Identification of Transcriptional Enhancer Factor-4 as a Transcriptional Modulator of CTP:Phosphocholine Cytidylyltransferase alpha *

Hiroyuki SugimotoDagger , Marica Bakovic§, Satoshi YamashitaDagger , and Dennis E. Vance||

From the Dagger  Department of Biochemistry, Gunma University School of Medicine, Maebashi 371-8511, Japan, the § Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G2W1, and the  Department of Biochemistry and CIHR Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G2S2, Canada

Received for publication, January 4, 2001, and in revised form, January 10, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCTalpha , CCTbeta 1, and CCTbeta 2. The mouse CCTalpha gene promoter is regulated by an enhancer element (Eb) located between -103 and -82 base pairs (5'-GTTTTCAGGAATGCGGAGGTGG-3') upstream from the transcriptional start site (Bakovic, M., Waite, K., Tang, W., Tabas, I., and Vance, D. E. (1999) Biochim. Biophys. Acta 1436, 147-165). To identify the Eb-binding protein(s), we screened a mouse embryo cDNA library by the yeast one-hybrid system and obtained 19 positive clones. Ten cDNA clones were identified as transcriptional enhancer factor-4 (TEF-4). The TEF-binding consensus sequence, 5'-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3', was identified within the Eb binding region. Gel-shift analysis using radiolabeled Eb fragment as a probe showed that cell extracts from yeast expressing hemagglutinin-tagged TEF-4 caused a marked band retardation that could be prevented with an anti-hemagglutinin antibody. When COS-7 cells were transfected with TEF-4, CCTalpha promoter-luciferase reporter activity and CCTalpha mRNA levels increased. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stimulated the CCTalpha promoter activity, whereas protein lacking the DNA binding domain, mTEA(-), did not. Unexpectedly, when the ATG core of the TEF-4 binding consensus within the Eb region was mutated, promoter activity was enhanced rather than decreased. Thus, TEF-4 might act as a dual transcriptional modulator as follows: as a suppressor via its direct binding to the Eb element and as an activator via its interactions with the basal transcriptional machinery. These results provide the first evidence that TEF-4 is an important regulator of CCTalpha gene expression.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In higher eukaryotes, CTP:phosphocholine cytidylyltransferase (CCT)1 is a rate-limiting and regulated enzyme in the synthesis of phosphatidylcholine (1-4). The first mammalian CCT was purified from rat liver (5, 6) and was used for isolation of the corresponding cDNA encoding a 367-amino acid (CCTalpha ) protein (7). The predicted CCTalpha structure consists of a nuclear localization signal, a catalytic domain, a helical lipid-binding domain, and a phosphorylation domain (1-4, 8-10). Recently, cDNAs for two other isoforms, CCTbeta 1 (10) and its splicing variant CCTbeta 2 (11), were cloned. All isoforms contain a highly homologous catalytic domain and an amphitropic helical domain that binds lipids (10). CCTalpha and CCTbeta 2 also contain a highly phosphorylated domain at their carboxyl terminus (1-4, 9, 11), whereas CCTbeta 1 lacks this domain. The roles of individual domains in the regulation of CCTalpha enzyme activity have been extensively studied. CCTalpha activity is modulated by the binding of specific lipids to the helical domain (1-4, 12, 13) and by phosphorylation at the carboxyl-terminal domain (1-4, 9, 14-17).

In addition to regulation at the protein level, CCTalpha is regulated at the transcriptional and post-transcriptional levels in various cells and tissues. CCTalpha mRNA was increased in colony-stimulating factor 1-stimulated macrophages (18), in hepatic tissues after partial hepatectomy (19), and during growth and development (20). However, the question of whether the elevation of CCTalpha mRNA levels was due to an increase in transcription or due to stabilization of the produced transcript needs to be further investigated (21-23).

Tang et al. (24) isolated the murine CCTalpha gene (Ctpct) and showed that the exon/intron organization of the gene closely resembles the functional domains of the enzyme. The gene is transcribed from two transcriptional start sites. The 5' promoter region lacks TATA/CAAT boxes, but contains GC-rich regions. Bakovic et al. (25) demonstrated that three GC regions are regulatory as follows: a "loose" Sp1 site at -31/-9, a cluster of three overlapping Sp1 sites (-88/-50), and a canonical Sp1-binding site (-148/-128). More recently, the same authors showed that transcription factors Sp1 (26, 27), Sp2 (28), and Sp3 (28) can competitively bind to these regions and that the promoter activity may depend on the relative abundance of those three factors (29, 30).

To identify the regulatory elements responsible for CCTalpha transcription, Bakovic et al. (25) prepared various promoter deletion constructs of Ctpct linked to the luciferase reporter. Subsequent functional assays revealed the presence of regions responsible for basal expression (-52/+38) as well as positive and negative regulatory elements (-201/-90). Furthermore, gel-shift assays indicated several unidentified proteins capable of binding to the region -103/-82, (GTTTTCAGGAATGCGGAGGTGG, Eb) and the neighboring region -130/-103.

In this study, we utilized the yeast one-hybrid system (31, 32) to clone cDNA encoding Eb-binding proteins. One of them was identified as a member of the transcription enhancer factor (TEF) family of proteins, TEF-4 (33, 34). TEF-4 is closely related to TEF-1 that was previously isolated as a regulatory protein of the SV40 enhancer (35). We report that TEF-4 regulates expression of the CCTalpha gene in COS-7 and 3T3-L1 cells.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Materials-- MATCHMAKER Yeast One-hybrid System for screening DNA-binding proteins, the Saccharomyces cerevisiae strain YM4271, and the 11-day mouse embryo MATCHMAKER cDNA library constructed in the pACT2 vector were purchased from CLONTECH (Palo Alto, CA). The promoter-less luciferase vector, pGL3-basic, carrying Photinus pyralis luciferase, the control pRL-CMV vector, carrying Renilla reniformis luciferase, and the Dual-luciferase Reporter Assay System were obtained from Promega (Madison, WI). FuGENETM6 transfection reagent, Dulbecco's modified Eagle's medium, and fetal bovine serum were from Roche Molecular Biochemicals, Sigma, and Life Technologies, Inc., respectively. COS-7 and 3T3-L1 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). 3-Aminotriazole (3-AT) was from Sigma.

Construction of Plasmids and Yeast Strains Containing the Eb Sequence-- The triple repeat of Eb (5'-GTTTTCAGGAATGCGGAGGTGG-3') flanked by an EcoRI site at the 5'-end and by StuI and XbaI sites at the 3'-end (5'-GAATTCGTTTTCAGGAATGCGGAGGTGGGTTTTCAGGAATGCGGAGGTGGGTTTTCAGGAATGCGGAGGTGGAGGCCTCTCGAGTCTAGA-3'), and its complementary sequence, were synthesized by the DNA core facility at the University of Alberta. Two strands (500 pmol each) were annealed in 100 µl of 25 mM Tris-HCl, pH 8.0, 0.5 mM MgCl2, and 25 mM NaCl by heating to 70 °C for 10 min followed by slow cooling to room temperature. The annealed sequence (3Eb) was digested and inserted between the EcoRI and XbaI sites of the yeast pHIS and pLacZ vectors (CLONTECH) to prepare pHIS-Eb and pLacZ-Eb plasmids, respectively. The correct orientation of 3Eb was ascertained by digestion with StuI and sequencing. pHIS-Eb and pLacZ-Eb were linearized with AflII and NcoI, respectively, and the linearized products were transformed into YM4271 yeast host. Cells were grown in SD/-His or SD/-Ura selective medium, and stable integrants for pHIS-Eb and pLacZ-Eb were isolated and named YHIS-Eb and YLacZ-Eb, respectively. The yeast strain YHIS-Eb was transformed with the pACT2 mouse embryo cDNA library and incubated in SD/-His/-Leu selective medium containing 45 mM 3-AT to suppress completely background growth due to leaky HIS3 expression in this reporter strain. The pACT2 cDNA library generates fusions of the cDNA for yeast GAL4 activation domain, the cDNA for a hemagglutinin tag, and a cDNA of encoded proteins. Positive one-hybrid yeast colonies were collected and propagated in 1 ml of SD/-His/-Leu medium at 30 °C overnight. pACT2 plasmids were then isolated using the YEASTMAKER plasmid isolation kit (CLONTECH) according to the manufacturer's instructions. The isolated plasmids were amplified using Electrocomp Transformation Kit (Invitrogen, Carlsbad, CA) and sequenced. A total of 19 positive pACT2 clones were obtained and examined. Ten clones were identified to encode TEF-4, presumably an Eb-binding protein, and two of the TEF-4 positive clones, named pY41b and pY51b, were used for further investigation.

Colony Lift Filter Assay for beta -Galactosidase Activity-- YLacZ-Eb yeast was transfected with positive TEF-4 plasmids, pY41b and pY51b, or control plasmid pYcont (pACT2 vector) and cultured in SD/-Leu selective media. If bound to the Eb region of the YLacZ-Eb promoter, TEF-4 expressed from pY41b and pY51b would expected to initiate transcription of the yeast beta -galactosidase. Formed colonies were streaked on plates with the same selective media and grown for 2 days. Filter paper was placed on the plates, lifted, frozen in liquid nitrogen to break the cell walls, and then thawed on a second filter paper impregnated with beta -galactosidase substrate, 5-bromo-4-chloro-3-indol-beta -D-galactopyranoside. The appearance of a blue color indicated a positive interaction of TEF-4 protein with the LacZ-Eb promoter. As a positive control for this assay we used Y53BLUE yeast strain carrying the p53 binding domain in front of the LacZ promoter transformed with the pGAD53m plasmid carrying the p53 protein. As negative controls we used a combination of YlacZ-Eb yeast with pGAD53m and pYcont plasmids or Y53BLUE yeast with pY41b and pY51b plasmids.

Preparation of Deleted and Mutated CCTalpha Promoter-Luciferase Reporters-- Various 5'-deleted CCTalpha promoter regions, LUC.C6 (-2068/-418), LUC.C7 (-1268/+38), LUC.C8 (-201/+38), LUC.D1 (-90/+38), LUC.D2 (-130/+38), and LUC.D3 (-52/+38), inserted into the promoter-less luciferase vector pGL3-basic (Promega) were prepared as described previously (25). To prepare mutated promoters, the ATG core of the TEF-4 binding region (Eb) was mutated to GCT or AGC (36). These mutations were created from a single-stranded -1268/+38 promoter isolated from LUC.C7 after ligation into pBluescript II SK (-) vector (Stratagene, La Jolla, CA) as described (37). The mutated promoter was either directly ligated back into pGL3-basic vector to prepare LUC.C7m and LUC.C7m2 or digested before ligation to prepare LUC.C8m as reported (25).

Construction of TEF-4 Mammalian Expression Plasmids and TEF-4 Deletion Mutants-- TEF-4 mammalian expression plasmid pcTEF-4 was prepared by digesting pY41b plasmid, encoding the full-length TEF-4, with EcoRI and XhoI and ligating the fragment into pcDNA3.1/V5-His vector (Invitrogen, Carlsbad, CA). To prepare a TEF-4 deletion mutant, the EcoRI/XhoI fragment obtained from pY41b was ligated into pBluescript II SK(-) to obtain TEF-4 SK(-). TEF-4 SK(-) was then digested with NcoI and AatII, and the sticky ends were blunted by T4 DNA polymerase and ligated to make a deleted TEF-4. This construct contained the TEA-DNA binding domain, encoding the first 171 amino acids plus 5 additional amino acids, CTTRR, at its carboxyl terminus. The sequence was then digested from the pBluescript vector and ligated into the pcDNA3.1/V5-His mammalian expression vector to make a pcmTEA(+) plasmid. To prepare a second TEF-4 mutant, TEF-4 SK(-) was digested with BalI and BamHI and then the sticky ends were blunted and ligated. This procedure generated a construct of TEF-4 without the TEA-DNA binding domain, i.e. lacking amino-terminal amino acids 5-133. The deleted TEF-4 fragment was isolated from the vector using EcoRI and XhoI and ligated into pcDNA3.1/V5-His to make the pcmTEA(-) expression plasmid.

Tissue Culture, Transfection, and Luciferase Assay-- COS-7 and 3T3-L1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells (1 × 105) were plated on 35-mm plates (Falcon-Becton Dickinson Labware, Franklin Lakes, NJ) and grown overnight. Three µl of FuGENETM6 was suspended in 100 µl of serum-free medium and mixed with 0.5 µg of the CCTalpha promoter-luciferase constructs (see above), 0.001 µg of pRL-CMV Renilla vector as a transfection control, and 0.5 µg of either pcTEF-4, pcmTEA(-), pcmTEA(+) or pcDNA control. Transfection was initiated by dropwise addition of DNA suspensions to the cell culture. Forty eight hours later cells were harvested, lysed in 200 µl of the Passive lysis buffer (Promega), and 10 µl of the cell lysate was used for the dual-luciferase assay according to the manufacturer's instructions. Luciferase activity was normalized for transfection efficiency by using the ratio of the activities obtained with the CCTalpha promoter deletion constructs (see above) and the pRL-CMV construct carrying the cytomegalovirus promoter-luciferase fusion.

Preparation of Cell and Nuclear Extracts-- TEF-4-positive yeast, Y41b, was cultured in 15 ml of SD/-His/-Leu medium containing 45 mM 3-AT. After 48 h, 12 ml of the late log-phase culture was transferred into 100 ml of the same medium and cultured for an additional 36 h. The cells were collected by centrifugation at 600 × g for 10 min and resuspended in 1 ml of homogenization buffer (0.1 M Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.01 M beta -mercaptoethanol, 20% glycerol). After vortexing with glass beads to break the cell walls, the cells were subjected 3 times to freezing and thawing and centrifuged at 10,000 × g for 5 min to remove the cell debris (38). The yeast transformed with pYcont were cultured in SD/-Leu medium and treated in a similar manner to obtain control cell extracts. Nuclear extracts from mammalian cells were prepared according to Andrews and Faller (39) with minor modification (25).

Preparation and Purification of Glutathione S-Transferase TEF-4 Fusion Protein (GST-TEF-4)-- Full-length TEF-4 cDNA was digested from pY41b with EcoRI and XhoI and ligated into the pGEX-KG (Promega) vector to obtain the pGST-TEF-4 expression plasmid. pGST-TEF-4 or control vector were transformed into Escherichia coli strain HB101 and grown in Luria broth overnight. The cultures were subsequently transferred to 10 volumes of growth media and grown for 1 h. Isopropyl-beta -D-(-)-thiogalactopyranoside was then added to the culture to a final concentration of 0.5 mM, and the incubation was continued for 3 h. Cells were collected by centrifugation, resuspended in 0.5 ml of lysis buffer (25 mM Hepes-NaOH, pH 7.5, 20 mM KCl, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 0.02% Triton X-100), sonicated, and centrifuged at 10,000 × g for 15 min. To obtain purified proteins, the supernatant was applied to a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). GST-TEF-4 and GST proteins were eluted according to the manufacturer's instructions.

Electromobility Gel-shift and Supershift Assays-- The opposite strands of Eb, mutated Eb (5'-GTGGTTTTCAGGAGCTCGGAGGTGGCATT-3', Ebm) and the SV-40 GT-IIC enhanson (5'-ACCAGCTGTGGAATGTGTGTCGA-3'), 500 pmol each, were annealed (70 °C, 10 min) in 100 µl of 25 mM Tris-HCl, pH 8.0, 0.5 mM MgCl2, and 25 mM NaCl and then cooled to room temperature. An aliquot (10 pmol) of the double-stranded oligonucleotides was 5'-end-labeled with [32P]ATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase and purified on a Sephadex G-25 column (Amersham Pharmacia Biotech). A DNA protein-binding reaction was performed for 30 min at room temperature in 40 µl of 1× binding buffer (40 mM Tris-HCl, pH 7.9, 4 mM MgCl2, 2 mM EDTA, 100 mM NaCl, 2 mM dithiothreitol, 200 µg/ml bovine serum albumin, 20% glycerol, and 0.2% Nonidet P-40) containing 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech), 1 µl of the radiolabeled probe (50,000-80,000 cpm), and one of the following: yeast cell extracts, mammalian nuclear extracts, purified GST-TEF-4 or GST proteins. In some cases, unlabeled double-stranded Eb (100-fold molar excess), anti-hemagglutinin antibody (Roche Molecular Biochemicals), or anti-GST antibody (Amersham Pharmacia Biotech) was included in the incubation mixture. The reaction was stopped by addition of 4 µl of 6× DNA loading buffer. The labeled probe was separated from DNA-protein complexes by electrophoresis on 6% nondenaturing polyacrylamide gels in 0.5× Tris borate/EDTA buffer (44.5 mM Tris-HCl, pH 8.3, 44.5 mM boric acid, and 1 mM EDTA) at 4 °C until the xylene cyanol dye reached 5 cm from the bottom of the gel. Autoradiography was carried out by exposure of the gel to Kodak X-Omat XAR2 film with an intensifying screen at -70 °C for 16-48 h.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-- COS-7 cells were transformed with pcTEF-4 or pcDNA as described above. After being cultured for 48 h, the cells were treated with Isogen (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions, and total RNA was extracted. One µg of total RNA was reverse-transcribed at 60 °C for 30 min and then subjected to 20 cycles of amplification (94 °C for 1 min, 56 °C for 1 min, and 60 °C for 1.5 min) using the one-step RT-PCR kit (Toyobo, Osaka, Japan). The primers used for CCTalpha were 5'-ATGCACAGTGTTCAGCCAA-3' (sense) and 5'-GGGCTTACTAAAGTCAACTTCAA-3' (antisense). Primers for glycero-3-phosphate dehydrogenase (G3PDH) were 5'-TCCACCACCCTGTTGCTGTA-3' (sense) and 5'-ACCACAGTCCATGCCATCAC-3' (antisense). The intensities of the CCTalpha bands were normalized to those of the G3PDH bands using the Quantity One software (PDI, Huntington Station, NY).

Statistical Analysis-- All values are expressed as means ± S.D. Group means were compared by Student's t test or the Cochran-Cox test after analysis of variance to determine the significance of difference between the individual means. Statistical significance was assumed at p < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cloning of the Putative Regulatory Proteins That Bind to the Eb Element of the CCTalpha Promoter-- A yeast integrant (YHIS-Eb) carrying three repeats of Eb in the front of the his3 coding region was constructed and used to screen for the Eb-binding proteins and promoter activation by the one-hybrid expression system. The Yhis-Eb yeast strain was transformed with the pACT2 mouse embryo cDNA library encoding fusions of the yeast gal4 activation domain, a cDNA of an unknown protein, and a hemagglutinin tag at its 5'-end. The yeast transformation efficiency was 1 × 104 colony-forming units/µg as determined on SD/-Leu selective medium. After screening 1.5 × 106 colonies, 19 positive clones were obtained that were able to grow on the SD/-His/-Leu selective medium containing 45 mM 3-AT. Of the cDNAs obtained from those yeast clones, 10/19 encoded transcriptional enhancer factor-4 (33, 34); 1/19 encoded ribosomal protein L22 (40); 1/19 encoded ribosomal protein S4 (41); 1/19 encoded phosphatidylinositol phospholipase Calpha (42); 1/19 encoded a mouse ortholog of the human FRAP-related protein (43); 1/19 encoded DNA polymerase I (44); and 4/19 encoded unknown cDNAs. Thus, the most frequently isolated clones were those encoding transcriptional enhancer factor TEF-4. Since we found that the TEF-binding consensus (35), 5'-(A/T)(A/G)(A/G)(A/T) ATG (C/T)(G/A)-3' was present in the Eb promoter region, we decided to focus on TEF-4 and further analyze its role in CCTalpha gene transcription. We selected the clones pY41b and pY51b, isolated from the yeast Y41b and Y51b, that encoded the full-length or almost full-length TEF-4 (pY41b from the position -24 and pY51b from the position +94 relative to the translation start codon).

To confirm that TEF-4 clones can drive transcription of other yeast genes carrying the Eb sequence in their promoters, the yeast integrant YLacZ-Eb carrying a 3-fold repeat of Eb followed by the beta -galactosidase lacZ gene was transformed with pY41b, pY51b plasmids, or pYcont (pACT2 vector), cultured on SD/-Leu selective medium, and tested for the expression of beta -galactosidase. Formed colonies were examined for beta -galactosidase by the colony-lift filter assay. The colonies of YLacZ-Eb transformed with pY41b and pY51b expressed beta -galactosidase and turned blue in the presence of the beta -galactosidase substrate. However, the colonies of YLacZ-Eb transformed with either pGAD53m encoding a fusion of p53 with the GAL4 activation domain (which does not bind to the Eb region) or the pYcont empty vector, did not yield blue colonies, as expected. We next used Y53BLUE yeast strain that carried the p53-binding sequence in front of lacZ as a positive control. Y53BLUE transformed with pGAD53m plasmid expressed beta -galactosidase and the colonies turned blue. When the Y53BLUE yeast was transformed with plasmids pY41b and pY51b encoding TEF-4, which does not bind to the p53 sites, or with the empty vector pYcont, no blue color was obtained. The untransformed YLacZ-Eb or Y53BLUE yeast produced a similar negative response. Together, these results indicate that pY41b and pY51b encodes TEF-4 that binds to the Eb region of heterologous yeast promoters and drives transcription of the his3 and lacZ genes. The results also confirm the validity of the TEF-4 clones and their expression.

Evaluation of TEF-4 Binding to the Eb Promoter Element-- To confirm the binding of TEF-4 to the Eb region, we performed gel-shift analysis using the radiolabeled Eb oligonucleotide as a probe. The labeled Eb fragment was incubated with cell extracts isolated from the yeast expressing TEF-4 (Y41b) or control (Ycont), and DNA-protein complexes were separated by electrophoresis and visualized by autoradiography (Fig. 1A). When Y41b extract was used, two slower migrating bands in addition to the faster moving probe were observed (Fig. 1A, lane 2). The intensities of the two slower bands were markedly decreased after competition with a large excess of unlabeled probe (Fig. 1A, lane 4), suggesting that both originated from a protein that binds to Eb. However, the lower band was also visible in the experiments with control yeast extracts (Fig. 2A, lane 3) indicating that only the upper band was specific for TEF-4/DNA binding. This was further confirmed by a competition experiment with an antibody specific for the hemagglutinin tag, which was part of the TEF-4 fusion protein. When various amounts of the anti-hemagglutinin antibody were added to the reaction mixture, the intensity of the upper band decreased, whereas the intensity of the lower band was not affected (Fig. 1B). These results clearly support the notion that the upper band was specific for the interaction of the TEF-4 fusion protein with Eb.



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Fig. 1.   Gel-shift and supershift analysis for the TEF-4/hemagglutinin tag or purified TEF-4/GST fusion protein binding with the Eb promoter element. A, the end-labeled Eb probe was incubated with 65 µg of the extract from yeast carrying pY41b (lanes 2 and 4) or pYcont (lane 3). A 100-fold molar excess of unlabeled probe was used for competition with the labeled Eb probe (lane 4). B, the end-labeled Eb probe was incubated with 65 µg of the extract from yeast carrying pY41b. Cell extracts were incubated without (lane 1) or with 2 (lane 2), 4 (lane 3), or 8 µg (lane 4) of anti-hemagglutinin antibody. C, the end-labeled Eb (lanes 1-7) or GT-IIC probe (lanes 8 and 9) were incubated with 1 µg of purified GST-TEF-4 (lanes 2, 4, 6, and 8) or purified GST (lanes 3, 5, 7, and 9). In lanes 4 and 5, the purified proteins were incubated with 5 µg of anti-GST antibody. In lanes 6 and 7, a 100-fold molar excess of unlabeled Eb probe was added. Bovine serum albumin was used so that the protein content in each lane was equal. The arrows indicate the positions of specific DNA-protein complexes. Each experiment was repeated twice with similar results.



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Fig. 2.   Gel-shift analysis using TEF-4 expressed in COS-7 cells or purified GST-TEF-4 with Eb or mutated Eb (Ebm) probe. A, the end-labeled Eb probe (lanes 1-5) or Ebm probe (lanes 6-8) was incubated with 20 µg of nuclear extract from COS-7 cells transfected with vector control (lanes 2, 4, and 7) or pcTEF-4 (lanes 3, 5, and 8). A 100-fold molar excess of unlabeled probe was used for competition with labeled Eb (lanes 4 and 5). B, the end-labeled Eb probe (lane 1) or Ebm probe (lanes 2 and 3) was incubated with 1 µg of purified GST (lane 2) or purified GST-TEF-4 (lanes 1 and 3). The arrows indicate the positions of specific DNA-protein complexes. Each experiment was repeated twice with similar results.

Antibodies specific for TEF-4 are not available. To confirm that the binding to Eb is specific for TEF-4 protein regions and not for the hemagglutinin tag, we cloned and purified a fusion protein, glutathione S-transferase-TEF-4 (GST-TEF-4). We also made pure GST and used it as a negative control in parallel gel-shift assays. As shown in Fig. 1C, migration of the Eb band was retarded only in the presence of purified GST-TEF-4 (lane 2) and not in the presence of GST (lane 3). The addition of an anti-GST antibody (lane 4) or cold Eb competitor (lane 6) prevented formation of the retarded bands. Purified GST-TEF-4 also caused a band retardation of the SV40 enhancer element GT-IIC (lane 8) that is known to be regulated by TEF proteins. Furthermore, GST-TEF-4 did not bind to the CCTalpha basal promoter fragment -90/+38 that does not contain a TEF-4 consensus sequence (data not shown). Taken together, we conclude that TEF-4 is responsible for the formation of the protein complexes within the Eb region of the CCTalpha promoter.

DNA Binding Properties of TEF-4 after Its Expression in Mammalian Cells-- To test whether or not TEF-4 could also bind to the Eb promoter element after expression in mammalian cells, we prepared a mammalian TEF-4 expression plasmid, pcTEF-4, and transfected COS-7 cells with pcTEF-4 or control vector pcDNA. We isolated cell nuclear extracts and performed gel-shift analysis using Eb and mutated Eb (Ebm in which the TEF-4 ATG-binding core was mutated to GCT) as probes. Nuclear extracts from COS cells transfected with pcTEF-4 produced two slowly migrating bands in addition to the rapidly migrating Eb band (Fig. 2A, lane 3). The intensities of the two retarded bands markedly decreased after competition with an excess of cold Eb probe (lanes 4 and 5). The upper band was also visible in the experiments with control nuclear extracts (lane 2), indicating that some endogenous nuclear proteins or the protein complex of TEFs with some other factors also bind to Eb. The migration of the lower band, however, coincided with the migration of the purified GST-TEF-4, suggesting that this band originated from the binding of TEF-4 overexpressed in COS-7 cells. When the mutated element Ebm was used instead, no protein binding was detected with nuclear extracts from TEF-4-expressing cells (Fig. 2A, lane 8) nor did the purified GST-TEF-4 fusion protein bind to the mutated probe (Fig. 2B, lane 3). These results suggested that both overexpressed TEF-4 and an endogenous nuclear protein from COS-7 cells can bind to the Eb promoter element by recognizing the same ATG core sequence within the TEF binding consensus.

Activation of Chimeric CCTalpha Promoter-Luciferase Reporters by TEF-4-- To examine whether or not TEF-4 can mediate transcription of the CCTalpha gene, we prepared various CCTalpha promoter deletion constructs linked to the luciferase reporter and transfected either COS-7 or 3T3-L1 cells with pcTEF-4 or pcDNA control. The expression of luciferase activity was determined by dual-luciferase assays and normalized for transfection efficiency after cotransfection with pRL-CMV Renilla vector. The Renilla luciferase activity of the CMV promoter-driven controls was constant and was not affected by TEF-4 throughout the experiments. As shown in Fig. 3A, when cells were transfected with CCTalpha promoter-luciferase constructs the luciferase activity was highest for the shortest promoter construct, -52/+38 (LUC.D3), and was lower for the longer -90/+38 (LUC.D1) construct. Luciferase activity increased again with the construct containing longer promoter region (-130/+38, LUC.D2) and with the construct -201/+38 (LUC.C8). These results suggested that important regulatory regions for CCTalpha transcription in COS-7 and 3T3-L1 cells resided at positions -52/+38 and -202/-52. The core promoter region -52/+38 was responsible for the constitutive expression (25), and further upstream regions (-202/-52), which were shown to be regulatory (25), were both negatively and positively modulated in those two cell lines. When the cells were cotransfected with TEF-4 expression plasmid, the luciferase activities for all promoter-reporter constructs increased 2-2.5-fold relative to the controls. Surprisingly, the maximal stimulatory effect of TEF-4 was observed with the core promoter construct LUC.D3 (-53/+38) lacking the consensus binding sites for TEF-4. The actual TEF-4-binding site Eb is located further upstream in the sequence between -103 and -82. These results indicate that the primary stimulatory effect of TEF-4 on the CCTalpha promoter was through its action on basal transcription and that the further upstream TEF-4 consensus binding site, if functional, might play a role that was not obvious from the above deletion analysis.



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Fig. 3.   Effect of TEF-4 on the expression of various CCTalpha gene promoter-luciferase reporter constructs. A, truncated CCTalpha promoters (LUC.D1, LUC.D2, LUC.D3, LUC.C6, LUC.C7, and LUC.C8) and mutated CCTalpha promoters (LUC.C7m and LUC.C8m in which the ATG core in the Eb region was mutated to GCT; and LUC.C7m2 in which the ATG core in the Eb region was mutated to AGC) were cloned into the luciferase reporter vector pGL3-basic. Luciferase plasmids (0.5 µg) and pRL-CMV (0.001 µg) were transfected into COS-7 cells with pcDNA (0.5 µg) (white bar) or pcTEF-4 (0.5 µg) (black bar). Reporter activities were measured 48 h after transfection and normalized for transfection efficiency as described under "Experimental Procedures." TEF-4 significantly enhanced the luciferase activity of each luciferase construct compared with vector control (p < 0.05). * and ** represent p < 0.03 and p < 0.01, respectively, for the LUC.C8m to LUC.C8, and LUC.C7m and LUC.C7m2 to LUC.C7 compared with the identical pcDNA or pcTEF-4 treatment. N.D. represents not detectable. B, LUC.D3 or LUC.C7 (0.5 µg) and pRL-CMV (0.001 µg) were transfected into COS-7 cells with pcDNA (0.5 µg) (lane 1, white bar), pcTEF-4 (0.5 µg) (lane 2, black bar), or mutated TEF-4, pcmTEA(-) (lane 3, hatched bar) or pcmTEA(+) (lane 4, striped bar). * and ** represent p < 0.03 and p < 0.01, respectively, compared with vector control. C, luciferase plasmids (LUC.D3, LUC.D1, LUC.D2, LUC.C8, and LUC.C8m) (0.5 µg) and pRL-CMV (0.001 µg) were transfected into 3T3-L1 cells with pcDNA (0.5 µg) (white bar) or pcTEF-4 (0.5 µg) (black bar). TEF-4 significantly enhanced the luciferase activity by each luciferase construct compared with vector control (p < 0.05). * represents p < 0.05 for LUC.C8m compared with LUC.C8 with the same pcDNA or pcTEF-4 treatment. Values are the means ± S.D. from three independent dishes. Each experiment was repeated three times with similar results.

To substantiate the notion that TEF-4 functionally interacts with the Eb region in mammalian cells, we prepared luciferase reporter constructs containing point mutations in the Eb region (ATG to GCT) of the LUC.C8 (-201/+38) and LUC.C7 (-1268/+38), named LUC.C8m and LUC.C7m, respectively. We also prepared a luciferase reporter containing point mutations in the Eb region (ATG to AGC) of the LUC.C7 (-1268/+38), named LUC.C7m2, to which TEF-5 was reported not to bind (36). When COS-7 cells were transfected with these mutated Eb constructs the luciferase activity increased above that of the wild-type constructs. Furthermore, TEF-4 expression enhanced the luciferase activity of the mutated constructs relative to that of the wild-type constructs (Fig. 3A). These results suggest that TEF-4 functionally binds to the Eb region of CCTalpha promoter where it acts as a transcriptional repressor. Taken together, deletion and mutation analyses show that TEF-4 can act as both a positive and a negative modulator of the CCTalpha promoter activity; the two functions combined predominantly produce a stimulatory effect.

To establish which domain(s) of TEF-4 is responsible for its stimulatory activity and binding to the CCTalpha promoter, we expressed TEF-4 deletion mutants pcmTEA(+) and pcmTEA(-) in COS-7 cells and measured luciferase activity from the LUC.D3 core promoter construct (stimulatory effect) and LUC.C7 (stimulatory and inhibitory effects) construct. The TEF-4 deletion mutant containing only the so-called TEA-DNA binding domain, pcmTEA(+), retained its stimulatory effect on luciferase expression from both LUC.D3 and LUC.C7 promoter-reporter constructs (Fig. 3B, lane 4). A second TEF-4 deletion mutant, lacking the DNA binding domain but containing the other regions of the protein, did not have any significant effect on the promoter activity (Fig. 3B, lane 3).

These results suggest that the DNA binding domain of TEF-4 is critical and sufficient for its function as a transcriptional modulator of the CCTalpha promoter which, as demonstrated in Fig. 3A, was predominantly through its effect on basal transcription. On the other hand, the full-length GST-TEF-4 fusion protein containing the TEA domain did not bind to the core promoter region (-90/+38) since this region does not have a consensus binding site for TEF-4 (data not shown). Thus, it appears that TEF-4 does not stimulate the basal activity through direct binding to the core promoter. Instead, we postulate that TEF-4 stimulates the expression of some other transcription factor(s) necessary for the basal CCTalpha gene transcription and/or directly interacts through its TEA domain with a nuclear protein(s) that is critical for CCTalpha transcription.

To confirm that the multiple effects of TEF-4 on the CCTalpha promoter activity are not restricted to COS cells, we performed similar functional assays in 3T3-L1 cells. The results are shown in Fig. 3C. The exogenous TEF-4 expressed in 3T3-L cells enhanced the basal activity of the deletion promoter series and of the TEF-4 consensus site mutant LUC.C8m as was the case in COS-7 cells.

TEF-4 Expression Increases CCTalpha mRNA Abundance-- COS-7 cells were transfected with pcTEF-4 or its control vector pcDNA to determine whether or not TEF-4 could also increase the amount of CCTalpha mRNA by acting on the natural promoter as it did on the promoter-luciferase reporter chimeras. As shown in Fig. 4, TEF-4 increased the CCTalpha mRNA level about 1.4-fold compared with that in control cells. These results confirm that TEF-4 can modulate the abundance of CCTalpha transcripts in cultured cells.



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Fig. 4.   TEF-4 increases CCTalpha mRNA abundance. A, COS-7 cells were transfected with pcDNA (0.5 µg) or pcTEF-4 (0.5 µg). Total mRNA was obtained 48 h after transfection, and 1 µg of total RNA was used for RT-PCR analysis of CCTalpha and G3PDH. B, the band intensities in A were quantified as described under "Experimental Procedures." The values for G3PDH mRNA were used to normalize the band density of CCTalpha mRNA. * represents p < 0.05 compared with the vector control. Values are means ± S.D. from three independent dishes. Each experiment was repeated three times with similar results.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Physiological Role of TEF-4 Is Unknown-- In the transcription enhancer factor family, TEF-1 was initially purified from HeLa cells, based on its binding affinity for the SV40 enhancer element GT-IIC (45), and then subsequently cloned and characterized (35). TEF-1 consists of 426 amino acids forming four separate structural domains as follows: a DNA-binding domain (TEA/ATTS domain) at its amino terminus, proline-rich and serine-threonine-tyrosine-rich domains in the middle region, and a zinc finger motif at its carboxyl terminus. The TEA region directly interacts with consensus DNA elements, and the proline-rich and zinc finger domains modulate TEF-1 binding to these elements (46). TEF-1 also contains potential phosphorylation sites for calmodulin-, cAMP-, and cGMP-dependent protein kinases (47) that could also modulate its function as a transcription factor.

TEF-4, initially named as the "embryonic TEA domain-containing factor" or ETF, was identified in neural precursor cells by subtraction screening with adult mouse brain mRNA (33). Based on its level and pattern of expression, TEF-4 has been implicated in the developmental regulation of neural (33), kidney (34), and other tissues (34), but its actual physiological role remains unknown. TEF-4 contains 66% sequence identity to TEF-1, mostly in the TEA region, hence other domains may be important for TEF-4-specific activity (33). Two other members of the family, TEF-3 (34) and TEF-5 (36), were identified by PCR amplification using degenerate primers corresponding to the highly conserved TEA domain. TEF factors activate promoters via binding to the consensus sequence (5'-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3') containing a conserved ATG core. Although it was known that TEF nuclear factors could activate viral promoters by binding to GT-IIC and SphI/SphII enhancer elements (48), until recently the actual targets for TEFs in the mammalian genome were largely unknown. TEF-1 was reported to stimulate transcription of alpha -myosin heavy chain in myocytes (49) and TEF-5 to regulate the cell-specific expression of chorionic somatomammotropin-B in placenta (36). There were no reports of promoters that could be targeted for regulation by TEF-4. Therefore, this is the first report demonstrating that a mammalian gene, CCTalpha , can be regulated by TEF-4 through modulation of its basal transcription and through binding to the TEF consensus site within the distal enhancer region.

TEF-4 Has Both Stimulatory and Repressive Actions on the CCTalpha Promoter-- Our initial studies demonstrated that unknown nuclear factors bound to the -103/-82, or Eb, region of the mouse CCTalpha promoter and modulated transcription (25). In the present study, we isolated an Eb-binding nuclear factor, TEF-4, by functional cloning using the yeast one-hybrid system. The sequence alignments suggested that the primary structure of the TEA domain of TEF-4 is indistinguishable from that of TEF-1 (Ref. 34 and data not shown). Indeed, we showed (Fig. 1C) that purified GST-TEF-4 binds to the GT-IIC motif present in the SV40 enhancer as previously shown for TEF-1 (35). Consequently, we might expect that other TEF members, TEF-1, TEF-3, and TEF-5, would also bind to the Eb sequence. However, only TEF-4 cDNA was functionally cloned in the present study suggesting that if the cDNAs for other TEFs were present in the library, the TEA domain was not the sole determinant of TEF-4 DNA binding affinity and function (46). The gel-shift and supershift experiments with yeast and mammalian proteins and purified GST-TEF-4 fusion protein clearly demonstrated that the Eb region within the CCTalpha promoter represents a true binding site for TEF-4. However, we did not test other members of the TEF family for their binding and regulatory properties. Thus, we do not exclude the possibility that other TEF members may also be relevant for the regulation of expression of CCTalpha .

Previous reports showed that overexpressed TEF-1 represses the expression of the GT-IIC-containing reporter genes in HeLa cells due to the presence of a limited amount of a putative coactivator (35, 50). Transfection of TEF-4 failed to induce any obvious effect on the luciferase gene expression from a tetramerized GT-IIC oligonucleotide ligated upstream of the thymidine kinase promoter-reporter (33). However, we report in this paper for the first time that TEF-4 is a functional transcription factor that is able to enhance the activity of the CCTalpha promoter-luciferase reporters and increase the level of CCTalpha mRNA in transfected cells. Previous studies have shown that the level of CCTalpha mRNA is increased upon stimulation of cells by colony-stimulating factor-1 (18), after partial hepatectomy (19), or during tissue growth and differentiation (20). Our data suggest that TEF-4, and perhaps other members of the family, might play a positive regulatory role in transcription of the CCTalpha gene under these stimulatory conditions.

The present results demonstrate that overexpression of TEF-4 significantly enhances the CCTalpha promoter activity but that the activation was moderate compared with the vector control. We demonstrated that TEF-4 might play multiple roles in the regulation of the CCTalpha promoter. We established that TEF-4 stimulates basal transcription since overexpressed TEF-4 increases the activity of the basal promoter construct -52/+38 that lacks the TEF-4 consensus binding site. By preparing mutated TEF-4 proteins, we demonstrated that the TEA-DNA binding domain is important and sufficient for its stimulatory activity. These results suggest that either TEF-4 stimulates transcription of a basic transcription factor(s) or that TEF-4 directly interacts via its TEA domain with pre-existing protein(s) or cofactor(s) required for basal transcription. Recently, it was found that the p160 family of nuclear receptor coactivators, SRC1, TIF2, and RAC3, act as bona fide coactivators of TEF-4 and other members of the TEF family of transcription factors (51). These coactivators are potential targets for the action of TEF-4 on the CCTalpha promoter.

Unexpectedly, when the TEF-4 binding region Eb within the CCTalpha promoter was mutated, the overexpressed TEF-4 did not decrease but further enhanced the luciferase activity (Fig. 3A). These data suggest that after binding to the Eb region TEF-4 acts as a transcriptional repressor and might explain why transactivation by TEF-4 was attenuated in longer promoter constructs and why the effect of TEF-4 on CCTalpha transcription was relatively modest. Repression of transcription by TEF proteins is known to occur. The cause for TEF-1 repression has been suggested to be due to titration by the overexpressed TEF-1 of another limiting transcriptional coactivator (35). It is well established that the E-box motif for TEF-1 and the M-CAT motif for Max are both required for expression of the cardiac alpha -myosin heavy chain gene in rat myocytes (52). Low concentrations of either TEF-1 or Max alone modestly activate expression of the gene and repress the gene at higher concentrations. However, when TEF-1 and Max were cotransfected, a synergistic transactivation of the alpha -myosin heavy chain gene occurred, which was explained not only by TEF-1 and Max binding to their respective DNA motifs but also by protein-protein interactions between them. In this respect it is noteworthy that TEF-1 repressed the human chorionic somatomammotropin B promoter in BeWo choriocarcinoma cells (53) via direct protein-protein interactions with TATA-binding proteins. However, it was established that another member of the family, TEF-5, specifically binds to several functional motifs in the human chorionic somatomammotropin-B gene enhancer and thus stimulates its activity (36).


    ACKNOWLEDGEMENTS

We thank Dr. Donna Douglas, Dr. Kazuaki Tatei, and Sandra Ungarian for excellent technical assistance and Prof. Jean Vance for helpful comments on the manuscript.


    FOOTNOTES

* This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan, and from the Canadian Institutes of Health Research.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: Dept. of Biochemistry and CIHR Group on Molecular and Cell Biology of Lipids, 328 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G2S2, Canada. Fax: 1-780-492-3383; E-mail: dennis.vance@ ualberta.ca.

Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M100090200


    ABBREVIATIONS

The abbreviations used are: CCT, CTP:phosphocholine cytidylyltransferase; 3-AT, 3-aminotriazole; Ctpct, CTP:phosphocholine cytidylyltransferase alpha  gene; G3PDH, glycero-3-phosphate dehydrogenase; RT-PCR, reverse transcriptase-polymerase chain reaction; TEF, transcriptional enhancer factor; GSTm, glutathione S-transferase; CMV, cytomegalovirus.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Kent, C. (1997) Biochim. Biophys. Acta 1348, 79-90[Medline] [Order article via Infotrieve]
2. Lykidis, A., and Jackowski, S. (2000) Prog. Nucleic Acid Res. Mol. Biol. 65, 361-393[Medline] [Order article via Infotrieve]
3. Johnson, J. E., and Cornell, R. B. (1999) Mol. Membr. Biol. 16, 217-235[CrossRef][Medline] [Order article via Infotrieve]
4. Vance, D. E. (1996) in Biochemistry Lipids, Lipoproteins, and Membranes (Vance, D. E. , and Vance, J. E., eds) , pp. 153-181, Elsevier Science Publishers B.V., Amsterdam
5. Weinhold, P. A., Rounsifer, M. E., and Feldman, D. A. (1986) J. Biol. Chem. 261, 5104-5110[Abstract/Free Full Text]
6. Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081[Abstract/Free Full Text]
7. Kalmar, G. B., Kay, R. J., Lachance, A., Aebersold, R., and Cornell, R. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6029-6033[Abstract]
8. Pelech, S. L., and Vance, D. E. (1982) J. Biol. Chem. 257, 14198-14202[Free Full Text]
9. Yang, W., and Jackowski, S. (1995) J. Biol. Chem. 270, 16503-16506[Abstract/Free Full Text]
10. Lykidis, A., Murti, K. G., and Jackowski, S. (1998) J. Biol. Chem. 237, 14022-14029[CrossRef]
11. Lykidis, A., Baburina, I., and Jackowski, S. (1999) J. Biol. Chem. 274, 26992-27001[Abstract/Free Full Text]
12. Boggs, K. P., Rock, C. O., and Jackowski, S. (1995) J. Biol. Chem. 270, 7757-7764[Abstract/Free Full Text]
13. Sohal, P. S., and Cornell, R. B. (1990) J. Biol. Chem. 265, 11746-11750[Abstract/Free Full Text]
14. MacDonald, J. I. S., and Kent, C. (1994) J. Biol. Chem. 269, 10529-10537[Abstract/Free Full Text]
15. Wang, Y., and Kent, C. (1995) J. Biol. Chem. 270, 17843-17849[Abstract/Free Full Text]
16. Arnold, R. S., DePaoli-Roach, A. A., and Cornell, R. B. (1997) Biochemistry 36, 6149-6156[CrossRef][Medline] [Order article via Infotrieve]
17. Houweling, M., Jamil, H., Hatch, G. M., and Vance, D. E. (1994) J. Biol. Chem. 269, 7544-7551[Abstract/Free Full Text]
18. Tessner, T. G., Rock, C. O., Kalmar, G. B., Cornell, R. B., and Jackowski, S. (1991) J. Biol. Chem. 266, 16261-16264[Abstract/Free Full Text]
19. Houweling, M., Cui, Z., Tessitore, L., and Vance, D. E. (1997) Biochim. Biophys. Acta 1346, 1-9[Medline] [Order article via Infotrieve]
20. Sesca, E., Perletti, G. P., Binasco, V., Chiara, M., and Tessitore, L. (1996) Biochem. Biophys. Res. Commun. 229, 158-162[CrossRef][Medline] [Order article via Infotrieve]
21. Houweling, M., Tijburg, L. B. M., Vaartjes, W. J., Batenburg, J. J., Kalmar, G. B., Cornell, R. B., and Van Golde, L. M. G. (1993) Eur. J. Biochem. 214, 927-933[Abstract]
22. Martina, H., Zimmermann, L. J., Wang, J., Kuliszewski, M., Liu, J., and Post, M. (1994) Am. J. Physiol. 267, L25-L32[Abstract/Free Full Text]
23. Hogan, M., Kuliszewski, M., Lee, W., and Post, M. (1996) Biochem. J. 314, 799-803[Medline] [Order article via Infotrieve]
24. Tang, W., Keesler, G. A., and Tabas, I. (1997) J. Biol. Chem. 272, 13146-13151[Abstract/Free Full Text]
25. Bakovic, M., Waite, K., Tang, W., Tabas, I., and Vance, D. E. (1999) Biochim. Biophys. Acta 1438, 147-165[Medline] [Order article via Infotrieve]
26. Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987) Cell 51, 1079-1090[Medline] [Order article via Infotrieve]
27. Courey, A. J., and Tjian, R. (1988) Cell 55, 887-898[Medline] [Order article via Infotrieve]
28. Kingsley, C., and Winoto, A. (1992) Mol. Cell. Biol. 12, 4251-4261[Abstract]
29. Bakovic, M., Waite, K., and Vance, D. E. (2000) J. Lipid Res. 41, 583-594[Abstract/Free Full Text]
30. Lania, L., Majello, B., and DeLuca, P. (1997) Int. J. Biochem. Cell Biol. 29, 1313-1323[CrossRef][Medline] [Order article via Infotrieve]
31. Li, J. J., and Herskowitz, I. (1993) Science 262, 1870-1874[Medline] [Order article via Infotrieve]
32. Wang, M. M., and Reed, R. R. (1993) Nature 364, 121-126[CrossRef][Medline] [Order article via Infotrieve]
33. Yasunami, M., Suzuki, K., Houtani, T., Sugimoto, T., and Ohkubo, H. (1995) J. Biol. Chem. 270, 18649-18654[Abstract/Free Full Text]
34. Jaquemin, P., Hwang, J.-J., Martial, J. A., Dolle, P., and Davidson, I. (1996) J. Biol. Chem. 271, 21775-21785[Abstract/Free Full Text]
35. Xiao, J. H., Davidson, I., Matthes, H., Garnier, J.-M., and Chambon, P. (1991) Cell 65, 551-568[Medline] [Order article via Infotrieve]
36. Jacquemin, P., Martial, J. A., and Davidson, I. (1997) J. Biol. Chem. 272, 12928-12937[Abstract/Free Full Text]
37. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492[Abstract]
38. Arndt, K. T., Styles, C., and Fink, G. R. (1987) Science 237, 874-880[Medline] [Order article via Infotrieve]
39. Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499-2499[Medline] [Order article via Infotrieve]
40. Fujita, Y., Okamoto, T., Noshiro, M., Kato, Y., Takada, K., Sato, J. D., Ozaki, T., Mckeehan, W., Crabb, J., and Whitney, R. (1994) Biochem. Biophys. Res. Commun. 199, 706-713[CrossRef][Medline] [Order article via Infotrieve]
41. Zinn, A. R., Bressler, S. L., Beer-Romero, P., Adler, D. A., Chapman, V. M., Page, D. C., and Disteche, C. M. (1991) Genomics 11, 1097-1101[Medline] [Order article via Infotrieve]
42. Adams, M. D., Fields, C., and Venter, J. C. (1992) Nature 357, 367-368[Medline] [Order article via Infotrieve]
43. Cimprich, K. A., Shin, T. B., Keith, C. T., and Schreiber, S. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2850-2855[Abstract/Free Full Text]
44. Song, C. Z., Hanada, K., Yano, K., Maeda, Y., Yamamoto, K., and Muramatsu, M. (1994) J. Biol. Chem. 269, 26976-26981[Abstract/Free Full Text]
45. Davidson, I., Xiao, J. H., Rosales, R., Staub, A., and Chambon, P. (1988) Cell 54, 931-942[Medline] [Order article via Infotrieve]
46. Hwang, J.-J., Chambon, P., and Davidson, I. (1993) EMBO J. 12, 2337-2348[Abstract]
47. Jiang, S.-W., Trujillo, M. A., Sakagashira, M., Wilke, R. A., and Eberhardt, N. L. (2000) Biochemistry 39, 3505-3513[CrossRef][Medline] [Order article via Infotrieve]
48. Ishiji, T., Lace, M. J., Parkkinen, S., Anderson, R. D., Haugen, T. H., Cripe, T. P., Xiao, J.-H., Davidson, I., Chambon, P., and Turek, L. P. (1992) EMBO J. 11, 2271-2281[Abstract]
49. Gupta, M. P., Gupta, M., and Zak, R. (1994) J. Biol. Chem. 269, 29677-29687[Abstract/Free Full Text]
50. Shimizu, N., Smith, G., and Izumo, S. (1993) Nucleic Acids Res. 21, 4103-4110[Abstract]
51. Belandia, B., and Parker, M. G. (2000) J. Biol. Chem. 275, 30801-30805[Abstract/Free Full Text]
52. Gupta, M. P., Amin, C. S., Gupta, M., Hay, N., and Zak, R. (1997) Mol. Cell. Biol. 17, 3924-3936[Abstract]
53. Jiang, S.-W., and Eberbardt, N. L. (1996) J. Biol. Chem. 271, 9510-9518[Abstract/Free Full Text]


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