gene, whose expression is restricted to specific embryonic tissues during development and to undifferentiated embryonal carcinoma cells in tissue culture. Analysis of the cis- and trans-acting elements required for the activity of the previously identified FGF-4 enhancer in F9 embryonal carcinoma cells showed that enhancer function depends on sequences that bind Sp1 and ubiquitous as well as F9-specific octamer-binding proteins. However, sequences immediately upstream of the octamer motif, which conform to a binding site for the high-mobility group (HMG) domain factor family, were also critical to enhancer function. We have identified a novel F9-specific factor, Fx, which specifically recognizes this motif. Fx formed complexes with either Oct-1 or Oct-3 in a template-dependent manner. The ability of different enhancer variants to form the Oct-Fx complexes correlated with enhancer activity, indicating that these complexes play an essential role in transcriptional activation of the FGF-4 gene. Thus, while FGF-4 enhancer function is octamer site dependent, its developmentally restricted activity is determined by the interaction of octamer-binding proteins with the tissue-specific factor Fx. PMID: 7969117 [PubMed - indexed for MEDLINE] NR167: Nippon Rinsho. 1994 Dec;52(12):3191-6. [Regulation of gene expression by cholesterol and macrophage scavenger receptor] [Article in Japanese] Kawabe Y, Itakura H, Kodama T. 3rd Department of Internal Medicine, University of Tokyo. Cellular cholesterol level is strictly regulated by the synthesis and the incorporation of cholesterol. Cholesterol synthetic enzymes and lipoprotein receptors are regulated mainly at the transcriptional level. Recently, sterol regulatory element binding proteins (SREBPs) which induce LDL receptor expression, were cloned and the mechanism of sterol-mediated gene regulation was proposed. Cholesterol synthesizing enzymes such as HMG-CoA reductase, farnesyl pyrophosphate synthase and squalene synthase are also regulated by the cellular cholesterol level, but it is suggested that SREBPs are not play critical role in their regulation. Scavenger receptors also uptake cholesterol into macrophage, but the receptors are not regulated by the cellular cholesterol level and this leads to the abnormal accumulation of cholesterol in macrophages. Publication Types: Review Review, Tutorial PMID: 7853709 [PubMed - indexed for MEDLINE] NR168: Kidney Int. 1994 Aug;46(2):358-64. Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in rats with subtotal nephrectomy. Pandak WM, Vlahcevic ZR, Heuman DM, Krieg RJ, Hanna JD, Chan JC. Department of Medicine, Medical College of Virginia, Richmond. Chronic renal failure is associated with hyperlipidemia and atherosclerosis. The mechanism responsible for the observed increase of serum cholesterol in chronic renal disease is not certain. The objective of the present study was to characterize the effect of induced renal failure on 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, the two rate determining enzymes of the cholesterol and bile acid biosynthetic pathways, respectively. Studies were carried out in rats with subtotal (75%) nephrectomy, which resulted in a marked elevation of blood urea nitrogen (371 +/- 44% of control, P < 0.001), and was accompanied by significant increases in the levels of serum cholesterol (133 +/- 7%, P < 0.005) and triglycerides (185 +/- 25, P < 0.01). In nephrectomized rats, an increase in the specific activity of HMG-CoA reductase (219 +/- 30% above control levels, P < 0.02) was observed. This increase occurred in the presence of elevated hepatic microsomal cholesterol concentrations (150 +/- 13% of controls, P < 0.01). Surprisingly, the increase in HMG-CoA reductase specific activity was not associated with parallel increases in HMG-CoA reductase steady-state mRNA levels and gene transcriptional activity. These uremic rats also exhibited a marked increase in the specific activity of cholesterol 7 alpha-hydroxylase (240 +/- 559% of controls, P < 0.05). There was no concomitant increase in cholesterol 7 alpha-hydroxylase steady-state mRNA levels or gene transcriptional activity. The factors responsible for the observed increases in HMG-CoA reductase and cholesterol 7 alpha-hydroxylase specific activity in renal failure remain to be determined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 7967347 [PubMed - indexed for MEDLINE] NR169: J Biol Chem. 1994 Jul 22;269(29):18767-72. Peroxisome proliferator-activated receptor mediates induction of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene by fatty acids. Rodriguez JC, Gil-Gomez G, Hegardt FG, Haro D. Unit of Biochemistry, School of Pharmacy, University of Barcelona, Spain. Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter-chloramphenicol acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in chloramphenicol acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE) in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in the PPREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism. PMID: 7913466 [PubMed - indexed for MEDLINE] NR170: J Biol Chem. 1994 Apr 1;269(13):10071-8. Regulation of cholesterol 7 alpha-hydroxylase gene expression in Hep-G2 cells. Effect of serum, bile salts, and coordinate and noncoordinate regulation with other sterol-responsive genes. Taniguchi T, Chen J, Cooper AD. Department of Medicine, Stanford University School of Medicine, California 94305. Regulation of cholesterol 7 alpha-hydroxylase mRNA level in Hep-G2 cells was studied and compared with that of two other sterol-responsive genes, those for the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In culture medium containing 10% fetal bovine serum (complete medium) for up to 24 h, the mRNA for cholesterol 7 alpha-hydroxylase gradually increased to 2-fold of the time 0 control. Culture of Hep-G2 cells in serum-free medium for 24 h resulted in stimulation of mRNA levels for LDL receptor (5-fold) and HMG-CoA reductase (6-fold). Surprisingly, the mRNA level for cholesterol 7 alpha-hydroxylase also increased 5-fold at 8 h and 4-fold at 24 h compared with the time 0 control. The addition of beta-migrating very low density lipoprotein (beta-VLDL) (40 micrograms/ml) and 25-hydroxycholesterol (5 micrograms/ml) prevented the increase in mRNA level for the LDL receptor, and HMG-CoA reductase and the levels were 10-26% of the control at 8 h. The effect with beta-VLDL was sustained for 24 h. With 25-hydroxycholesterol, both LDL receptor and HMG-CoA reductase mRNA returned to base line by 24 h. In contrast, beta-VLDL increased cholesterol 7 alpha-hydroxylase mRNA level above the serum-free control within 8 h (+32%), and this was sustained for 24 h (+47%). There was a slight induction of cholesterol 7 alpha-hydroxylase mRNA levels by 25-hydroxycholesterol at 8 h (+18%); but by 24 h, its level was below that of the control (-47%). There was no induction of cholesterol 7 alpha-hydroxylase mRNA levels by beta-VLDL or 25-hydroxycholesterol when the cells were grown in complete medium. As determined by nuclear run-on assay, the increase in the transcriptional rate of the cholesterol 7 alpha-hydroxylase gene in cells grown in serum-free medium (3.9-fold of the rate in complete medium) and incubated with beta-VLDL (+68% above serum-free control) at 8 h, was comparable with the increase in mRNA levels (3.5-fold and +32%, respectively). When bile salts were added to serum-free medium and cells cultured for up to 24 h, chenodeoxycholate and glycochenodeoxycholate caused a marked suppression of the level of cholesterol 7 alpha-hydroxylase mRNA, while cholate and its conjugates did not.(ABSTRACT TRUNCATED AT 400 WORDS) PMID: 8144506 [PubMed - indexed for MEDLINE] NR171: Hepatology. 1994 Apr;19(4):941-7. Effects of different bile salts on steady-state mRNA levels and transcriptional activity of cholesterol 7 alpha-hydroxylase. Pandak WM, Vlahcevic ZR, Heuman DM, Redford KS, Chiang JY, Hylemon PB. Section of Gastroenterology, McGuire Veterans Administration Medical Center, Richmond, Virginia. Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid synthesis pathway, is down-regulated by taurocholate by way of negative feedback control at the level of gene transcription. The molecular basis of regulation of cholesterol 7 alpha-hydroxylase by other hydrophobic bile salts and under more physiological conditions is not known. The aim of this study was to investigate the molecular basis of regulation of cholesterol 7 alpha-hydroxylase by several naturally occurring bile salts in rats with intact enterohepatic circulation. Male Sprague-Dawley rats were pair-fed for 14 days normal chow (control), cholestyramine (5% of diet), cholic acid (1%), chenodeoxycholic acid (1%) or deoxycholic acid (0.25%). When rats were killed, livers were harvested and HMG-CoA reductase specific activity and cholesterol 7 alpha-hydroxylase specific activities, steady-state mRNA levels and transcriptional activity were determined and compared with those of control rats fed normal chow. Compared with results in paired controls, cholestyramine feeding led to an approximate threefold increase in HMG-CoA reductase specific activity. Feeding of hydrophobic bile salts profoundly decreased the specific activity of HMG-CoA reductase. Cholestyramine led to a three-fold increase in cholesterol 7 alpha-hydroxylase specific activity, steady-state mRNA levels and gene transcriptional activity. The feeding of cholic (1%), chenodeoxycholic (1%) and deoxycholic acid (0.25%) led to significant decreases in cholesterol 7 alpha-hydroxylase specific activities (62%, 84% and 97%, respectively), steady-state mRNA levels (72%, 29% and 61%, respectively) and transcriptional activities (44%, 43% and 54%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 8138269 [PubMed - indexed for MEDLINE] NR172: J Biol Chem. 1994 Mar 4;269(9):6645-50. Non-sterol compounds that regulate cholesterogenesis. Analogues of farnesyl pyrophosphate reduce 3-hydroxy-3-methylglutaryl-coenzyme A reductase levels. Bradfute DL, Simoni RD. Department of Biological Sciences, Stanford University, California 94305-5020. Farnesyl acetate and ethyl farnesyl ether, two analogues of farnesyl pyrophosphate, stimulate post-transcriptional down-regulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids. Farnesyl acetate and ethyl farnesyl ether reduce translation of HMG-CoA reductase mRNA and enhance degradation of the enzyme, the same regulatory effects attributed to the putative non-sterol regulatory metabolite (Goldstein, J.L., and Brown, M.S. (1990) Nature 343, 425-430). HMGal, a fusion protein consisting of the membrane domain of HMG-CoA reductase linked to Escherichia coli beta-galactosidase, is subject to the same regulated degradation as HMG-CoA reductase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). At 10 micrograms/ml (37.8 microM), farnesyl acetate and ethyl farnesyl ether trigger a 50-80% reduction in HMGal activity. Farnesyl acetate reduces the synthesis of HMG-CoA reductase and HM-Gal by 60-80%, but neither farnesyl compound affects HMG-CoA reductase mRNA levels. Farnesyl acetate and ethyl farnesyl ether stimulated the degradation of HMG-CoA reductase and HMGal, reducing the half-lives of the enzymes by 40-70%. In addition to their regulatory effects on HMG-CoA reductase, these farnesyl compounds also directly disrupt sterol synthesis. PMID: 8120018 [PubMed - indexed for MEDLINE] DR173: EMBO J. 1994 Jan 15;13(2):416-24. Functional differences between the two splice variants of the nucleolar transcription factor UBF: the second HMG box determines specificity of DNA binding and transcriptional activity. Kuhn A, Voit R, Stefanovsky V, Evers R, Bianchi M, Grummt I. German Cancer Research Center, Heidelberg. The nucleolar transcription factor UBF consists of two proteins, UBF1 and UBF2, which originate by alternative splicing. Here we show that deletion of 37 amino acids within the second of five HMG box motifs in UBF2 is important for the dual role of UBF as transcriptional activator and antirepressor. UBF1 is a potent antirepressor and transcriptional activator, whereas the ability of UBF2 to counteract histone H1-mediated repression and to stimulate ribosomal gene transcription both in vivo and in vitro is at least one order of magnitude lower. The difference in transcriptional activity between UBF1 and UBF2 is due to their different binding to the ribosomal gene promoter and enhancer. Apparently, the presence of an intact HMG box2 modulates the sequence-specific binding of UBF to rDNA control elements. However, the interaction of UBF with rDNA does not entirely depend on sequence recognition. Both UBF isoforms bind efficiently to four-way junction DNA, indicating that they recognize defined DNA structures rather than specific sequences. The results demonstrate that the HMG boxes are functionally diverse and that HMG box2 plays an important role in specific binding of UBF to rDNA. PMID: 8313887 [PubMed - indexed for MEDLINE]