Cloning and Characterization of a Novel Zinc Finger Transcriptional Repressor

We have identified a novel transcriptional repressor, AEBP2, that binds to a regulatory sequence (termed AE-1) located in the proximal promoter region of the aP2 gene that encodes the adipose fatty acid-binding protein. Sequence analysis of AEBP2 cDNA revealed that it encodes a protein containing three Gli-Krüppel (Cys2-His2)-type zinc fingers. Northern blot analysis revealed two transcripts (4.5 and 3.5 kilobases) which were ubiquitously expressed in every mouse tissue examined. In co-transfection assays, AEBP2 repressed transcription from the homologous aP2 promoter containing multiple copies of the AE-1 sequence. Moreover, a chimeric construct encoding a fusion AEBP2 protein with the Gal4 DNA-binding domain was able to repress the transcriptional activity of a heterologous promoter containing the Gal4-binding sequence. The transcriptional repression function of AEBP2 was completely abolished when one of the conserved histidine residues and a flanking serine residue in the middle zinc finger were replaced with an arginine residue. The defective transcriptional repression function of the mutant derivative was due neither to lack of expression nor to a failure to localize to the nucleus. Moreover, both the wild-type and mutant derivative of either the histidine-tagged recombinant AEBP2 proteins or the in vitro translated Gal4-AEBP2 fusion proteins were equally able to bind to the target DNA. These results suggest that a portion of the zinc finger structure may play a direct role in transcriptional repression function, but not in DNA binding. We have identified a novel transcriptional repressor, AEBP2, that binds to a regulatory sequence (termed AE-1) located in the proximal promoter region of the aP2 gene that encodes the adipose fatty acid-binding protein. Sequence analysis of AEBP2 cDNA revealed that it encodes a protein containing three Gli-Krüppel (Cys2-His2)-type zinc fingers. Northern blot analysis revealed two transcripts (4.5 and 3.5 kilobases) which were ubiquitously expressed in every mouse tissue examined. In co-transfection assays, AEBP2 repressed transcription from the homologous aP2 promoter containing multiple copies of the AE-1 sequence. Moreover, a chimeric construct encoding a fusion AEBP2 protein with the Gal4 DNA-binding domain was able to repress the transcriptional activity of a heterologous promoter containing the Gal4-binding sequence. The transcriptional repression function of AEBP2 was completely abolished when one of the conserved histidine residues and a flanking serine residue in the middle zinc finger were replaced with an arginine residue. The defective transcriptional repression function of the mutant derivative was due neither to lack of expression nor to a failure to localize to the nucleus. Moreover, both the wild-type and mutant derivative of either the histidine-tagged recombinant AEBP2 proteins or the in vitro translated Gal4-AEBP2 fusion proteins were equally able to bind to the target DNA. These results suggest that a portion of the zinc finger structure may play a direct role in transcriptional repression function, but not in DNA binding. The adipose P2 (aP2 or422) 1The abbreviations used are: aP2, adipose P2; AE-1, adipocyte enhancer 1; C/EBPα, CCAAT/enhancer-binding protein α; AEBP, adipocyte enhancer-binding protein; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; NLS, nuclear localization signal; CMV, cytomegalovirus; TK, thymidine kinase. gene, which encodes the adipose fatty acid-binding protein, is thought to be an important gene in triglyceride metabolism during adipocyte differentiation (1Cornelius P. MacDougald O.A. Lane M.D. Annu. Rev. Nutr. 1994; 14: 99-129Crossref PubMed Scopus (575) Google Scholar, 2MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (943) Google Scholar, 3Spiegelman B.M. Flier J. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). The abundance of aP2 mRNA is greatly enhanced during adipocyte differentiation (4Hunt C.R. Ro H.-S. Min H.-Y. Dobson D.E. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3786-3790Crossref PubMed Scopus (268) Google Scholar). The AE-1 sequence (nucleotides −159 to −125) in the proximal promoter region of the aP2 gene functions in aP2 gene expression as either a positive or a negative regulatory element. Mutation at the AE-1 site affects its ability to bind specific nuclear factors and diminishes the promoter function of the aP2 gene in adipocytes (5Herrera R. Ro H.-S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar). At least one protein, C/EBPα, binds to the AE-1 sequence and functions as a transcriptional activator foraP2 gene expression during adipocyte differentiation (5Herrera R. Ro H.-S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar, 6Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly T.J. Lane M.D. Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (467) Google Scholar). Other AE-1-binding proteins (termed AEBP) in 3T3 preadipocytes have been implicated as transcriptional repressors in the regulation ofaP2 gene expression (5Herrera R. Ro H.-S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar, 7Distel R.J. Ro H.-S. Rosen B.S. Groves D.L. Spiegelman B.M. Cell. 1987; 49: 835-844Abstract Full Text PDF PubMed Scopus (332) Google Scholar, 8Ro H.-S. Roncari D.A.K. Mol. Cell. Biol. 1991; 11: 2303-2306Crossref PubMed Scopus (21) Google Scholar). To clone a cDNA encoding a protein that interacts at the AE-1 site, we expressed cDNAs from a 3T3-L1 preadipocyte cell library with a Uni-Zap XR vector (Stratagene) and screened for the clones by the Affinity Screening procedure using random concatamers of the AE-1 sequence. Three independent phage plaques were isolated that produce fusion proteins interacting specifically with the AE-1 sequence. Further analysis revealed that one cDNA clone encodes mRNA whose expression is down-regulated during adipocyte differentiation. This cDNA and its encoded protein, AEBP1, has been previously characterized in detail. AEBP1 is a novel carboxypeptidase, and the carboxypeptidase activity of AEBP1 is involved in aP2 repression. AEBP1, by binding to the regulatory AE-1 site, acts as a negative regulator ofaP2 gene expression (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). A new member of the family of AE-1-binding proteins, being reported here, is a zinc finger protein which is able to repress reporter gene expression through both homologous and heterologous promoters. Overexpression of AEBP2 in cells with a reporter construct, which is driven by the aP2promoter containing a multiple copies of the AE-1 sequence, resulted in repression of transcriptional activity. Furthermore, an AEBP2 fusion protein with the DNA-binding domain of the yeast transcriptional activator Gal4 was able to repress the expression of a reporter gene driven by a heterologous promoter with five copies of the Gal4-binding sequence. Significantly, the repression function of AEBP2 was completely abolished, without affecting its DNA binding ability and expression level, when one of the conserved histidine residues in the middle zinc finger motif was mutated. These results indicate that the middle zinc finger motif of AEBP2 is critical for the transcriptional function, and suggest that some of the zinc finger motif may not be involved in DNA binding. To clone a cDNA encoding a protein that interacts at the AE-1 site (nucleotides −159 to −125 of the aP2 gene, Refs. 8Ro H.-S. Roncari D.A.K. Mol. Cell. Biol. 1991; 11: 2303-2306Crossref PubMed Scopus (21) Google Scholar and 9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar) in 3T3-L1 preadipocytes, cDNAs from a 3T3-L1 preadipocytes library were expressed with a Uni-Zap XR vector (Stratagene) and screened for an expression clone by the affinity screening procedure (10Singh H. LeBowitz J.H. Baldwin Jr., A.S. Sharp P.A. Cell. 1988; 52: 415-423Abstract Full Text PDF PubMed Scopus (419) Google Scholar) with random concatamers of the AE-1 sequence as described previously (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). The 303-residue AEBP2 sequence was compared with the non-redundant GenBankTM data base using the National Center for Biotechnology Information program tblastn. Sequences with significant matches around the zinc finger domain include the human GLI protein and other related zinc finger transcription factors (smallest sum probability values less than 10−10) with GenBankTM accession numbers X07385, U60762, U57454,AF026305, U42462, D14827, AB007297, AB007295, AB007298, AB007296,D14828, and U42461. The AEBP2 cDNA was subcloned into the mammalian expression vector pRc/CMV (Invitrogen) to construct the AEBP2 expression plasmid pRc/CMVAEBP2. The Gal4-AEBP2 fusion expression plasmid was constructed by subcloning the AEBP2 open reading frame (from amino acids 7 to 247) into the Gal4 fusion vector pG4 as described (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). For this construction, we utilized another AEBP2 cDNA (clone 20) whose 5′ end corresponds to nucleotide 104 of the full-length AEBP2 cDNA. The AEBP2 coding sequence was subcloned as a SmaI-XmnI fragment into pG4 in both orientations to construct pG4-AEBP2 and pG4-AEBP2(−), respectively. Total RNA from different mouse tissues was prepared with the RNA STAT-60 Solution (TEL-TEST “B,” Inc.) according to the manufacturer's protocol. Twenty micrograms of total RNA from each tissue were loaded on a formaldehyde denaturating 1% agarose gel and blotted onto MSI nylon transfer membrane (Micron Separations Inc.) as described (11Xu L. He G.-P. Li A. Ro H.-S. Nucleic Acids Res. 1994; 22: 646-655Crossref PubMed Scopus (25) Google Scholar). The filter was hybridized in the QuikHyb solution (Stratagene) with 32P-labeled AEBP2 cDNA probe in a hybridization oven (Hybaid) for 3 h at 65 °C. The filter was washed twice with 2 × SSC, 0.1% SDS for 15 min at room temperature and twice with 0.2 × SSC, 0.1% SDS for 30 min at 65 °C before exposure to x-ray film overnight at −70 °C. Recombinant AEBP2 protein was made by utilizing the expression vector pET-16b (Novagen) as described previously (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). EMSA was carried out by incubating the recombinant protein or the in vitro produced Gal4 fusion proteins, which were made by the TNT-coupled wheat germ extract system (Promega), with 0.25 fmol of 32P-end-labeled AE-1 sequence in a buffer containing 100 mm KCl, 10 mm Tris-HCl, pH 7.9, 50 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 5% glycerol, and 200 μm ZnCl2. After 30 min incubation at room temperature, the mixture was loaded on a 4% polyacrylamide nondenaturing gel in 6.75 mm Tris-HCl, pH 7.9, 1 mm EDTA, pH 8.0, 3.3 mm sodium acetate, pH 7.9, and 2.5% glycerol, and electrophoresed at 15 V/cm at 4 °C. Both the gel and the running buffer contained 200 μmZnCl2. For the competition assay, 50 fmol of unlabeled oligonucleotides, either specific AE-1 or nonspecific SP1 and AP3, were added to the reaction prior to the addition of the probe. The transient transfection assay was carried out by the Polybrene procedure (12Kawai S. Nishizawa M. Mol. Cell. Biol. 1984; 4: 1172-1178Crossref PubMed Scopus (329) Google Scholar) as described previously (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). Transfection of NIH 3T3 cells was performed with 2 μg of the reporter plasmid paP2(3AE-1/−120)CAT and 5 μg of the AEBP2 expression plasmid pRc/CMVAEBP2. The transfection was also performed with 5 μg each of the reporter plasmids pGALTKCAT or pTKCAT and the fusion Gal-AEBP2 expression plasmid pG4-AEBP2 or the control plasmid pG4-AEBP2(−). All transfections also included 1 μg of pHermes-lacZ. β-Galactosidase activity was assayed 48 h after transfection to normalize for transfection efficiency, and CAT activity was assayed as described (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). A 3T3-L1 preadipocyte cDNA expression library (Uni-Zap XR vector; Stratagene) was screened with random concatamers of the AE-1 sequence by the affinity screening procedure (10Singh H. LeBowitz J.H. Baldwin Jr., A.S. Sharp P.A. Cell. 1988; 52: 415-423Abstract Full Text PDF PubMed Scopus (419) Google Scholar) as described previously (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). Three independent phage plaques (A1, A2, and A8) produced fusion proteins interacting specifically with the AE-1 sequence. Further screening of the library with the partial cDNA sequence from A2 resulted in the isolation of a longer cDNA, which we named AEBP2 (adipocyte enhancer-binding protein 2, differing from AEBP1 (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar)). Sequence analysis revealed that this cDNA contains an open reading frame of 303 amino acid residues from the first ATG located at nucleotide to a located at nucleotide The in the open reading frame from the first ATG has a of in and of the AEBP2 cDNA produced a protein with a of not Moreover, a protein with a of was with the recombinant AEBP2 protein not on the of the vitro translated and the for the site M. J. Biol. 1989; PubMed Scopus Google the first ATG is the The cDNA has a region with a and A of AEBP2 cDNA is in The protein contains three sequence related to the Gli-Krüppel (Cys2-His2)-type zinc finger All three zinc encoded by the cDNA of AEBP2 the sequence for this of zinc finger Sequence of the zinc of AEBP2 with of other zinc finger proteins containing three zinc finger is in the zinc finger a site for a conserved cell regulatory in the open reading frame The site is by the zinc finger domain and a region with of amino acids that may be a nuclear localization at the site may the function of the and of the protein into the P.A. Cell. 1991; 64: Full Text PDF PubMed Scopus Google Scholar). Northern hybridization was to the expression of AEBP2 in a of mouse transcripts and kilobases) with different of abundance were in the tissues of AEBP2 RNA were in the In to other the contained the of AEBP2 RNA In some tissues and RNA with were also and The of expression of AEBP2 mRNA in different tissues is not AEBP2 transcripts were also in and and in the not The of AEBP2 expression the that AEBP2 may have an important function in mouse We have reported previously that the AE-1 site as a in the regulation ofaP2 gene expression (5Herrera R. Ro H.-S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar, 8Ro H.-S. Roncari D.A.K. Mol. Cell. Biol. 1991; 11: 2303-2306Crossref PubMed Scopus (21) Google Scholar, 9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). The AE-1 sequence binds either the transcriptional activator (5Herrera R. Ro H.-S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar) or the transcriptional AEBP1 (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). AEBP2, as another AE-1-binding protein, may also in this recombinant AEBP2 protein was used to the binding of AEBP2 as described previously (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google Scholar). we that AEBP2 binds to the AE-1 in a The be by an of the unlabeled AE-1 but not by SP1 or by binding The binding reaction for or binding was in the of not without an zinc finger as by one of the conserved histidine residues cDNA was with at to and the were by activity and the histidine and serine residues with an arginine the binding activity not These results suggest that the zinc finger motif is for DNA and that the DNA binding activity may be in the other two fingers. To AEBP2 is able to transcription through with the AE-1 site, we used a reporter construct in which the chloramphenicol gene expression is driven by the aP2promoter containing an AE-1 site (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google with an AEBP2 expression plasmid In transient transfection we have not of AEBP2 on expression of the CAT gene driven by the aP2 promoter to which contains the AE-1 site at −159 to This lack of may be due to a positive at nucleotide −125 to (5Herrera R. Ro H.-S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar). transcriptional activity be by the of binding site of a transcription we have the transcription function of AEBP2 with a reporter containing a multiple copies of the AE-1 sequence. transfection analysis with the CAT reporter plasmid in which the CAT gene is driven by promoter to with three copies of the AE-1 sequence of the that AEBP2 has repression activity. The CAT activity from cells with was compared with the CAT activity in cells with the control plasmid pRc/CMV the in vitro binding assay, we that the middle zinc finger is for binding to the AE-1 sequence. We the in the middle zinc finger has on the repression function of We constructed an expression plasmid that the mutant derivative of AEBP2, and the transcriptional repression function using the reporter plasmid in the repression activity of this mutant of AEBP2 was that the middle zinc finger may be critical for the repression To the transcriptional function of AEBP2, we used a AEBP2 fusion protein with an added DNA-binding of the yeast Gal4 transcription as an and the reporter plasmid which contains five copies of the Gal4-binding sequence of the of the thymidine promoter the CAT gene expression (9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google in the transient transfection After co-transfection with a CAT activity was from the cells with the Gal4-AEBP2 expression plasmid in to the activity in the control cells with the control plasmid pG4-AEBP2(−), in which the AEBP2 sequence is in the in to the Gal4 DNA-binding domain in the CAT activity was when a reporter plasmid a Gal4-binding site, was used 4 and repression by AEBP2 localization to the promoter and the repression activity is specific and is not due to transcriptional or to a nonspecific of the RNA Furthermore, results suggest that the repression function of AEBP2 may be by an repression than by a repression which by competition with a positive in the binding to the DNA in the of the homologous promoter assay, the in the middle zinc finger of AEBP2 resulted in repression activity when Gal4-AEBP2 was using a heterologous promoter with the Gal4-binding site To the wild-type and the mutant derivative of the Gal4-AEBP2 fusion proteins were equally expressed in the the fusion proteins were by with the in both Gal4-AEBP2 and fusion proteins were equally The lack of repression function for the mutant derivative is not due to its failure to localize to the nucleus. of wild-type and mutant derivative of the Gal4-AEBP2 fusion proteins were in the blot analysis of a nuclear isolated from the cells not Moreover, this not the binding of the Gal4-AEBP2 fusion protein to the Gal4 binding both the wild-type and the mutant derivative were equally able to bind to the Gal4 binding sequence in the in vitro binding assay using the in vitro translated fusion proteins These results indicate that the middle zinc finger motif of AEBP2 is important for its repression In this we have characterized a novel transcriptional (termed containing three copies of the Gli-Krüppel (Cys2-His2)-type zinc finger and that at least one of the zinc is important for the repression The zinc finger which was identified as DNA binding structure in the RNA transcription P. PubMed Scopus Google Scholar, J. A.D. A. J. 4: PubMed Scopus Google is one of the transcription finger proteins function as either or and of proteins, in addition to the zinc finger motif whose transcriptional role is not other of transcriptional have been characterized by the of or amino R. 1989; PubMed Scopus Google Scholar). For the domain in the protein has been to be for its repression function J. Nature. PubMed Scopus Google Scholar, J. 1991; PubMed Scopus Google Scholar, P. J. M. Genes Dev. 1991; PubMed Scopus Google Scholar). The an conserved located in the of than of zinc finger proteins, also has been characterized as a repression motif Friedman H. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, R. A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). Other which have been implicated as repression include a acid in the transcription S. Mol. Cell. Biol. PubMed Scopus Google the from the first of the of the proteins to a J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google the domain in the He Y. 1996; Google a domain the amino acids and containing in the protein J. M. H. Mol. Cell. Biol. 1996; PubMed Scopus Google and a region in the transcription Y. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). A repression domain was also the zinc finger region of J. Y. Nucleic Acids Res. 1995; PubMed Scopus Google Scholar). other zinc finger transcription AEBP2 not of the repression described the function of AEBP2 was abolished, without affecting its DNA binding when the middle zinc finger was by one of the conserved histidine a zinc finger motif is critical for a transcription function has been a domain which the DNA-binding domain has been in a zinc finger motif J. Y. Nucleic Acids Res. 1995; PubMed Scopus Google Scholar). 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Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). AEBP2 may gene expression to one of transcription factors that the transcription through an repression AEBP2 may a in the regulation of aP2 gene The in the middle zinc finger may an of the of the protein to with either the transcription or a that of the zinc finger of transcription factors involved in the transcriptional repression of gene expression J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and cell affecting the expression of the Cell. Full Text PDF PubMed Scopus Google P. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). At least one positive Refs. R. Ro H.-S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar and 6Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly T.J. Lane M.D. Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (467) Google Scholar) and two negative 9He G.P. Muise A. Li A.W. Ro H.-S. Nature. 1995; 378: 92-96Crossref PubMed Scopus (137) Google and transcription factors involved in the regulation of aP2 gene expression through the AE-1 In to AEBP2, which is expressed both in preadipocytes and He and H.-S. is expressed in the of adipocyte differentiation (1Cornelius P. MacDougald O.A. Lane M.D. Annu. Rev. Nutr. 1994; 14: 99-129Crossref PubMed Scopus (575) Google Scholar, 2MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (943) Google Scholar, 3Spiegelman B.M. Flier J. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar) and AEBP1 expression is abolished in the of adipocyte differentiation. A. and H.-S. in specific may to the in the of gene by either or at different of In the and of the transcriptional function, and DNA-binding of the novel transcription AEBP2 have revealed that it is a new zinc finger transcriptional that is able to bind specifically to the AE-1 site located in the proximal promoter region of gene, and that the repression function, but not the DNA binding an middle zinc finger of AEBP2 mRNA expressed in mouse AEBP2 may as a transcriptional regulator transcripts were in both and We and R. A. for and on the A. Li for in the screening and of the J. for