Positive Regulation of the Human Macrophage Stimulating Protein Gene Transcription
IDENTIFICATION OF A NEW HEPATOCYTE NUCLEAR FACTOR-4 (HNF-4) BINDING ELEMENT AND EVIDENCE THAT INDICATES DIRECT ASSOCIATION BETWEEN NF-Y and HNF-4*

Atsuhisa UedaDagger §, Fumihiko Takeshita§, Shigeo YamashiroDagger , and Teizo YoshimuraDagger

From the Dagger  Immunopathology Section, Laboratory of Immunobiology, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 and § The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We previously reported that the transcription of the human macrophage stimulating protein (MSP) gene was positively regulated by the binding of NF-Y to the CAATT sequence in the promoter region of this gene. Here we confirmed our previous results and further characterized the MSP promoter. Luciferase assay with deletion constructs showed the importance of the region, +32 to +39, for the promoter activity in Hep3B cells. Two nuclear protein-DNA probe (+15 to +40) complexes, C1 and C2, were detected by electrophoretic mobility shift assay. C2 was specific to hepatoma cells and contained hepatocyte nuclear factor-4 (HNF-4). DNase I footprinting with recombinant HNF-4 located another HNF-4-binding site in the distal region, -89 to -54. Mutations in the CAATT or the proximal HNF-4-binding site significantly reduced the promoter activity in Hep3B cells and HNF-4-transfected HeLa cells, whereas mutations in the distal HNF-4-binding site had no effect. The close proximity between the CAATT and the proximal HNF-4-binding site suggested that a direct contact between NF-Y and HNF-4 might be important. Protein-protein interaction between the A-subunit of NF-Y and HNF-4 was detected by a yeast two-hybrid system. The binding of in vitro translated HNF-4 to immobilized NF-YA and in vitro translated NF-YA to immobilized HNF-4 was also detected. These results suggest the binding of HNF-4 to the proximal HNF-4-binding site directs the basal transcription of the MSP gene, and the maximal promoter activity may depend on the direct association between HNF-4 and NF-Y.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Macrophage stimulating protein (MSP)1 is a hepatocyte-derived serum protein that has high amino acid sequence similarity to hepatocyte growth factor/scatter factor (1-3). MSP was originally purified as a protein that made mouse resident peritoneal macrophages capable of responding to the chemoattractant C5a (4). However, recent study shows that MSP itself is a chemoattractant for mouse resident macrophages (5). The receptor for MSP is a receptor tyrosine kinase, Ron/Stk (6-10). Identification of the MSP receptor revealed that keratinocytes are another target cell of MSP (10), and MSP appears to be involved in the migration of keratinocytes during the healing process after skin injury. We and others (2, 3) previously reported that MSP mRNA was predominantly and constitutively expressed in the liver, probably in hepatic cells.

The transcriptional mechanisms of genes specifically expressed in hepatic cells, such as albumin, transthyretin, alpha 1-antitrypsin, or fibrinogen, have been previously investigated (11). In most of the cases, transcription of these genes is positively regulated by liver-specific transcriptional factors such as CCAAT/enhancer-binding protein alpha  (C/EBPalpha ) (12), hepatocyte nuclear factor (HNF)-1 (13), HNF-3 (14), and HNF-4 (15). Recently, we cloned the 5'-flanking region of the human MSP gene and found that the transcription of the human MSP gene was regulated by positive and negative regulatory elements (PRE and NRE) (16). The PRE was located in the region between 34 and +2 and was essential for the maximal transcription of this gene. This sequence carried the "CCAAT" sequence and was the binding site for the ubiquitously expressed trans-activator, MSP-PRE-binding protein-1, and MSP-PRE-binding protein-2 (identical to NF-Y/CCAAT-binding factor) (16). The NRE was located in the region between -141 and -34 and appeared to be responsible for the tissue-specific expression of this gene.

Waltz et al. (17) also investigated the mechanisms of the human MSP gene transcription. There were significant differences between the data obtained by two groups. Waltz et al. (17) identified putative transcription initiation sites at -75 and -76 (corresponding to -27 and -28 in out study), located on the CCAAT sequence, by using RNA from HepG2 cells transfected with the pL5CAT (-1348/+1) chimeric construct, but they could not obtain primer extension products from RNA isolated from untransfected HepG2 cells. Waltz et al. (17) also reported that the binding of HNF-4 to the region between -135 and -105 (corresponding to -98 and -69 in our study) activated the transcription of this gene (17). The binding of HNF-4 to the region was shown by EMSA and supershift. trans-Activity of HNF-4 was demonstrated with a CAT construct, pL5CAT5 (-135/-105), constructed by ligating the sequence between -135 and -105 to the upstream of the herpes simplex virus thymidine kinase promoter in pBLCAT5. The thymidine kinase promoter contained TATA box, whereas the wild-type MSP promoter does not contain TATA box. Thus, the presence of TATA box in pL5CAT 5 (-135/-105) could influence the outcome of their experiments.

After the publication of our results (16), we further characterized the PRE and NRE by DNase I protection assay. Two distinct footprints were detected with Hep3B cell nuclear extracts. One was the region between -33 and -4 that included the CCAAT sequence, and the other was the region between -80 and -65. In contrast, a broad region between -104 and -4 was protected with HeLa or HOS nuclear extracts. By EMSA and supershift assay, we confirmed that HNF-4 bound to the region between -80 and -65 as shown by Waltz et al. (17). Co-transfection of HOS cells with an HNF-4 expression vector and a vector containing 1 kilobase pair of the MSP promoter fused to the CAT gene dose-dependently increased CAT activity, suggesting that the binding of HNF-4 to this region was important for the tissue-specific expression of MSP mRNA. However, it was surprising that the deletion of the HNF-4 binding sequence from the pMD1k or mutation on the HNF-4 binding sequence did not affect the promotor activity of the MSP gene in Hep3B, HepG2, and HNF-4-transfected HOS cells.2 Thus, although some of our results agreed with that reported by Waltz et al. (17), we could not explain tissue-specific expression of MSP mRNA simply by the binding of HNF-4 to the previously reported HNF-4-binding site. There appear to be other mechanisms that regulate tissue-specific expression of MSP mRNA.

As described above, the 5'-region of the MSP gene does not contain TATA box. In promoters that do not have TATA box, an initiator (Inr) is functionally analogous to TATA and capable of directing basal transcription by RNA polymerase II (18, 19). In the promoter region of the MSP gene, a sequence similar to the weak consensus sequence for the Inr (-3YYCAYYYYY+6) (18) was found between +23 and +31. In our preliminary study, deletion of the region between -5 and +49 resulted in a significant decrease (1/20) in the CAT activity in Hep3B cells. In contrast, deletion of the region between +37 and +49 had no effect, suggesting that the region between -5 and +36 could be functionally important for the promoter activity.

In the present study, we first repeated CAT assay to confirm our previous results that suggested that the transcription of the MSP gene was regulated by PRE and NRE. After the confirmation of our previous results, we further analyzed the MSP promoter located between -5 and +49 and found that the binding of HNF-4 to the sequence between +32 and +39 was critical for the basal transcription of this gene, and the maximal promoter activity depended on the cooperation between HNF-4 and NF-Y.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Reagents-- Dulbecco's modified Eagle's medium, minimum essential medium, RPMI 1640, Dulbecco's phosphate-buffered saline, and isopropyl beta -D-thiogalactopyranoside were from Life Technologies, Inc. Fetal calf serum (FCS) was from HyClone, Logan, UT. L-[35S]Methionine was from Amersham Pharmacia Biotech. alpha -[32P]CTP was from ICN Biomedicals, Inc., Costa Mesa, CA. Oligonucleotides were from Operon Technologies, Inc., Alameda, CA, and BioServe, Laurel, MD. TnT-coupled rabbit reticulocyte lysate system and luciferase assay system were from Promega, Madison, WI. pcDNA3 was from Invitrogen, Carlsbad, CA. pET32a and His·Bind Resin columns were from Novagen, Madison, WI. Matchmaker Two-Hybrid System 2 was from CLONTECH, Palo Alto, CA.

Cell Culture and Luciferase Assay-- HeLa, HOS (osteosarcoma), and A172 (glioblastoma) cells were cultured in Dulbecco's modified Eagle's medium containing 10% FCS. Hep3B and HepG2 hepatoma cells were cultured in minimum essential medium containing 10% FCS. Jurkat cells were cultured in RPMI 1640 containing 10% FCS. All cell lines were maintained at 37 °C in 5% CO2. Cells were transiently transfected with 15 µg of each luciferase construct and 5 µg of pSV-beta galactosidase plasmid DNA per 100-mm tissue culture plate by a calcium phosphate precipitation technique (20). After 24 h incubation with each DNA-calcium phosphate precipitate, the cells were rinsed with phosphate-buffered saline and cultured in fresh medium for another 48 h. The cells were harvested, and luciferase activity was measured by the luciferase assay system. The resulting luciferase activity was corrected based on the beta -galactosidase activity in the same cell extract.

Plasmid Construction-- CAT constructs, pMD5.3k, pMD1.5k, pMD1k, pMD223, pMD141, pMD34, and pM2, were described previously (16). To obtain additional 5'-deletion CAT constructs, pMD121, pMD101 and pMD81, DNA fragments covering from -121 to +49, -101 to +49, or -81 to +49 were amplified by PCR with sense oligonucleotide primers that corresponded to the region from -121 to -102 (primer -121, 5'-GGCTGCAGTGGGACCTGAGGCCTGGCCC-3'), from -101 to -82 (primer -101, 5'-GGCTGCAGCTCATGGCTCCTGTCACCAG-3'), and from -81 to -62 (primer -81, 5'-GGCTGCAGTCTCAGGTCAGGGTCCAGC-3') with an antisense probe from +30 to +49 (5'-GGTCTAGACCTTCTGGCTGGAGGCTGCA-3'). PstI or XbaI linkers were incorporated in the 5'-ends of these primers. The pMD1k was used as the PCR template. The PCR products were subcloned into the PstI-XbaI site of the pCAT basic vector after digestion with PstI and XbaI.

To obtain the pGLMD1k containing the wild-type MSP promoter, the 1-kilobase pair 5'-flanking region, between -967 and +49, of the human MSP gene was PCR-amplified with a sense primer D967 (5'-GGGGTACCCGGACCGCGAAGGGAATA-3') and an antisense primer 49 (5'-GGAGATCTCCTTCTGGCTGGAGGCTGCA-3') by using the pMD5k plasmid (16) as a template. KpnI or BglII linkers were incorporated in the 5'-end of these primers. This PCR product was gel-purified, digested with BglII and KpnI, and then ligated into the BglII-KpnI site of the pGL3-basic luciferase reporter vector (Promega, Madison, WI). To obtain 3'-deletion mutants, pGLM38, pGLM31, pGLM26, and pGLM15, DNA fragments covering from -967 to +38, -967 to +31, -967 to +26, or -967 to +15 were amplified by PCR with a sense oligonucleotide primer, D967, with an antisense probe that corresponded to the region from +19 to +38 (primer 38, 5'-GGGAGATCTGAGGCTGCACTGTGACCCCA-3'), from +12 to +31 (primer 31, 5'-GGGAGATCTCACTGTGACCCCACCACAGC-3'), from +7 to +26 (primer 26, 5'-GGGAGATCTGACCCCACCACAGCCCATCC-3'), and from -5 to +15 (primer 15, 5'-GGGAGATCTCAGCCCATCCGGGAAGTTGT-3'). The PCR products were gel-purified, digested with BglII and KpnI, and then ligated into the BglII-KpnI site of the pGL3-basic plasmid.

Mutated constructs, pmDH, pmY, pm1PH, pm2PH, pm3PH, pm4PH, and pm5PH, were obtained by a two-step PCR mutagenesis method (21) with chemically synthesized mutated sense oligonucleotides, mDH (5'- CTGTCACCAGGTCgacGGTCAGGG-3'), mY (5'-GCCACCgAaTcCCGTAGGGA-3'), and m1PH (5'-GGTTTCACAACTTCCCGGATGGGCTGTGGcccGGTC-3'), m2PH (5'-TGGTGGGGatcCAGTGCAGCCTCCAGCCAG-3'), m3PH (5'-TGGTGGGGTCACAGctgAGCCTCCAGCCAG-3'), m4PH (5'-TGGTGGGGTCACAGTGCAggaTCCAGCCAG-3'), and m5PH (5'-CACAGTGCAGCCTCCctgCAGAAGGAGATCT-3').

In Vitro Protein-Protein Binding Assay-- Rat NF-YA (22), NF-YB (23), and human TFIIB (24) cDNAs were cloned by reverse transcriptase-PCR, and subcloned into the EcoRI site of the pcDNA3 and pET32a. The coding region of the human HNF-4 cDNA was amplified by PCR from the pMT7HNF-4 (generous gift from Dr. F. M. Sladek, University of California, Riverside, CA) and subcloned into the pcDNA3 (pcHNF4) and pET32a. The murine p65 expression vector CMV-LDp65 and human p50 expression vector CMV-399 (this vector coded amino acids 1 to 399 of p50) were provided by Dr. N. Rice (NCI-FCRDC, Frederick, MD). In vitro coupled transcription and translation were performed with the TnT-coupled rabbit reticulocyte lysate system according to a protocol provided by the supplier. Briefly, 1 µg of each pcDNA3 vector containing a different cDNA and T7 RNA polymerase were added to a 50-µl reaction mix and then the mix was incubated at 30 °C for 2 h. For radiolabeling, L-[35S]methionine was used at final activity of 0.8 µCi/µl.

Recombinant proteins were induced in the BL21(DE3) Escherichia coli strain by addition of 0.3 mM isopropyl beta -D-thiogalactopyranoside for 16 h at room temperature. The bacteria were lysed by sonication in a lysis buffer containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9. After ultracentrifugation, the clear bacterial lysates were applied onto a His·Bind Resin column. The bound proteins were eluted with 300 mM imidazole and dialyzed against buffer B (10 mM Tris-HCl, pH 7.9, 100 mM KCl, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride).

For protein-protein interaction assay, histidine-tagged HNF-4, NF-YA, and NF-YB were immobilized on His·Bind Resin in buffer B at 4 °C. The resin was packed in Pasteur pipettes, and the columns were washed with 10 volumes of the buffer. The columns were loaded with in vitro translated 35S-labeled HNF-4, NF-YB, NF-kappa B p50, or RelA and then rinsed with binding buffer (10 mM Tris-HCl, pH 7.9, 60 mM KCl, 4 mM MgCl2, 2% polyethylene glycol 8000). The columns were washed extensively with the binding buffer containing 30 mM imidazole. After the final wash, the bound proteins were eluted with 300 mM imidazole and analyzed by SDS-PAGE followed by autoradiography.

DNase I Footprinting-- To prepare the coding strand DNA carrying the MSP promoter region (-141 to +49), the pMD141 plasmid (16) was digested with HindIII, and the overhangs were filled in with [32P]dCTP, followed by a digestion with EcoRI. The resulting 350-base pair fragment was purified after gel electrophoresis. To prepare the noncoding strand DNA, the pGLMD141 was digested with NcoI, and the overhangs were filled in with [32P]dCTP, followed by a digestion with PstI. The resulting 243-base pair fragment was purified after gel electrophoresis. DNase I digestion was performed with 4 ng of the coding or noncoding strand DNA and bacterially expressed recombinant HNF-4 protein by the method described by Lefevre et al. (25), and the digested samples were analyzed on 8% polyacrylamide gels.

Electromobility Shift Assay (EMSA) and Supershift Assay-- EMSA was carried out as previously reported (16, 20). Briefly, the probes were prepared by annealing each sense oligonucleotide with antisense oligonucleotide shown in Table I. The binding reaction was performed at 20 °C for 30 min in 10 ml of 1× binding buffer (12 mM HEPES, pH 8.0, 60 mM KCl, 2.5 mM EDTA, 14% glycerol, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) containing 10 fmol of each probe end-labeled with [gamma -32P]ATP, 1 µg of dI-dC, and 3 µg of each nuclear extract. For the competition assay, 10-, 20-, or 50-fold excess amounts of appropriate unlabeled probes were added to the binding reaction mixtures. For supershift assay, nuclear extract was preincubated with 1 µl of normal rabbit serum or antiserum for 20 min before mixing with the probe. Gel electrophoresis was carried out at 200 V for 2 h after a prerun at 200 V for 30 min.

Yeast Two-hybrid Assay-- Yeast two-hybrid assay originally developed by Fields and Song (26) was performed with the Matchmaker Two-Hybrid System 2 according to the direction supplied from the manufacturer. Briefly, the full-length cDNAs for NF-YA, NF-YB, NF-YC, and TFIIB were obtained by reverse transcriptase-PCR. The cDNAs for HNF-4, NF-YA, NF-YB, and NF-YC were ligated into the NcoI-EcoRI site, and the TFIIB cDNA was ligated into the BamHI-EcoRI site of the pAS2-1 vector carrying the GAL4 DNA-binding domain (DNA-BD) (27) or the pACT2 carrying the GAL4 transcriptional activation domain (AD) (28). To investigate interactions between two transcription factors, a yeast strain, Y187 (MATa, trp 1, leu 2, URA3::GAL1USA-GAL1TATA-LACZ), was transformed with various combinations of the pAS2-1 vector coding different GAL4 DNA-BD fusion proteins and the pACT2 coding different GAL4 transcriptional AD fusion proteins. beta -Galactosidase activity in the yeast was assayed by colony-lift filter assay as qualitative assay and by liquid culture assay as quantitative assay (29, 30). For colony-lift assay, yeast colonies were streaked out onto synthetic dropout selection agar plates without leucine or tryptophan, and the plates were incubated for 3 days at 30 °C. Sterile VWR filters presoaked in Z buffer/5-bromo-4-chloro-3-indolyl beta -D-galactosidase were placed on the plates. The filters were lifted carefully and submerged in liquid nitrogen for 10 s. The filters were thawed at room temperature and placed on presoaked filters and then incubated at 30 °C until blue color appeared. For liquid culture assay, yeast colonies were inoculated into 5 ml of synthetic dropout without leucine or tryptophan and cultured overnight at 30 °C with shaking. Two ml of the overnight cultures were inoculated into 8 ml of YPD liquid, and the cells were grown at 30 °C for 3-5 h with shaking until the A600 reached 0.5-0.8. One and a half ml of the cultures were transferred into 1.5-ml microcentrifuge tubes, and the tubes were centrifuged at 14,000 rpm for 30 s. After removal of the supernatants, cells were washed and finally resuspended in 0.3 ml of Z buffer. One hundred µl of the cell suspensions were transferred to fresh microcentrifuge tubes and placed in liquid nitrogen until the cells were frozen. The cells were thawed at 37 °C, and 700 µl of Z buffer containing beta -mercaptoethanol were added to the tubes. One hundred sixty µl of o-nitrophenyl beta -D-galactopyranoside (4 mg/ml in Z buffer) were added to each tube, and the tubes were incubated at 30 °C. When the yellow color developed, the reaction was stopped by addition of 0.4 ml of 1 M Na2CO3. The tubes were centrifuged, and the supernatants were subjected to spectrophotometry at A420.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Tissue-specific Promoter Activity on the PRE of the MSP Gene-- As described in the Introduction, our previous results on the cis-element responsible for the positive regulation of the MSP gene transcription were significantly different from that by Waltz et al. (17). Therefore, we first repeated CAT assay with previously constructed CAT constructs containing different lengths of the 5'-flanking region of the MSP gene obtained by progressive deletion of the 5'-end (16) and also with newly created CAT constructs (Fig. 1B). A significant CAT activity was detected in the extracts of Hep3B cells transfected with pMD5.3k. Progressive deletion of the 5'-flanking region from -5.3k (pMD5.3k) to -34 (pMD34) resulted in a gradual increase in CAT activity. In contrast, no significant CAT activity was found in HeLa cells transfected with either pMD5.3k or other constructs containing the 5'-flanking regions progressively deleted up to -141. However, when the 5'-flanking region was deleted to -121, -101, or -81, CAT activity was increased 2.5-fold. Further deletion from -81 to -34 resulted in another 2-fold increase in CAT activity. These results suggested that the region between -141 and -34 might negatively regulate the transcription of the MSP gene. In both cells, deletion to +2 (pM2) resulted in a significant decrease in CAT activity. The region between -34 and +2 carries the CAATT sequence we previously identified as an NF-Y-binding site. Removal of the remaining proximal MSP gene sequence (pCAT-basic) resulted in a total loss of CAT activity, suggesting that the proximal 5'-flanking region between +2 and +49 may also be responsible for the positive regulation of the MSP gene transcription. Thus, these results further support our hypothesis that the transcription of the MSP gene is regulated by PRE and NRE.


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Fig. 1.   Schematic representation of 5'-deletion CAT constructs and 3'-deletion luciferase constructs and activities in Hep3B and HeLa cells. A, nucleotide sequence of the MSP gene promoter region (16). Two HNF-4-binding sites and an NF-Y-binding site are underlined. The major transcriptional start site is shown as +1. Arrows indicate minor transcriptional start site. * indicates the transcription start site reported by Waltz et al. (17). B, Hep3B and HeLa cells were transiently transfected with the CAT constructs containing different lengths of the 5'-flanking region of the MSP gene obtained by progressive deletion of the 5'-end. C, the wild-type MSP promoter construct (pGLMD1k) and 3'-deletion constructs were transfected into Hep3B and HeLa cells. Cells were harvested after 48 h incubation, and then CAT or luciferase assay was performed on each cell extract. Results are expressed as the ratio of luciferase activity in each cell extract relative to the CAT or luciferase activity in the cell extract from Hep3B cells transfected with 15 µg of pMD223 or 15 µg of pGLMD1k. Data are means ± 1 S.D. from four independent experiments.

To characterize the putative regulatory element located in the region between +2 and +49, we constructed additional luciferase plasmid constructs (Fig. 1C). High luciferase activities were detected in Hep3B cells transfected with either pGLMD1k or pGLM38. However, luciferase activities were significantly decreased when the 3'-sequence was further deleted to +31 (pGLM31). A similar loss in luciferase activity was also detected with pGLM26 or pGLM15. No significant luciferase activity was detected when these constructs were transfected into HeLa cells (Fig. 1C). These results suggested that the region between +31 and +38 was important for the maximal promoter activity of the human MSP gene in Hep3B cells.

Characterization of Binding Protein to the Proximal PRE-- To characterize nuclear proteins that could bind to the region between +31 and +38, EMSA was performed with a nucleotide probe covering the sequence from +16 to +45. Two specific nuclear protein-DNA complexes, C1 and C2 (Fig. 2A, lane 1), that were competed by addition of excess amounts of unlabeled probe (lanes 2-4), were detected with the nuclear extract from Hep3B cells. Shifted bands that migrated to the region of the C1 were detected with the nuclear extracts from HepG2, HeLa, HOS, A172, and Jurkat cells (Fig. 2B, lanes 2-6), but the C2 was formed only with the nuclear extracts from Hep3B and HepG2 cells.


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Fig. 2.   Characterization of nuclear proteins that bind to the proximal promoter region (+16 to +45) of the human MSP gene. A, 32P-labeled +16/+45 probe was incubated with the nuclear extracts of Hep3B cells. Competition assays were performed with 10-, 20-, and 50-fold excess amounts (molar) of the unlabeled probe. Arrows indicate specific DNA-nuclear protein complexes. B, EMSA was performed by using nuclear extracts from other cell lines (HepG2, HeLa, HOS, A172, and Jurkat cells). C, oligonucleotide probes containing the consensus binding sequences for CEBP/alpha , HNF-1, HNF-3, and HNF-4 were used as competitors. 32P-Labeled +16/+45 probe was incubated with the nuclear extracts of Hep3B cells in the presence of these competitors (20- or 50-fold molar excess). D, supershift assay. The nuclear extract from Hep3B cells was premixed with an antiserum against HNF-4 (lane 2) or normal rabbit serum (lane 3).

Competition assay was performed by using several nucleotide probes that carried the binding sequences for liver-specific trans-activator, CEBP/alpha , HNF-1, HNF-3, and HNF-4 (Table I). As shown in Fig. 2C, addition of excess amounts of unlabeled CEBP/alpha (lanes 5 and 6), HNF-1 (lanes 8 and 9), or HNF-3 probes (lanes 11 and 12) did not affect the formation of the C2. However, addition of excess amounts of unlabeled HNF-4 probe inhibited the formation of the C2 (lanes 14 and 15), suggesting that the region, +16 to +45, is a binding site for HNF-4. Unlabeled HNF-4 probe also inhibited the formation of the C1 (lanes 14 and 15).

                              
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Table I
Oligonucleotides used in the EMSAs

Supershift assay was performed with a specific antibody against HNF-4. As shown in Fig. 2D, anti-HNF-4 antibody inhibited the formation of the C2 (lane 2), whereas normal rabbit serum did not affect the formation of the C2 (lane 3). The formation of C1 was not inhibited by either anti-HNF-4 or normal rabbit serum (lanes 2 and 3, not clearly shown in the photograph).

Identification of Proximal and Distal HNF-4-binding Sites in the MSP Promoter Region-- The binding of HNF-4 to the promoter region of the MSP gene was further investigated by DNase I protection assay. The experiments were performed with a recombinant HNF-4 protein expressed in E. coli. As shown in Fig. 3, two regions were protected from DNase I digestion. One was in the proximal region, between +20 and +41 on the noncoding strand (antisense) and between +14 and +38 on the coding strand (sense), corresponding to the HNF-4-binding site described above. The protection of this region was not previously clear with Hep3B cell nuclear extracts (data not shown). Another region in the distal region, between -89 and -69 on the coding strand (sense) and between -54 and -81 on the non-coding strand (antisense), was also protected from DNase I digestion. This distal HNF-4-binding site is identical to the one previously reported by Waltz et al. (17) as an element responsible for the liver-specific expression of MSP/HGFL. The binding of HNF-4 to the distal HNF-4-binding site was confirmed by EMSA with Hep3B nuclear extract and also by supershift assay with an antiserum against HNF4 (data not shown).


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Fig. 3.   DNase I protection assay. Prior to DNase I digestion, 32P-labeled DNA fragments were incubated with 100 ng, 1 µg, or 10 µg of recombinant HNF-4. The regions protected from DNase I digestion are indicated by open boxes on the sides of the figure. NF-Y binding sequences ("CCAAT" on the coding strand and "ATTGG" on the non-coding strand) are indicated by closed boxes.

Effects of Mutations in the Proximal HNF-4-binding Site on the Formation of C2 and HNF-4-induced Transcriptional Activity-- To investigate the DNA sequence required for the protein binding to the proximal HNF-4-binding site, mutated oligonucleotide probes were synthesized and tested in EMSA (Fig. 4A). The mutations from "+24TCA+26" to "+24ATC+26" and from "+35CC+36" to "+35GA+36" resulted in significant decreases in the competitive activity (Fig. 4A, M2 and M4). In contrast, the other mutations, M1 (+16TGG+18 to +16CCC+18), M3 (+30TGC+32 to +30CTG+32), and M5 (+40CAG+42 to +40CTG+42), did not affect the HNF-4 binding. These results suggested that the base pairs, +24 to +26 and +35 and +36, were critical for HNF-4 binding.


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Fig. 4.   Effects of mutations in the proximal HNF-4-binding site on the formation of C2 and HNF-4-induced transcriptional activity. A, the formation of the probe +16/+45-nuclear protein complexes, C1 and C2 (HNF-4), was inhibited by 10-, 20-, or 50-fold excess amounts of the mutated probes, M1, M3, and M5. B, the mutations M1, M2, M3, M4, and M5 were introduced in the proximal HNF-4-binding site of the wild-type MSP promoter construct, pGLMD1k. Fifteen µg of pGLMD1k plasmid DNA or mutated constructs were transfected into Hep3B and HeLa cells. Three µg of pMT7HNF4 were used for the transfection of HeLa cells. Cells were harvested after a 48-h incubation, and then luciferase assay was performed on each cell extract. Results are expressed as the ratio of luciferase activity in each cell extract relative to the luciferase activity in extract from HeLa cells transfected with 15 µg of pGLMD1k. Data are means ± 1 S.D. from four independent experiments. The mutated base pairs are shown by underline.

We next constructed luciferase plasmids carrying the same mutations, M1, M2, M3, M4, and M5, on the proximal HNF-4-binding site of pGLMD1k (pm1PH, pm2PH, pm3PH, pm4PH, and pm5PH) (Fig. 4B). When HeLa cells were co-transfected with HNF-4 expression vector (pMT7HNF4) and pGLMD1k containing the wild-type MSP promoter, the luciferase activity was 15-fold increased. Introduction of M1 or M3 did not affect the luciferase activity. However, introduction of M2, M4, or M5 resulted in significant decreases in HNF-4-induced enhancer activities. These results indicated that the base pairs, +24 to +26 and +35 to+36, were critical for not only HNF-4 binding but also HNF-4-induced enhancer activity. Since M5 did not affect the binding of HNF-4, the decreased luciferase activity with the pm5PH was probably due to other mechanisms including decreased mRNA stability.

Synergistic trans-Activity between NF-Y and HNF-4-- Our previous study (16) and the present study identified three cis-elements in the promoter region of the MSP gene, two HNF-4-binding sites and one CCAAT box (binding site for NF-Y). To investigate the roles of these three cis-elements and two trans-activators in the transcription of the MSP gene, we performed luciferase assays by transfecting HeLa cells with HNF-4 expression vector, pMT7HNF-4, and various luciferase constructs shown in Fig. 5. When HeLa cells were co-transfected with pMT7HNF-4 and pGLMD1K containing the wild-type MSP promoter, the luciferase activity was 15-fold increased. Mutation in the proximal HNF-4 site (pmPH) resulted in 75% loss of the luciferase activity in HNF-4-transfected HeLa cells and 71% loss in Hep3B cells. However, mutation in the distal HNF-4-binding site (pmDH) did not affect the activity. Mutation in the CCAAT sequence (pmY) resulted in 60% loss of the activity in HNF-4-transfected HeLa cells and 75% loss in Hep3B cells. When both the CCAAT sequence and the proximal HNF-4 site were mutated (pmYmPH), the luciferase activity was decreased to the basal level (pmDHmYmPH). The luciferase activities obtained with either pmDHmY or pmDHmYmPH were not significantly different from that obtained with pmY or pmYmPH, respectively, in both HNF-4-transfected HeLa cells and Hep3B cells. There was no significant luciferase activity in HeLa cells that was not transfected with the pMT7HNF-4. These results strongly suggest that the binding of NF-Y to the CCAAT sequence and the binding of HNF-4 to the proximal HNF-4-binding site, not to the distal HNF-4-binding site, synergistically activate the transcription of the MSP gene in hepatocytes.


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Fig. 5.   Schematic representation of mutated luciferase constructs and luciferase activities in Hep3B, HeLa, and HNF-4-transfected HeLa cells. Mutated bases were introduced in the distal HNF-4-binding site, NF-Y-binding site, or proximal HNF-4-binding site of the wild-type MSP promoter construct, pGLMD1k. Fifteen µg of pGLMD1k plasmid DNA or mutated constructs were transfected into Hep3B and HeLa cells. Three µg of pMT7HNF-4 were used for the transfection of HeLa cells. Cells were harvested after 48 h incubation, and then luciferase assay was performed on each cell extract. Results are expressed as the ratio of luciferase activity in each cell extract relative to the luciferase activity in the extract from HeLa cells transfected with 15 µg of pGLMD1k. Data are means ± 1 S.D. from four independent experiments.

HNF-4 Contacts with NF-YA and TFIIB in a Y187 Yeast Strain-- Cooperative activity between the CCAAT sequence and the proximal HNF-4-binding site, not the distal HNF-4-binding site, leads to an hypothesis that a direct protein-protein interaction between NF-Y and HNF-4 might be required for the maximal promoter activity of the MSP gene. To test whether HNF-4 interacts with NF-Y or the basal transcriptional complexes, we used a yeast two-hybrid system. HNF-4 was expressed as a fusion protein with GAL4 activating domain (AD) by cloning HNF-4 cDNA into a yeast shuttle vector, pACT2. NF-YA, NF-YB, NF-YC, TFIIB, and TFIID (TATA-binding protein) were expressed as fusion proteins with GAL4-binding domain (BD) by cloning the cDNAs into an yeast shuttle vector, pAS2-1. Interaction between HNF-4 fused to AD and a protein fused to BD reconstitutes GAL4 function in vivo and results in the expression of lacZ gene in Y187 yeast strain. SV40 large T antigen-AD and murine p53-BD were used as positive control (31). The protein-protein interaction between p53 and T antigen was detected as shown in Fig. 6A. When the HNF-4-AD expression vector was co-transfected with pAS2-1 BD expression vector, the HNF-4-AD possessed no intrinsic activation activity (almost same to that of the pAS2-1 vector alone). However, when HNF-4-AD expression vector was co-transfected with NF-YA-BD or TFIIB-BD expression vector, significant levels of galactosidase activities were detected (Fig. 6A). NF-YB-BD, NF-YC-BD, and p53-BD failed to interact with HNF-4-AD. Expression of T antigen-AD, HNF-4-AD, p53-BD, NF-YA-BD, NF-YB-BD, NF-YC-BD, TFIIB-BD, and TFIID-BD in the yeast was confirmed by Western blot analysis (data not shown). The galactosidase activities were also quantified by liquid cultures of the yeast colonies (Fig. 6B). The interaction between HNF-4 and NF-YA was lower than that between p53 and large T antigen but appeared to be stronger than that between HNF-4 and TFIIB.


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Fig. 6.   Association of HNF-4 with TFIIB and NF-YA in a yeast two-hybrid system. A, two-hybrid analysis of HNF-4 and p53, NF-YA, NF-YB, NF-YC, or TFIIB. Pairs of fusion proteins containing either GAL4 activation domain (AD) or GAL4 DNA-binding domain (BD) were expressed in a Y187 yeast strain. Cells were streaked on leucine- and tryptophan-deficient medium plates containing glucose. After a 4-day incubation at 30 °C, colonies were lifted onto filters. SV40 large T antigen-AD and murine p53-BD were used as positive control. B, beta -galactosidase levels that reflect the extent of interaction between the chimeric AD and BD fusion proteins are displayed as the fold increase over levels observed with pACT2 and pAS2-1. Data are means ± 1 S.D. from five independent transformed yeast colonies.

Protein-Protein Interaction between HNF-4 and NF-YA in Vitro-- We also characterized the binding between NF-YA and HNF-4 by using recombinant proteins in vitro. As shown in Fig. 7A, lane 1, in vitro translated 35S-labeled NF-YA (upper panel) bound to the column to which bacterially expressed HNF-4 was immobilized and was eluted from the column (lower panel). 35S-Labeled HNF-4 also bound to the column (lane 2). Homodimerization of HNF-4 was previously characterized by others (11). 35S-Labeled NF-kappa B p50 and 35S-labeled RelA failed to bind to the column (lanes 3 and 4). Conversely, 35S-labeled HNF-4 bound to the columns to which bacterially expressed NF-YA or HNF-4 was immobilized (Fig. 7B, lanes 2 and 4) but not to the column to which bacterially expressed NF-YB was immobilized (Fig. 7B, lane 3).


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Fig. 7.   The binding of HNF-4 to NF-YA in vitro. A, autoradiograph of in vitro translated 35S-NF-YA, 35S-HNF-4, 35S-p50, or 35S-RelA after SDS-PAGE (upper panel). These 35S-labeled proteins were loaded onto a His·Bond resin column to which histidine-tagged recombinant HNF-4 was immobilized. The bound proteins were eluted from the column and analyzed by SDS-PAGE, and then autoradiography was performed (lower panel). B, in vitro translated 35S-labeled HNF-4 was loaded onto His·Bond columns to which histidine-tagged recombinant NF-YA, recombinant NF-YB, or recombinant HNF-4 was immobilized. The bound proteins were eluted from the columns, analyzed by SDS-PAGE, and then radioautography was performed.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, we identified two HNF-4 binding elements in the 5'-flanking region of the human MSP gene. The direct repeat 1 (DR1) "(AGGTCA)N(AGGTCA)" is the consensus DNA sequence for HNF-4 binding (32). This sequence is highly conserved in the sequence of the distal HNF-4-binding site (-83AGGTCT)C(AGGTCA-71) and weakly conserved in the sequence of the proximal HNF-4 binding site (+21GGGTCA)C(AGTGCA+33). The mutations from +24TCA+26 to +24ATC+26 (M2) and +34GCC+36 to +34GGA+36 (M4) resulted in a loss of HNF-4 binding activity and a decrease in the HNF-4-induced enhancer activity. On the other hand, the mutations from "+19TGG+21" to "+19CCC+21" (M1) and "+30TGC+32" to "+30CTG+32" (M3) did not affect either HNF-4 binding or the enhancer activity. Waltz et al. (17) investigated the effects of mutated bases on the binding activity of the distal HNF-4-binding site. Oligoprobes containing mutations between -78 and -70 were not able to compete with the wild-type oligoprobe for HNF-4 binding. Thus, the sequences responsible for HNF-4 binding appear to be different between the distal and proximal HNF-4-binding sites.

We previously detected four different transcription initiation sites, T (+1), G (+7), G (+20), and A (+26), by primer extension assay (16). One of the transcription initiation sites, A (+26), is located in the proximal HNF-4-binding site. As we previously reported, the promoter region of the MSP gene does not contain TATA box. In the genes that lack TATA box, an element called an initiator (Inr) is functionally analogous to TATA (18, 19). The Inr overlaps the transcription initiation site of numerous mammalian protein-coding genes. The Inr has a weak consensus sequence (5'-PyPyCAPyPyPyPyPy-3' or 5'-PyPyAN(T/A)PyPy-3', where Py is pyrimidine) and the functional Inr-binding protein would interact with a relatively low affinity (19). The binding affinity of HNF4 to the proximal HNF-4-binding site was also low compared with that between HNF-4 and the distal HNF-4-binding site (data not shown). Thus, the characteristics of the proximal HNF-4-binding site are similar to that of the Inr.

Transcription of genes is initiated by RNA polymerase II with other basal transcription factors including TFIIA, -B, -D, -E, -F, -G/J, -H, and -I. Before RNA synthesis begins, RNA polymerase II and basal factors bind to a promoter in an ordered series of steps and form a preinitiation complex (PIC) (33). Since HNF-4 binds very closely to the transcription initiation sites, we speculated a possible protein-protein interaction between HNF-4 and PIC. Recently, a protein-protein interaction between HNF-4 and TFIIB was reported (34). TFIIB is a member of the PIC and directly associated with TFIID that binds to TATA box (also known as TATA-binding protein). TFIIB plays a critical role in determining the transcription start site (35). We confirmed a direct interaction between TFIIB and HNF-4 by a two-hybrid system in yeast (Fig. 6, A and B).

We speculated another possible protein-protein interaction between HNF-4 and NF-Y. NF-Y consists of three subunits, NF-YA, NF-YB, and NF-YC (22, 23). NF-Y binds to the CCAAT sequence in the 5'-flanking sequences of many genes and regulates the transcription of those genes (22). The CCAAT sequence was found near upstream of the TATA sequence of some genes. The binding of NF-Y to the CCAAT sequence was also essential for the transcription of some TATA-less genes (36). These reports suggested a direct association of NF-Y to the PIC. In the present study, we have shown that HNF-4 is capable of directly binding to the A-subunit of NF-Y, suggesting that the binding of HNF-4 to both TFIIB and NF-Y is important for the positioning of PIC and the initiation of the transcription of the MSP gene.

The distal HNF-4-binding site resides in the NRE located between -141 and -34 (16). When the NRE was deleted from the wild-type MSP promoter, NF-Y-induced CAT activity could be detected in HeLa and HOS cells. Therefore, we previously speculated that the binding of some repressor proteins to the NRE negatively regulated the transcription of the MSP gene in non-hepatic cells. In the present study, several additional CAT plasmids were constructed, and CAT functional assay was performed with HeLa cells. The previously reported NRE appears to be divided into two NREs. One is located between -141 and -121 (NRE1), and another is between -81 and -34 (NRE2). Since HNF-4 binds to the 5'-end of the NRE2, HNF-4 could inhibit the binding of putative repressors to the region. We observed a wide area including the NRE protected from DNase Idigestion with the nuclear extracts from HeLa and HOS cells. However, we were unable to demonstrate the binding of repressor proteins to this region by EMSA (data not shown). Further study is necessary to understand the mechanisms involved in tissue-specific transcription of the MSP gene.

We detected two different nuclear protein-DNA complexes by EMSA with an oligonucleotide carrying the proximal HNF-4-binding site sequence and the nuclear extracts of Hep3B and HepG2 cells (Fig. 2A, C1 and C2). The C2 was specific to Hep3B and HepG2 cells and contained HNF-4, whereas shifted bands that migrated to the region of the C1 were also detected with the nuclear extracts of nonhepatic cells. This ubiquitously expressed protein could bind to the several mutated probes M1, M3, and M5, but not to M2 and M4, showing the same binding pattern as HNF-4. The binding of a ubiquitously expressed protein to an HNF-4-binding site on the apolipoprotein CIII promoter (termed element B) was previously reported. The binding of HNF-4 and CIIIB1 to this element activates the apoCIII promoter (37). Conversely, the binding of chicken ovalbumin upstream promoter-transcriptional factor 1 (38) or apoAI regulatory protein 1 (also known as chicken ovalbumin upstream promoter-transcriptional factor TFII) (39) to the element negatively regulates the promoter activities. These factors might be involved in the formation of the C1. As we reported previously (16) and also in the present article, CAT activities were increased even in HeLa cells after removal of the NRE from the promoter region of the MSP gene. There appear to be other proteins that bind to the proximal HNF-4-binding site and function as a trans-activator for the MSP gene transcription.

In summary, we identified two HNF-4-binding sites in the promoter region of the MSP gene. The proximal HNF-4-binding site, not the distal HNF-4-binding site, and the CCAAT sequence that we previously characterized as an NF-Y-binding site cooperatively regulated the transcription of the MSP gene. Finally, we presented evidence that indicated direct association between NF-YA and HNF-4.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Frances M. Sladek and Nancy Rice for generous gifts of the pMT7HNF-4, rat HNF-4 expression vector, and an antiserum against HNF-4, and the murine p65 expression vector CMV-LDp65 and human p50 expression vector CMV-399, respectively. We are also grateful to Dr. Jun Fukushima for helpful discussion.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Bldg. 560, Rm. 12-71, NCI-FCRDC, Frederick, MD 21702. Tel.: 301-846-1560; Fax: 301-846-6145; E-mail:yoshimur{at}mail.ncifcrf.gov.

1 The abbreviations used are: MSP, macrophage stimulating protein; C/EBP, CCAAT/enhancer-binding protein; HNF, hepatocyte nuclear factor; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; NRE, negative regulatory element; PRE, positive regulatory element; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; PCR, polymerase chain reaction; PIC, preinitiation complex; Inr, initiator; BD, binding domain; AD, activation domain.

2 A. Ueda and T. Yoshimura, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Skeel, A., Yoshimura, T., Showalter, S. D., Tanaka, S., Appella, E., and Leonard, E. J. (1991) J. Exp. Med. 173, 1227-1234[Abstract]
  2. Yoshimura, T., Yuhki, N., Wang, M.-H., Skeel, A., and Leonard, E. J. (1993) J. Biol. Chem. 268, 15461-15468[Abstract/Free Full Text]
  3. Han, S., Stuart, L. A., and Degen, S. J. F. (1991) Biochemistry 30, 9768-9780[Medline] [Order article via Infotrieve]
  4. Leonard, E. J., and Skeel, A. (1976) Exp. Cell Res. 102, 434-438[Medline] [Order article via Infotrieve]
  5. Skeel, A., and Leonard, E. J. (1994) J. Immunol. 152, 4618-4623[Abstract/Free Full Text]
  6. Ronsin, C., Muscatelli, F., Mattei, M.-G., and Breathnach, R. (1993) Oncogene 8, 1195-1202[Medline] [Order article via Infotrieve]
  7. Iwama, A., Okamoto, K., Sudo, T., Matsuda, Y., and Suda, T. (1994) Blood 83, 3160-3169[Abstract/Free Full Text]
  8. Wang, M.-H., Skeel, A., Leonard, E. J., Ronsin, C., Gesnel, M.-C., Breathnach, R., and Coupey, L. (1994) Science 266, 117-119[Medline] [Order article via Infotrieve]
  9. Gaudino, G., Follenzi, A., Naldini, L., Collesi, C., Santoro, M., Gallo, K. A., Godowski, P. J., and Comoglio, P. M. (1994) EMBO J. 13, 3524-3532[Abstract]
  10. Wang, M.-H., Iwama, A., Skeel, A., Suda, T., and Leonard, E. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3933-3937[Abstract/Free Full Text]
  11. Lai, E., and Darnell, J. E., Jr. (1991) Trends Biochem. Sci. 16, 427-430[CrossRef][Medline] [Order article via Infotrieve]
  12. Landschulz, W. H., Johnson, P. F., Adashi, E. Y., Graves, B. J., and McKnight, S. L. (1988) Gene Dev. 2, 786-800[Abstract]
  13. Frain, M., Swart, G., Monaci, P., Nicosia, A., Stampfli, S., Frank, R., and Cortese, R. (1989) Cell 59, 145-157[Medline] [Order article via Infotrieve]
  14. Lai, E., Prezioso, V. R., Smith, E., Litvin, O., Costa, R. H., and Darnell, J. E., Jr. (1990) Gene Dev. 4, 1427-1436[Abstract]
  15. Sladek, F. M., Zhong, W., Lai, E., and Darnell, J. E., Jr. (1990) Gene Dev. 4, 2353-2365[Abstract]
  16. Ueda, A., and Yoshimura, T. (1996) J. Biol. Chem. 271, 20265-20272[Abstract/Free Full Text]
  17. Waltz, S. E., Gould, F. K., Air, E. L., McDowell, S. A., and Degen, S. J. F. (1996) J. Biol. Chem. 271, 9024-9032[Abstract/Free Full Text]
  18. Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113[Medline] [Order article via Infotrieve]
  19. Kaufmann, J., and Smale, S. T. (1994) Gene Dev. 8, 821-829[Abstract]
  20. Ueda, A., Okuda, K., Ohno, S., Shirai, A., Igarashi, T., Matsunaga, K., Fukushima, J., Kawamoto, S., Ishigatsubo, Y., and Okubo, T. (1994) J. Immunol. 153, 2052-2063[Abstract/Free Full Text]
  21. Baowei, C., and Alan, E. D. (1994) BioTechniques 17, 657-659[Medline] [Order article via Infotrieve]
  22. Vuorio, T., Maity, S. N., and de Crombrugghe, B. (1990) J. Biol. Chem. 265, 22480-22486[Abstract/Free Full Text]
  23. Maity, S. N., Vuorio, T., and de Crombrugghe, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5378-5382[Abstract]
  24. Maliks, S., Hisatake, K., Sumimoto, H., Horikoshi, M., and Roeder, R. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9553-9557[Abstract]
  25. Lefevre, C., Imagawa, M., Dana, S., Grindlay, J., Bodner, M., and Karin, M. (1987) EMBO J. 6, 971-981[Abstract]
  26. Fields, S., and Song, O. (1989) Nature 340, 245-246[CrossRef][Medline] [Order article via Infotrieve]
  27. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell 75, 805-816[Medline] [Order article via Infotrieve]
  28. Li, L., Elledge, S. J., Peterson, C. A., Bales, E. S., and Legerski, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5012-5016[Abstract]
  29. Guarente, L. (1983) Methods Enzymol. 101, 181-191[Medline] [Order article via Infotrieve]
  30. Bartel, P. L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993) in Cellular Interactions in Development (Hartley, D. A., ed), pp. 153-179, Oxford University Press, Oxford
  31. Iwabuchi, K., Li, B., Bartel, P., and Fields, S. (1993) Oncogene 8, 1693-1696[Medline] [Order article via Infotrieve]
  32. Jiang, G., Nepomuceno, L., Hopkins, K., and Sladek, F. M. (1995) Mol. Cell. Biol. 15, 5131-5143[Abstract]
  33. Kollmar, R., and Farnham, P. J. (1993) Proc. Soc. Exp. Biol. Med. 203, 127-139[Abstract]
  34. Malik, S., and Karathanasis, S. K. (1996) Mol. Cell. Biol. 16, 1824-1831[Abstract]
  35. Pinto, I., Wu, W.-H., Na, J., and Hampsey, N. (1994) J. Biol. Chem. 269, 30569-30573[Abstract/Free Full Text]
  36. Pugh, B. F., and Tjian, R. (1991) Genes Dev. 5, 1935-1945[Abstract]
  37. Ogami, K., Kardassis, D., Cladaras, C., and Zannis, V. I. (1991) J. Biol. Chem. 266, 9640-9646[Abstract/Free Full Text]
  38. Wang, L.-H., Tsai, S. Y., Cook, R. G., Beattie, W. G., Tsai, M.-J., and O'Malley, B. W. (1989) Nature 340, 163-166[CrossRef][Medline] [Order article via Infotrieve]
  39. Ladias, J. A., and Karathanasis, S. K. (1991) Science 251, 561-565[Medline] [Order article via Infotrieve]


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