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• W2052808935 abstract "We have isolated two novel Krüppel-like zinc finger proteins containing the evolutionarily conserved Krüppel-associated box (KRAB), KRAZ1 and KRAZ2, and demonstrated that they repress transcription when heterologously targeted to DNA. Their repression activity appeared to be mediated by the putative corepressor KAP-1 (KRAB-associated protein-1), because KRAZ1/2 bind to KAP-1, but KRAB mutants of KRAZ1/2 that are unable to interact with KAP-1 lack repression activity, and KAP-1 has intrinsic repressor activity and potentiates KRAZ1/2-mediated repression. We dissected the KAP-1 protein into a KRAB-interacting domain and a region necessary for repression. Using a mammalian two-hybrid assay, we further demonstrated that KAP-1 deletions lacking repression activity fused to the VP16 transactivation domain strongly activated transcription when coexpressed with KRAZ1. In contrast, VP16-KAP-1 fusions retaining repression activity resulted in repression. These results provide the first evidence that KAP-1 functionally interacts with KRAB in mammalian cells and seems to exert repressor activity in the DNA-bound KRAB-KAP-1 complex, and they further support the hypothesis that KAP-1 functions as a corepressor for the large class of KRAB-containing zinc finger proteins. We have isolated two novel Krüppel-like zinc finger proteins containing the evolutionarily conserved Krüppel-associated box (KRAB), KRAZ1 and KRAZ2, and demonstrated that they repress transcription when heterologously targeted to DNA. Their repression activity appeared to be mediated by the putative corepressor KAP-1 (KRAB-associated protein-1), because KRAZ1/2 bind to KAP-1, but KRAB mutants of KRAZ1/2 that are unable to interact with KAP-1 lack repression activity, and KAP-1 has intrinsic repressor activity and potentiates KRAZ1/2-mediated repression. We dissected the KAP-1 protein into a KRAB-interacting domain and a region necessary for repression. Using a mammalian two-hybrid assay, we further demonstrated that KAP-1 deletions lacking repression activity fused to the VP16 transactivation domain strongly activated transcription when coexpressed with KRAZ1. In contrast, VP16-KAP-1 fusions retaining repression activity resulted in repression. These results provide the first evidence that KAP-1 functionally interacts with KRAB in mammalian cells and seems to exert repressor activity in the DNA-bound KRAB-KAP-1 complex, and they further support the hypothesis that KAP-1 functions as a corepressor for the large class of KRAB-containing zinc finger proteins. A large number of studies on transcriptional factors has revealed that functional domains of many transcriptional factors are modular. They can be structurally and functionally separated into DNA-binding domains and effector (activation or repression) domains. DNA-binding domains can be classified according to common structural motifs, which are well conserved throughout evolution (1Pabo C.O. Sauer R.T. Annu. Rev. Biochem. 1992; 61: 1053-1095Crossref PubMed Scopus (1221) Google Scholar). One of such motifs is the C2H2-type zinc finger repeat that has been estimated to be present in several hundreds of genes, thus being identified as a major family of DNA-binding proteins (2Bellefroid E.J. Lecocq P.J. Benhida A. Poncelet D.A. Belayew A. Martial J.A. DNA. 1989; 8: 377-387Crossref PubMed Scopus (195) Google Scholar, 3Chowdhury K. Deutsch U. Gruss P. Cell. 1987; 48: 771-778Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 4Klug A. Schwabe J.W. FASEB J. 1995; 9: 597-604Crossref PubMed Scopus (533) Google Scholar). The vast majority of the C2H2-type zinc finger proteins (ZFPs) 1The abbreviations used are: ZFP, zinc finger protein; KRAB, Krüppel-associated box; PCR, polymerase chain reaction; nt, nucleotide(s); DBD, DNA-binding domain; HA, hemagglutinin; NLS, nuclear localization signal; 6MT, six repeats of the Myc epitope; AD, activation domain; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; TIF, transcriptional intermediary factor. 1The abbreviations used are: ZFP, zinc finger protein; KRAB, Krüppel-associated box; PCR, polymerase chain reaction; nt, nucleotide(s); DBD, DNA-binding domain; HA, hemagglutinin; NLS, nuclear localization signal; 6MT, six repeats of the Myc epitope; AD, activation domain; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; TIF, transcriptional intermediary factor. are classified as Krüppel-like on the basis of the fact that they share a highly conserved stretch of seven amino acids (the H/C link) connecting multiple tandem repeats of the zinc finger domain (5Schuh R. Aicher W. Gaul U. Cote S. Preiss A. Maier D. Seifert E. Nauber U. Schroder C. Kemler R. Cell. 1986; 47: 1025-1032Abstract Full Text PDF PubMed Scopus (324) Google Scholar). In contrast to the DNA-binding domains, analysis of the structure or targets of effector domains has been hampered by the lack of amino acid sequence homology or structural motifs common among them. Nevertheless, some effector domains can be loosely categorized according to the primary amino acid content. It has been reported that activation domains are often rich in acidic amino acids and/or proline and glutamine (6Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2186) Google Scholar, 7Johnson P.F. McKnight S.L. Annu. Rev. Biochem. 1989; 58: 799-839Crossref PubMed Scopus (826) Google Scholar). Less is known about repression domains; however, some of them are rich in alanine, proline, or charged amino acids (8Hanna-rose W. Hansen U. Trends Genet. 1996; 12: 229-234Abstract Full Text PDF PubMed Scopus (280) Google Scholar).The Krüppel-associated box (KRAB) was first identified as an evolutionarily conserved motif consisting of 75 amino acids that has been assumed to be present in about one-third of the Krüppel-like ZFPs (9Bellefroid E.J. Poncelet D.A. Lecocq P.J. Revelant O. Martial J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3608-3612Crossref PubMed Scopus (344) Google Scholar). It is found almost exclusively in the N terminus of the Krüppel-like ZFPs that contain zinc finger domains in their C terminus, and is subdivided into KRAB-A and B domain (9Bellefroid E.J. Poncelet D.A. Lecocq P.J. Revelant O. Martial J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3608-3612Crossref PubMed Scopus (344) Google Scholar). Recently several KRAB domains have been shown to act as potent repressors when heterologously tethered to the promoter (10Margolin J.F. Friedman J.R. Meyer W.K. Vissing H. Thiesen H.J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (508) Google Scholar, 11Witzgall R. O'Leary E. Leaf A. Onaldi D. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4514-4518Crossref PubMed Scopus (311) Google Scholar, 12Pengue G. Calabro V. Bartoli P.C. Pagliuca A. Lania L. Nucleic Acids Res. 1994; 22: 2908-2914Crossref PubMed Scopus (119) Google Scholar, 13Vissing H. Meyer W.K. Aagaard L. Tommerup N. Thiesen H.J. FEBS Lett. 1995; 369: 153-157Crossref PubMed Scopus (126) Google Scholar). The KRAB-A domain present in every KRAB domain, but not the B domain, is responsible for such transcriptional repression (10Margolin J.F. Friedman J.R. Meyer W.K. Vissing H. Thiesen H.J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (508) Google Scholar, 11Witzgall R. O'Leary E. Leaf A. Onaldi D. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4514-4518Crossref PubMed Scopus (311) Google Scholar). The KRAB domain is rich in charged amino acids and predicted to fold into two amphipathic α helices, which might thus serve as a protein-protein interaction surface (9Bellefroid E.J. Poncelet D.A. Lecocq P.J. Revelant O. Martial J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3608-3612Crossref PubMed Scopus (344) Google Scholar). Unlike other alanine- or proline-rich repression domains, the KRAB domain is highly conserved in a subfamily of the Krüppel-like ZFPs sharing a common amino acid sequence and a predicted secondary structure, suggesting that the KRAB domains in many different ZFPs may share a common cellular target(s) (10Margolin J.F. Friedman J.R. Meyer W.K. Vissing H. Thiesen H.J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (508) Google Scholar).This prediction was recently substantiated by the isolation and characterization of a novel protein that interacts with KRAB, KRAB-associated protein-1 (KAP-1) (14Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (526) Google Scholar). KAP-1 was also identified as TIF1β or KRIP-1 (15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 16Kim S.S. Chen Y.M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (243) Google Scholar) (hereafter referred to as KAP-1) and found to interact with several different KRAB domains but not with KRAB-A mutants deficient in repression, to enhance KRAB-mediated repression and to repress transcription when directly targeted to DNA (14Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (526) Google Scholar, 15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 16Kim S.S. Chen Y.M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (243) Google Scholar). KAP-1 is a member of the RBCC subfamily of the RING finger proteins that contain the RING finger motif defined as C3HC4 finger and followed by one or two B box-type fingers and a putative coiled-coil domain (14Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (526) Google Scholar, 15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 16Kim S.S. Chen Y.M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (243) Google Scholar, 17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar). Although the functions of these domains are so far unknown, they have been thought to be involved in nucleic acid-protein and/or protein-protein interactions (18Freemont P.S. Ann. N. Y. Acad. Sci. 1993; 684: 174-192Crossref PubMed Scopus (378) Google Scholar, 19Saurin A.J. Borden K.L. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-214Abstract Full Text PDF PubMed Scopus (609) Google Scholar). In the C-terminal portion, KAP-1 also contains a C4HC3 zinc finger called PHD finger followed by a domain similar to the bromodomain (14Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (526) Google Scholar, 15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 16Kim S.S. Chen Y.M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (243) Google Scholar, 17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar). The PHD finger is often found in concert with the bromodomain and is also present in a number of proteins implicated in chromatin-mediated transcriptional regulation, thereby suggesting that it might be involved in interactions with chromatin components (20Aasland R. Gibson T.J. Stewart A.F. Trends Biochem. Sci. 1995; 20: 56-59Abstract Full Text PDF PubMed Scopus (747) Google Scholar). The bromodomain has also been identified in several proteins that regulate transcription in the context of large multiprotein complexes and/or through interaction with, or modification of, chromatin (21Haynes S.R. Dollard C. Winston F. Beck S. Trowsdale J. Dawid I.B. Nucleic Acids Res. 1992; 20: 2603Crossref PubMed Scopus (319) Google Scholar, 22Tamkun J.W. Deuring R. Scott M.P. Kissinger M. Pattatucci A.M. Kaufman T.C. Kennison J.A. Cell. 1992; 68: 561-572Abstract Full Text PDF PubMed Scopus (766) Google Scholar, 23Jeanmougin F. Wurtz J.M. Le Douarin B. Chambon P. Losson R. Trends Biochem. Sci. 1997; 22: 151-153Abstract Full Text PDF PubMed Scopus (228) Google Scholar). Thus, it has been proposed to mediate the protein-protein interactions influencing the assembly and/or activity of such complexes, or to be involved in interactions with chromatin (21Haynes S.R. Dollard C. Winston F. Beck S. Trowsdale J. Dawid I.B. Nucleic Acids Res. 1992; 20: 2603Crossref PubMed Scopus (319) Google Scholar).A highly related protein to KAP-1 in the overall structure including the conserved domains is TIF1α, which was originally identified as a putative coactivator of the nuclear receptor (24LeDouarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (574) Google Scholar). However, it was subsequently reported to interact with KRAB and repress transcription when directly recruited to the promoter (15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar). Therefore, KAP-1 and TIF1α have been postulated to constitute a new family of transcriptional intermediary factors and function as mediators of transcriptional repression for the large class of KRAB-containing ZFPs (17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar). However, there is no compelling evidence directly indicating that KAP-1 or TIF1α indeed interacts with KRAB in mammalian cells and exerts the repression function in a physiological context of the resultant DNA-bound KRAB-KAP-1/TIF1α complex.In this report, we describe the isolation and characterization of two novel KRAB-ZFPs, KRAZ1 and KRAZ2, which repress transcription and interact with KAP-1. We further investigated the functional interaction of KRAZ1/2 with KAP-1 using a mammalian two-hybrid assay based on the detailed molecular characterization of the functional domains in KAP-1. Our results present the first evidence that KAP-1 functionally interacts with KRAB in mammalian cells and that KAP-1 seems to function as a corepressor of KRAB-ZFPs in the complex with the DNA-bound KRAB domains.DISCUSSIONIt has recently been thought to be a universal molecular mechanism that DNA-binding transcriptional repressors exert their repression activity by recruitment of corepressors in many systems from yeast andDrosophila to mammals (14Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (526) Google Scholar). Our study, together with the previous reports (14Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (526) Google Scholar, 15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 16Kim S.S. Chen Y.M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (243) Google Scholar), revealed that KAP-1 functions as a universal corepressor for the large class of KRAB-ZFPs, because it fulfills the criteria for corepressors: first, KAP-1 interacts in mammalian cells as well as in vitro with several different KRAB domains including novel ones we isolated, but not with KRAB mutants defective in repression. Second, KAP-1 has intrinsic repressor activity, because KAP-1 efficiently represses transcription when targeted to the promoter. Third, overexpression of KAP-1 potentiates KRAB-mediated repression in a manner dependent on the interaction with KRAB. Fourth, KAP-1 seems to exert the repressor activity in a physiological context of the DNA-bound KRAB-KAP-1 complex. These findings further support the model that KRAB-ZFPs bind to cis-regulatory sequences by their putative DNA-binding domains, zinc fingers, recruit the corepressors including KAP-1 to the promoter by their KRAB domains, and thereby repress transcription (14Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (526) Google Scholar, 15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 16Kim S.S. Chen Y.M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (243) Google Scholar).We demonstrated that amino acids 239–421 of KAP-1, which almost correspond to the coiled-coil region, are essential and sufficient for the interaction with KRAB in vitro, but the RING-B1/B2 region is dispensable (Fig. 6). However, involvement of the RING-B1/B2 region in the in vivo interaction with KRAB seems to be complicated but important. The RING finger is essentially dispensablein vivo and also in vitro, but the B1/B2 finger is required only in vivo but not in vitro. These results are in agreement with the report by Moosmann et al.(15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar) suggesting that the coiled-coil region of KAP-1/TIF1β or TIF1α is essential but not sufficient for the interaction with KRAB in a yeast two-hybrid assay. They clearly indicated that the B1/B2 finger of KAP-1/TIF1β is required for the functional interaction, whereas the RING finger is dispensable (15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar). Nevertheless, the RING finger is suggested to contribute to the efficient interaction with KRAB in vivo by the observation in our mammalian two-hybrid assay that activation by VP16-KAP1 145–421 is consistently lower than that of VP16-KAP1 N421 (Fig. 8 A, compare lanes 4 and14). Moreover, the competition experiment revealed that KAP-1 145C lacking the RING finger could partially compete with VP16-KAP-1 N487 for binding to KRAZ1, and KAP-1 239C, 145–421, or 239–421 could not (data not shown). It might thus also be possible that a third molecule(s) is required for the stable interaction between KAP-1 and KRAB in vivo and that the RING-B1/B2 finger region of KAP-1 is necessary for the interaction with such a third molecule(s).Amino acids 396–562 of KAP-1, which reside between the coiled-coil and the PHD finger, are considered to be a core domain essential for repression but not sufficient for full repression. Several explanations can be made why the N-terminal portion (the RBCC domain) or the most C-terminal portion (the bromo-like domain) should be required for full repression in addition to the core domain. First, the N- or C-terminal region may contribute to the functional conformation or stability of the KAP-1 protein. Second, KAP-1 might possess two or more independent repression modules that are composed of the N- or C-terminal region together with the core domain. The core domain may thus be shared with both repression modules and would be essential, whereas the N- or C-terminal region would be dispensable as long as the other module remains functional. Third, KAP-1 can function only within a multiprotein complex, and the N or C terminus is required for efficient incorporation of KAP-1 into such a complex through specific protein-protein interaction that is the possible function of these domains, or more simply, there might be a definite threshold of molecular mass necessary for proper formation of such a multiprotein complex.Several hypotheses have been proposed for the mechanism(s) underlying KRAB/KAP-1-mediated repression (15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar, 36Pengue G. Lania L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1015-1020Crossref PubMed Scopus (52) Google Scholar). One is the reorganization of chromatin into a repressive state by heterochromatin formation, because in the yeast two-hybrid assays, both KAP-1 and TIF1α were found to be associated with mHP1α and mMOD1, which are mouse homologs of Drosophila heterochromatin protein 1 that repress transcription by the formation of heterochromatin or a similar structure (17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar). However, we observed that KAP-1 N562 lacking the putative mHP1α/mMOD1-interacting domain (amino acids 571–579) (17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar) could repress transcription more effectively (17.8-fold) than KAP-1 554C containing this site (7.7-fold) (Figs. 5 and 7 A), in agreement with the finding that a mutation of TIF1α within the HP1-interacting domain that disrupts the interaction in yeast did not affect repression activity in transfected mammalian cells (17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar). Thus, the formation of repressed chromatin by direct interaction with mHP1α and/or mMOD1 does not seem to be a principle mechanism of KAP-1/TIF1β- and TIF1α-mediated repression, although lack of interaction with mHP1α/mMOD1 in KAP-1 N562 remains to be confirmed.At present, the most favorable model for KRAB/KAP-1-mediated repression is active repression (8Hanna-rose W. Hansen U. Trends Genet. 1996; 12: 229-234Abstract Full Text PDF PubMed Scopus (280) Google Scholar), in which KRAB or KAP-1 interacts in an inhibitory manner with specific proteins of the basal transcription machinery, thereby interfering with the assembly and/or function of them (15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 36Pengue G. Lania L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1015-1020Crossref PubMed Scopus (52) Google Scholar). Several lines of evidence support the active repression model rather than the formation of repressive chromatin (15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar, 36Pengue G. Lania L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1015-1020Crossref PubMed Scopus (52) Google Scholar). First, KRAB/KAP-1-mediated repression is quite efficient even in short term transient transfection assays as in this study, as well as in anin vitro transcription experiment (36Pengue G. Lania L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1015-1020Crossref PubMed Scopus (52) Google Scholar), in which the bulk of the reporter DNA templates would not be fully assembled into chromatin. Second, it was recently demonstrated that KRAB is able to repress transcription by RNA polymerase II and III, but neither by RNA polymerase I nor T7 RNA polymerase in mammalian cells, suggesting that KRAB exerts its repression activity by interfering with some component(s) for RNA polymerase II and III transcription rather than by altering the chromatin structure into a repressive state (37Moosmann P. Georgiev O. Thiesen H.J. Hagmann M. Schaffner W. Biol. Chem. 1997; 378: 669-677Crossref PubMed Scopus (72) Google Scholar). There are two potential molecular mechanisms proposed for active repression, direct and quenching repression (8Hanna-rose W. Hansen U. Trends Genet. 1996; 12: 229-234Abstract Full Text PDF PubMed Scopus (280) Google Scholar). For KRAB/KAP-1-mediated repression, direct repression seems to be more likely than quenching repression, because KRAB has been demonstrated to repress transcription from a number of different polymerase II-dependent promoters (12Pengue G. Calabro V. Bartoli P.C. Pagliuca A. Lania L. Nucleic Acids Res. 1994; 22: 2908-2914Crossref PubMed Scopus (119) Google Scholar, 15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar) and suppress the activating function of various transcriptional activators (36Pengue G. Lania L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1015-1020Crossref PubMed Scopus (52) Google Scholar), and moreover, KRAB is able to repress basal promoter activity in addition to activated transcription in anin vitro transcription experiment (36Pengue G. Lania L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1015-1020Crossref PubMed Scopus (52) Google Scholar).The active repression mechanism has been considered to involve specific inhibitory protein-protein interactions; however, it is also possible that active repression is accomplished without direct interaction with any components of the transcription machinery, but rather by modification of them. With regard to this possibility, the recent report by Fraser et al. (38Fraser R.A. Heard D.J. Adam S. Lavigne A.C. Le Douarin B. Tora L. Losson R. Rochette-Egly C. Chambon P. J. Biol. Chem. 1998; 273: 16199-16204Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) seems to be instructive. They showed that TIF1α possesses intrinsic protein kinase activity responsible for autophosphorylation as well as for phosphorylation of TFIIEα, TAFII28, and TAFII55. Although functional involvement of such phosphorylation in transcriptional repression and whether KAP-1/TIF1β also has similar activity should be further studied, it is intriguing that KRAB/KAP-1-mediated repression can be regulated through phosphorylation of specific components in the basal transcription complex. It should also be noted that active direct repression and repression by chromatin reorganization are not mutually exclusive (15Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar). The possible existence of multiple repression modules in KAP-1 (this study) and TIF1α (17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar) might imply repression through both mechanisms.It was recently proposed that TIF1α and KAP-1/TIF1β constitute a new family of transcriptional intermediary factors (TIFs) that might play a dual role in the control of transcription at the chromatin level (17Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (465) Google Scholar), because TIF1α was originally identified as a protein enhancing transcription by the nuclear receptor in yeast (24LeDouarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (574) Google Scholar), and KAP-1/TIF1β was recently reported to activate transcription by glucocorticoid receptor and C/EBPβ (39Chang C.J. Chen Y.L. Lee S.C. Mol. Cell. Biol. 1998; 18: 5880-5887Crossref PubMed Google Scholar), also because they share common properties not only to repress transcription when targeted to the promoter but also to interact with mHP1α/mMOD1 and with the KRAB repressor domains. Therefore, it seems possible that TIFs might function as corepressors or coactivators in a way dependent on the chromatin status, the promoter context, or the combination of certain types of transcription factors that bring them to the promoter.It is clearly necessary to elucidate the mechanism(s) and identify the molecular target(s) in KRAB/KAP-1-mediated repression. In addition, it is also important to investigate the biological significance of KRAB/KAP-1-mediated repression. In the mammalian two-hybrid assay, we clearly demonstrated that repression of transcription by GAL4-KRAB can be converted to strong activation when coexpressed with the VP16-fused KAP1 deletions that contain the KRAB-interacting domain but no repression activity, suggesting that these VP16-KAP1 deletions would function as dominant negative mutants with regard to the physiological function of KRAB-ZFPs and/or KAP-1. Therefore, functional investigation using these deletions as a molecular probe in mammalian cells as well as in transgenic animals will facilitate the elucidation of the physiological importance of transcriptional repression by the large class of KRAB-ZFPs and their corepressor KAP-1. A large number of studies on transcriptional factors has revealed that functional domains of many transcriptional factors are modular. They can b" @default.
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