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W1991293300 abstract "Sp1-like proteins are characterized by three conserved C-terminal zinc finger motifs that bind GC-rich sequences found in promoters of numerous genes essential for mammalian cell homeostasis. These proteins behave as transcriptional activators or repressors. Although significant information has been reported on the molecular mechanisms by which Sp1-like activators function, relatively little is known about mechanisms for repressor proteins. Here we report the functional characterization of BTEB3, a ubiquitously expressed Sp1-like transcriptional repressor. GAL4 assays show that the N terminus of BTEB3 contains regions that can act as direct repressor domains. Immunoprecipitation assays reveal that BTEB3 interacts with the co-repressor mSin3A and the histone deacetylase protein HDAC-1. Gel shift assays demonstrate that BTEB3 specifically binds the BTE site, a well characterized GC-rich DNA element, with an affinity similar to that of Sp1. Reporter and gel shift assays in Chinese hamster ovary cells show that BTEB3 can also mediate repression by competing with Sp1 for BTE binding. Thus, the characterization of this protein expands the repertoire of BTEB-like members of the Sp1 family involved in transcriptional repression. Furthermore, our results suggest a mechanism of repression for BTEB3 involving direct repression by the N terminus via interaction with mSin3A and HDAC-1 and competition with Sp1 via the DNA-binding domain. Sp1-like proteins are characterized by three conserved C-terminal zinc finger motifs that bind GC-rich sequences found in promoters of numerous genes essential for mammalian cell homeostasis. These proteins behave as transcriptional activators or repressors. Although significant information has been reported on the molecular mechanisms by which Sp1-like activators function, relatively little is known about mechanisms for repressor proteins. Here we report the functional characterization of BTEB3, a ubiquitously expressed Sp1-like transcriptional repressor. GAL4 assays show that the N terminus of BTEB3 contains regions that can act as direct repressor domains. Immunoprecipitation assays reveal that BTEB3 interacts with the co-repressor mSin3A and the histone deacetylase protein HDAC-1. Gel shift assays demonstrate that BTEB3 specifically binds the BTE site, a well characterized GC-rich DNA element, with an affinity similar to that of Sp1. Reporter and gel shift assays in Chinese hamster ovary cells show that BTEB3 can also mediate repression by competing with Sp1 for BTE binding. Thus, the characterization of this protein expands the repertoire of BTEB-like members of the Sp1 family involved in transcriptional repression. Furthermore, our results suggest a mechanism of repression for BTEB3 involving direct repression by the N terminus via interaction with mSin3A and HDAC-1 and competition with Sp1 via the DNA-binding domain. basic transcription element-binding protein Chinese hamster ovary glutathione S-transferase zinc finger transforming growth factor β-inducible early response gene basic transcription element trichostatin A histone deacetylase-1 full-length amino acid(s) polyacrylamide gel electrophoresis DNA binding domain Kruppel-like factor Sp1-like proteins, defined by the presence of three highly homologous C-terminal zinc finger motifs and variant N-terminal domains, are emerging as important regulators of cell homeostasis. Promoters containing Sp1-like sites are essential for the expression of numerous genes necessary for cell cycle progression (1Muller C. Yang R. Beck-von-Peccoz L. Idos G. Verbeek W. Koeffler H.P. J. Biol. Chem. 1999; 274: 11220-11228Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 2Li J.M. Nichols M.A. Chandrasekharan S. Xiong Y. Wang X. J. Biol. Chem. 1995; 270: 26750-26753Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 3Prowse D.M. Bolgan L. Molnar A. Dotto G.P. J. Biol. Chem. 1997; 272: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), DNA synthesis (4Zhao L. Chang L.S. J. Biol. Chem. 1997; 272: 4869-4882Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and other cell processes (5Birnbaum M.J. Wright K.L. van Wijnen A.J. Ramsey-Ewing A.L. Bourke M.T. Last T.J. Aziz F. Frenkel B. Rao B.R. Aronin N. Biochemistry. 1995; 34: 7648-7658Crossref PubMed Scopus (35) Google Scholar, 6Lin S.Y. Black A.R. Kostic D. Pajovic S. Hoover C.N. Azizkhan J.C. Mol. Cell. Biol. 1996; 16: 1668-1675Crossref PubMed Scopus (252) Google Scholar, 7Zwicker J. Liu N. Engeland K. Lucibello F.C. Muller R. Science. 1996; 271: 1595-1597Crossref PubMed Scopus (147) Google Scholar, 8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and studies have shown that certain Sp1-like proteins induce apoptosis (9Tachibana I. Imoto M. Adjei P. Gores G.J. Subramaniam M. Spelsberg T. Cook T. Urrutia R. J. Clin. Invest. 1997; 99: 2365-2374Crossref PubMed Scopus (192) Google Scholar), cell growth inhibition (10Cook T. Gebelein B. Mesa K. Mladek A. Urrutia R. J. Biol. Chem. 1998; 273: 25929-25936Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 12Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar), differentiation (13Billon N. Carlisi D. Datto M.B. van Grunsven L.A. Watt A. Wang X.F. Rudkin B.B. Oncogene. 1999; 18: 2872-2882Crossref PubMed Scopus (122) Google Scholar, 14Denver R.J. Ouellet L. Furling D. Kobayashi A. Fujii-Kuriyama Y. Puymirat J. J. Biol. Chem. 1999; 274: 23128-23134Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), and carcinogenesis (15El Rouby S. Newcomb E.W. Oncogene. 1996; 13: 2623-2630PubMed Google Scholar). In addition, the disruption of some Sp1-like genes in mice shows that these proteins are critical for normal development (12Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar, 16Marin M. Karism A. Visser P. Grosveld F. Philipsen S. Cell. 1997; 89: 619-628Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 17Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar, 18Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar). Thus, understanding how Sp1-like proteins bind DNA and regulate transcription is important to uncover the molecular mechanisms underlying a large number of cellular events.The existence of at least 17 different Sp1-like proteins offers a significant challenge for understanding how individual members regulate gene expression in a tissue-, cell-, and promoter-specific manner. One mechanism leading to specificity among Sp1-like proteins is a differential pattern of expression. For instance, Sp1, TIEG2, and BTEB11 are ubiquitously expressed, whereas the KLF proteins are restricted to certain tissues. Specificity among Sp1-like proteins is also dictated by recognition of DNA. For example, the Sp proteins preferentially bind GC sites (19Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar, 20Sogawa K. Imataka H. Yamasaki Y. Kusume H. Abe H. Fujii-Kuriyama Y. Nucleic Acids Res. 1993; 21: 1527-1532Crossref PubMed Scopus (179) Google Scholar) whereas the KLF subgroup prefers the CA site (21Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar, 22Matsumoto N. Laub F. Aldabe R. Zhang W. Ramirez F. Yoshida T. Terada M. J. Biol. Chem. 1998; 273: 28229-28237Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 23Miller I.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (646) Google Scholar). Interestingly, co-expressed Sp1-like proteins exhibiting similar binding specificity, such as Sp1 and Sp3, but often display opposite transcriptional regulatory properties (24Birnbaum M.J. van Wijnen A.J. Odgren P.R. Last T.J. Suske G. Stein G.S. Stein J.L. Biochemistry. 1995; 34: 16503-16508Crossref PubMed Scopus (177) Google Scholar, 25Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 270: 20-28Abstract Full Text Full Text PDF Scopus (96) Google Scholar). Thus, activators and repressors that bind to the same sequence may have evolved to turn on and off a discrete set of promoters by competing for this site. In this regard, emerging evidence indicates that Sp1-like proteins are able to compete for DNA binding, such as has been reported for BTEB1/Sp1/GKLF (8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 26Sogawa K. Kikuchi Y. Imataka H. Fujii-Kuriyama Y. J. Biochem. 1993; 114: 605-609Crossref PubMed Scopus (49) Google Scholar) and BKLF/EKLF (27Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Together, the results of these mechanistic studies on competition between Sp1-like proteins validate many of the predictions derived from sequence homology data, suggesting that these proteins regulate similar sequences in vivo.The molecular mechanisms behind the function of the Sp1-like activators have been extensively reported in the literature. For example, both Sp1 and EKLF have been shown to activate transcription by interacting with co-activators (13Billon N. Carlisi D. Datto M.B. van Grunsven L.A. Watt A. Wang X.F. Rudkin B.B. Oncogene. 1999; 18: 2872-2882Crossref PubMed Scopus (122) Google Scholar, 28Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (978) Google Scholar, 29Ryu S. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7137-7142Crossref PubMed Scopus (37) Google Scholar, 30Ryu S. Zhou S. Ladurner A.G. Tjian R. Nature. 1999; 397: 446-450Crossref PubMed Scopus (298) Google Scholar, 31Perkins A. Int. J. Biochem. Cell Biol. 1999; 31: 1175-1192Crossref PubMed Scopus (56) Google Scholar, 32Zhang W. Bieke J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar). In contrast, the molecular mechanisms involved in the function of Sp1-like repressor proteins are less defined. The Sp1-like proteins involved in transcriptional repression include the TIEG proteins (9Tachibana I. Imoto M. Adjei P. Gores G.J. Subramaniam M. Spelsberg T. Cook T. Urrutia R. J. Clin. Invest. 1997; 99: 2365-2374Crossref PubMed Scopus (192) Google Scholar, 10Cook T. Gebelein B. Mesa K. Mladek A. Urrutia R. J. Biol. Chem. 1998; 273: 25929-25936Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Cook T. Gebelein B. Belal M. Mesa K. Urrutia R. J. Biol. Chem. 1999; 274: 29500-29504Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), Sp3 (24Birnbaum M.J. van Wijnen A.J. Odgren P.R. Last T.J. Suske G. Stein G.S. Stein J.L. Biochemistry. 1995; 34: 16503-16508Crossref PubMed Scopus (177) Google Scholar, 25Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 270: 20-28Abstract Full Text Full Text PDF Scopus (96) Google Scholar), BKLF and BKLF2/KLF8 (21Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar, 34van Vliet J. Turner J. Crossley M. Nucleic Acids Res. 2000; 28: 1955-1962Crossref PubMed Scopus (129) Google Scholar), GKLF (8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and Ap2-rep (35Imhof A. Schuierer M. Werner O. Moser M. Roth C. Bauer R. Buettner R. Mol. Cell. Biol. 1999; 19: 194-204Crossref PubMed Google Scholar). However, only BKLF and a closely related protein, BKLF2/KLF8, have been shown to associate with co-repressors (34van Vliet J. Turner J. Crossley M. Nucleic Acids Res. 2000; 28: 1955-1962Crossref PubMed Scopus (129) Google Scholar, 36Turner J. Crossley M. EMBO J. 1998; 17: 5129-5140Crossref PubMed Scopus (277) Google Scholar). Because it is likely that these proteins bind similar sequences, an important question that remains is whether the mechanism of repression is similar between all Sp1-like repressors or whether Sp1-like subfamilies are defined by distinct mechanisms of repression.In this study, we have pursued this question by characterizing the human transcriptional repressor BTEB3, a novel Sp1-like protein that is significantly related to BTEB1. A partial sequence of this protein was recently published with the name of NSLP1 from our laboratory (37Cook T. Gebelein B. Urrutia R. Ann. N. Y. Acad. Sci. 1999; 880: 94-102Crossref PubMed Scopus (120) Google Scholar), but in light of its sequence similarity and functional conservation with BTEB1, we have renamed it BTEB3. This study focuses on the characterization of BTEB3 as a transcriptional repressor with two mechanisms of action: competition with the activator Sp1 and interaction with the co-repressor mSin3A and the histone deacetylase protein HDAC-1. These results expand our understanding of the functional properties of BTEB-related Sp1-like proteins and suggest that these proteins have evolved at least in part to balance the activating function of Sp1. Our data also provide evidence that different subfamilies of Sp1-like transcriptional repressor proteins, such as BTEBs and BKLFs, may function via association with distinct co-repressors.DISCUSSIONHere, we have characterized the transcriptional repressor and DNA binding activities of BTEB3, a novel member of the BTEB subfamily of Sp1-like proteins. We demonstrated that the BTEB3 N-terminal repressor activity was TSA-sensitive and associated with mSin3A and HDAC-1in vivo (Fig. 2). However, TSA did not completely reduce BTEB3-mediated repression, a phenomenon also observed for the well characterized mSin3A-interacting protein Mad1 (46Laherty C.D. Yang W.-M. Sun J.-M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Interestingly, mSin3A lacking the HDAC-1 interacting domain can still repress (46Laherty C.D. Yang W.-M. Sun J.-M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar,47Ayer D.E. Trends Cell Biol. 1999; 9: 193-198Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), which indicates that other mechanisms participate in mSin3a-mediated repression. Thus, in a similar manner, other histone deacetylase-independent co-repressors or chromatin remodeling complexes may contribute to BTEB3 repression activity.The N terminus of BTEB3 contains three separable repressor domains (R1, R2, and R3) (Fig. 3). R1 is separated from R2 by a linker and exhibits homology to the R1 domains of TIEG proteins, which bind to mSin3A (50Zhang J.-S. Moncrieffe M.C. Kaczynski J. Ellenrieder V. Prendergast F.G. Urrutia R. Mol. Cell. Biol. 2001; 21: 5041-5049Crossref PubMed Scopus (155) Google Scholar), suggesting that it may function as a distinct repressor domain in vivo. In contrast, since a linker does not separate R2 and R3, these may comprise a single repression domain. Fig. 4 shows that each domain silences transcription of a BTE-containing promoter via the native DNA binding domain to the same extent as the full-length protein. This is different from the data of the GAL4 assay (Fig. 3), which show that each domain is less potent than the entire N terminus. This discrepancy may be due to different basal promoters present in the reporter constructs (promoter context). In addition, different DNA binding domains and associated proteins may influence the function of these domains. Currently, it is difficult to define which of these possibilities is operating in vivo. Fig. 4Ddemonstrates that the repression domains of BTEB3 bind mSin3A, but not HDAC-1, supporting a model in which BTEB3 directly interacts with mSin3A, to recruit HDAC-1. These data are consistent with results showing that mSin3A and HDAC-1 co-precipitate with BTEB3 (Fig.2C). Thus, BTEB3 represses transcription by a mechanism involving the recruitment of the HDAC-1·mSin3A complex via three N-terminal repression domains.Since Sp1 is ubiquitously expressed, displacing it from promoters by competition may be a significant mechanism of repression. Fig. 5 shows that BTEB3 binds the BTE element with an affinity comparable to Sp1, suggesting that competition between the two proteins is possible. Fig.6 shows that BTEB3 does compete with Sp1 to antagonize its activation function. These data support a model in which competition with Sp1 via the zinc finger domain can contribute to the repressor function of BTEB3. To evaluate the contribution of each mechanism to the overall repression of BTEB3, we created a BTEB3 mutant containing the zinc fingers and evaluated its ability to compete with and antagonize Sp1. Fig. 6A shows that this domain can compete with Sp1 similar to the full-length protein. It also retains its ability to repress a BTE-containing promoter, although to a lesser degree than the full-length protein (Fig. 6C). Since the BTEB3 zinc finger domain alone is not able to interact with co-repressor proteins (Fig.2C) but retains partial repression activity, this further supports the idea that BTEB3 represses transcription by competing with Sp1. Thus, it is likely that, in vivo, the competition for DNA binding as well as direct repressor function are acting together to suppress transcription at promoters activated by Sp1. However, the contribution of these mechanisms to the overall repression activity may vary according to promoter context, co-repressor availability, or signaling. This model is attractive since a potent repression mechanism of this type may be required to counterbalance Sp1-mediated activation, the major GC binding activity detected in most mammalian cells. Indeed, several Sp1-like proteins may function according to this model, including BTEB1 (26Sogawa K. Kikuchi Y. Imataka H. Fujii-Kuriyama Y. J. Biochem. 1993; 114: 605-609Crossref PubMed Scopus (49) Google Scholar), Sp3 (25Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 270: 20-28Abstract Full Text Full Text PDF Scopus (96) Google Scholar), GKLF (8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and TIEG2. 2T. Cook and R. Urrutia, unpublished results. In addition, like BTEB3, some of these proteins also contain an N-terminal repressor activity. Thus, it is likely that both of these mechanisms of regulation may be a common motif among a subset of Sp1-like proteins.Recently, BTEB3 has been reported to display activator function (41Martin K. Cooper W. Metcalfe J. Kemp P. Biochem. J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar,42Song A. Chen Y. Thamatrakoln K. Storm T. Krensky A. Immunity. 1999; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this regard, although we do not rule that this protein may have activator function, the detailed characterization reported here strongly supports a repressor role for this gene. Interestingly, other Sp1-like proteins including BTEB1 (45Imataka H. Sogawa K. Yasumoto K. Kikuchi Y. Sasano K. Kobayashi A. Hayami M. Fujii-Kuriyama Y. EMBO J. 1992; 11: 3663-3671Crossref PubMed Scopus (307) Google Scholar, 48Kobayashi A. Sogawa K. Imataka H. Fujii-Kuriyama Y. J. Biochem. (Tokyo). 1995; 117: 91-95Crossref PubMed Scopus (32) Google Scholar) and TIEG2 (10Cook T. Gebelein B. Mesa K. Mladek A. Urrutia R. J. Biol. Chem. 1998; 273: 25929-25936Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Cook T. Gebelein B. Belal M. Mesa K. Urrutia R. J. Biol. Chem. 1999; 274: 29500-29504Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 49Asano H. Li S.X. Stamatoyannopoulos G. Mol. Cell. Biol. 1999; 19: 3571-3579Crossref PubMed Google Scholar) display both activator and repressor function depending on the promoter context. Indeed, the activation previously reported for BTEB3 was observed in a different promoter context and includes the SV40 (41Martin K. Cooper W. Metcalfe J. Kemp P. Biochem. J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar), β-globin (40Asano H. Li X.S. Stamatoyannopoulos G. Blood. 2000; 95: 3578-3584Crossref PubMed Google Scholar), and RANTES (42Song A. Chen Y. Thamatrakoln K. Storm T. Krensky A. Immunity. 1999; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) promoters. In addition, the cell lines used for these studies may also express other factors that have an effect on both the DNA binding and transcriptional regulatory activity of BTEB3. These differences in the systems need to be taken into consideration when evaluating the data and will require further investigation to better understand BTEB3-mediated transcriptional control.In summary, the fact that BTEB3 can repress transcription through at least two mechanisms makes it a good candidate for a repressor that can counterbalance the potent activator function of Sp1 on GC promoters. More importantly, we show that BTEB3 represses transcription by a mechanism different from that of BKLF proteins, suggesting that Sp1-like proteins can be classified into subgroups based on the mode of repression. However, it remains possible that these proteins interact with different co-repressors under different conditions and future experiments may better clarify these data. Thus, our results support the hypothesis that Sp1-like proteins with similar DNA binding domains but different transcription regulatory properties have evolved to provide fine-tune gene expression regulation on certain GC-rich sequences present in mammalian promoters. Sp1-like proteins, defined by the presence of three highly homologous C-terminal zinc finger motifs and variant N-terminal domains, are emerging as important regulators of cell homeostasis. Promoters containing Sp1-like sites are essential for the expression of numerous genes necessary for cell cycle progression (1Muller C. Yang R. Beck-von-Peccoz L. Idos G. Verbeek W. Koeffler H.P. J. Biol. Chem. 1999; 274: 11220-11228Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 2Li J.M. Nichols M.A. Chandrasekharan S. Xiong Y. Wang X. J. Biol. Chem. 1995; 270: 26750-26753Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 3Prowse D.M. Bolgan L. Molnar A. Dotto G.P. J. Biol. Chem. 1997; 272: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), DNA synthesis (4Zhao L. Chang L.S. J. Biol. Chem. 1997; 272: 4869-4882Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and other cell processes (5Birnbaum M.J. Wright K.L. van Wijnen A.J. Ramsey-Ewing A.L. Bourke M.T. Last T.J. Aziz F. Frenkel B. Rao B.R. Aronin N. Biochemistry. 1995; 34: 7648-7658Crossref PubMed Scopus (35) Google Scholar, 6Lin S.Y. Black A.R. Kostic D. Pajovic S. Hoover C.N. Azizkhan J.C. Mol. Cell. Biol. 1996; 16: 1668-1675Crossref PubMed Scopus (252) Google Scholar, 7Zwicker J. Liu N. Engeland K. Lucibello F.C. Muller R. Science. 1996; 271: 1595-1597Crossref PubMed Scopus (147) Google Scholar, 8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and studies have shown that certain Sp1-like proteins induce apoptosis (9Tachibana I. Imoto M. Adjei P. Gores G.J. Subramaniam M. Spelsberg T. Cook T. Urrutia R. J. Clin. Invest. 1997; 99: 2365-2374Crossref PubMed Scopus (192) Google Scholar), cell growth inhibition (10Cook T. Gebelein B. Mesa K. Mladek A. Urrutia R. J. Biol. Chem. 1998; 273: 25929-25936Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 12Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar), differentiation (13Billon N. Carlisi D. Datto M.B. van Grunsven L.A. Watt A. Wang X.F. Rudkin B.B. Oncogene. 1999; 18: 2872-2882Crossref PubMed Scopus (122) Google Scholar, 14Denver R.J. Ouellet L. Furling D. Kobayashi A. Fujii-Kuriyama Y. Puymirat J. J. Biol. Chem. 1999; 274: 23128-23134Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), and carcinogenesis (15El Rouby S. Newcomb E.W. Oncogene. 1996; 13: 2623-2630PubMed Google Scholar). In addition, the disruption of some Sp1-like genes in mice shows that these proteins are critical for normal development (12Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar, 16Marin M. Karism A. Visser P. Grosveld F. Philipsen S. Cell. 1997; 89: 619-628Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 17Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar, 18Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar). Thus, understanding how Sp1-like proteins bind DNA and regulate transcription is important to uncover the molecular mechanisms underlying a large number of cellular events. The existence of at least 17 different Sp1-like proteins offers a significant challenge for understanding how individual members regulate gene expression in a tissue-, cell-, and promoter-specific manner. One mechanism leading to specificity among Sp1-like proteins is a differential pattern of expression. For instance, Sp1, TIEG2, and BTEB11 are ubiquitously expressed, whereas the KLF proteins are restricted to certain tissues. Specificity among Sp1-like proteins is also dictated by recognition of DNA. For example, the Sp proteins preferentially bind GC sites (19Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar, 20Sogawa K. Imataka H. Yamasaki Y. Kusume H. Abe H. Fujii-Kuriyama Y. Nucleic Acids Res. 1993; 21: 1527-1532Crossref PubMed Scopus (179) Google Scholar) whereas the KLF subgroup prefers the CA site (21Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar, 22Matsumoto N. Laub F. Aldabe R. Zhang W. Ramirez F. Yoshida T. Terada M. J. Biol. Chem. 1998; 273: 28229-28237Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 23Miller I.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (646) Google Scholar). Interestingly, co-expressed Sp1-like proteins exhibiting similar binding specificity, such as Sp1 and Sp3, but often display opposite transcriptional regulatory properties (24Birnbaum M.J. van Wijnen A.J. Odgren P.R. Last T.J. Suske G. Stein G.S. Stein J.L. Biochemistry. 1995; 34: 16503-16508Crossref PubMed Scopus (177) Google Scholar, 25Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 270: 20-28Abstract Full Text Full Text PDF Scopus (96) Google Scholar). Thus, activators and repressors that bind to the same sequence may have evolved to turn on and off a discrete set of promoters by competing for this site. In this regard, emerging evidence indicates that Sp1-like proteins are able to compete for DNA binding, such as has been reported for BTEB1/Sp1/GKLF (8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 26Sogawa K. Kikuchi Y. Imataka H. Fujii-Kuriyama Y. J. Biochem. 1993; 114: 605-609Crossref PubMed Scopus (49) Google Scholar) and BKLF/EKLF (27Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Together, the results of these mechanistic studies on competition between Sp1-like proteins validate many of the predictions derived from sequence homology data, suggesting that these proteins regulate similar sequences in vivo. The molecular mechanisms behind the function of the Sp1-like activators have been extensively reported in the literature. For example, both Sp1 and EKLF have been shown to activate transcription by interacting with co-activators (13Billon N. Carlisi D. Datto M.B. van Grunsven L.A. Watt A. Wang X.F. Rudkin B.B. Oncogene. 1999; 18: 2872-2882Crossref PubMed Scopus (122) Google Scholar, 28Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (978) Google Scholar, 29Ryu S. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7137-7142Crossref PubMed Scopus (37) Google Scholar, 30Ryu S. Zhou S. Ladurner A.G. Tjian R. Nature. 1999; 397: 446-450Crossref PubMed Scopus (298) Google Scholar, 31Perkins A. Int. J. Biochem. Cell Biol. 1999; 31: 1175-1192Crossref PubMed Scopus (56) Google Scholar, 32Zhang W. Bieke J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar). In contrast, the molecular mechanisms involved in the function of Sp1-like repressor proteins are less defined. The Sp1-like proteins involved in transcriptional repression include the TIEG proteins (9Tachibana I. Imoto M. Adjei P. Gores G.J. Subramaniam M. Spelsberg T. Cook T. Urrutia R. J. Clin. Invest. 1997; 99: 2365-2374Crossref PubMed Scopus (192) Google Scholar, 10Cook T. Gebelein B. Mesa K. Mladek A. Urrutia R. J. Biol. Chem. 1998; 273: 25929-25936Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Cook T. Gebelein B. Belal M. Mesa K. Urrutia R. J. Biol. Chem. 1999; 274: 29500-29504Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), Sp3 (24Birnbaum M.J. van Wijnen A.J. Odgren P.R. Last T.J. Suske G. Stein G.S. Stein J.L. Biochemistry. 1995; 34: 16503-16508Crossref PubMed Scopus (177) Google Scholar, 25Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 270: 20-28Abstract Full Text Full Text PDF Scopus (96) Google Scholar), BKLF and BKLF2/KLF8 (21Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar, 34van Vliet J. Turner J. Crossley M. Nucleic Acids Res. 2000; 28: 1955-1962Crossref PubMed Scopus (129) Google Scholar), GKLF (8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and Ap2-rep (35Imhof A. Schuierer M. Werner O. Moser M. Roth C. Bauer R. Buettner R. Mol. Cell. Biol. 1999; 19: 194-204Crossref PubMed Google Scholar). However, only BKLF and a closely related protein, BKLF2/KLF8, have been shown to associate with co-repressors (34van Vliet J. Turner J. Crossley M. Nucleic Acids Res. 2000; 28: 1955-1962Crossref PubMed Scopus (129) Google Scholar, 36Turner J. Crossley M. EMBO J. 1998; 17: 5129-5140Crossref PubMed Scopus (277) Google Scholar). Because it is likely that these proteins bind similar sequences, an important question that remains is whether the mechanism of repression is similar between all Sp1-like repressors or whether Sp1-like subfamilies are defined by distinct mechanisms of repression. In this study, we have pursued this question by characterizing the human transcriptional repressor BTEB3, a novel Sp1-like protein that is significantly related to BTEB1. A partial sequence of this protein was recently published with the name of NSLP1 from our laboratory (37Cook T. Gebelein B. Urrutia R. Ann. N. Y. Acad. Sci. 1999; 880: 94-102Crossref PubMed Scopus (120) Google Scholar), but in light of its sequence similarity and functional conservation with BTEB1, we have renamed it BTEB3. This study focuses on the characterization of BTEB3 as a transcriptional repressor with two mechanisms of action: competition with the activator Sp1 and interaction with the co-repressor mSin3A and the histone deacetylase protein HDAC-1. These results expand our understanding of the functional properties of BTEB-related Sp1-like proteins and suggest that these proteins have evolved at least in part to balance the activating function of Sp1. Our data also provide evidence that different subfamilies of Sp1-like transcriptional repressor proteins, such as BTEBs and BKLFs, may function via association with distinct co-repressors. DISCUSSIONHere, we have characterized the transcriptional repressor and DNA binding activities of BTEB3, a novel member of the BTEB subfamily of Sp1-like proteins. We demonstrated that the BTEB3 N-terminal repressor activity was TSA-sensitive and associated with mSin3A and HDAC-1in vivo (Fig. 2). However, TSA did not completely reduce BTEB3-mediated repression, a phenomenon also observed for the well characterized mSin3A-interacting protein Mad1 (46Laherty C.D. Yang W.-M. Sun J.-M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Interestingly, mSin3A lacking the HDAC-1 interacting domain can still repress (46Laherty C.D. Yang W.-M. Sun J.-M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar,47Ayer D.E. Trends Cell Biol. 1999; 9: 193-198Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), which indicates that other mechanisms participate in mSin3a-mediated repression. Thus, in a similar manner, other histone deacetylase-independent co-repressors or chromatin remodeling complexes may contribute to BTEB3 repression activity.The N terminus of BTEB3 contains three separable repressor domains (R1, R2, and R3) (Fig. 3). R1 is separated from R2 by a linker and exhibits homology to the R1 domains of TIEG proteins, which bind to mSin3A (50Zhang J.-S. Moncrieffe M.C. Kaczynski J. Ellenrieder V. Prendergast F.G. Urrutia R. Mol. Cell. Biol. 2001; 21: 5041-5049Crossref PubMed Scopus (155) Google Scholar), suggesting that it may function as a distinct repressor domain in vivo. In contrast, since a linker does not separate R2 and R3, these may comprise a single repression domain. Fig. 4 shows that each domain silences transcription of a BTE-containing promoter via the native DNA binding domain to the same extent as the full-length protein. This is different from the data of the GAL4 assay (Fig. 3), which show that each domain is less potent than the entire N terminus. This discrepancy may be due to different basal promoters present in the reporter constructs (promoter context). In addition, different DNA binding domains and associated proteins may influence the function of these domains. Currently, it is difficult to define which of these possibilities is operating in vivo. Fig. 4Ddemonstrates that the repression domains of BTEB3 bind mSin3A, but not HDAC-1, supporting a model in which BTEB3 directly interacts with mSin3A, to recruit HDAC-1. These data are consistent with results showing that mSin3A and HDAC-1 co-precipitate with BTEB3 (Fig.2C). Thus, BTEB3 represses transcription by a mechanism involving the recruitment of the HDAC-1·mSin3A complex via three N-terminal repression domains.Since Sp1 is ubiquitously expressed, displacing it from promoters by competition may be a significant mechanism of repression. Fig. 5 shows that BTEB3 binds the BTE element with an affinity comparable to Sp1, suggesting that competition between the two proteins is possible. Fig.6 shows that BTEB3 does compete with Sp1 to antagonize its activation function. These data support a model in which competition with Sp1 via the zinc finger domain can contribute to the repressor function of BTEB3. To evaluate the contribution of each mechanism to the overall repression of BTEB3, we created a BTEB3 mutant containing the zinc fingers and evaluated its ability to compete with and antagonize Sp1. Fig. 6A shows that this domain can compete with Sp1 similar to the full-length protein. It also retains its ability to repress a BTE-containing promoter, although to a lesser degree than the full-length protein (Fig. 6C). Since the BTEB3 zinc finger domain alone is not able to interact with co-repressor proteins (Fig.2C) but retains partial repression activity, this further supports the idea that BTEB3 represses transcription by competing with Sp1. Thus, it is likely that, in vivo, the competition for DNA binding as well as direct repressor function are acting together to suppress transcription at promoters activated by Sp1. However, the contribution of these mechanisms to the overall repression activity may vary according to promoter context, co-repressor availability, or signaling. This model is attractive since a potent repression mechanism of this type may be required to counterbalance Sp1-mediated activation, the major GC binding activity detected in most mammalian cells. Indeed, several Sp1-like proteins may function according to this model, including BTEB1 (26Sogawa K. Kikuchi Y. Imataka H. Fujii-Kuriyama Y. J. Biochem. 1993; 114: 605-609Crossref PubMed Scopus (49) Google Scholar), Sp3 (25Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 270: 20-28Abstract Full Text Full Text PDF Scopus (96) Google Scholar), GKLF (8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and TIEG2. 2T. Cook and R. Urrutia, unpublished results. In addition, like BTEB3, some of these proteins also contain an N-terminal repressor activity. Thus, it is likely that both of these mechanisms of regulation may be a common motif among a subset of Sp1-like proteins.Recently, BTEB3 has been reported to display activator function (41Martin K. Cooper W. Metcalfe J. Kemp P. Biochem. J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar,42Song A. Chen Y. Thamatrakoln K. Storm T. Krensky A. Immunity. 1999; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this regard, although we do not rule that this protein may have activator function, the detailed characterization reported here strongly supports a repressor role for this gene. Interestingly, other Sp1-like proteins including BTEB1 (45Imataka H. Sogawa K. Yasumoto K. Kikuchi Y. Sasano K. Kobayashi A. Hayami M. Fujii-Kuriyama Y. EMBO J. 1992; 11: 3663-3671Crossref PubMed Scopus (307) Google Scholar, 48Kobayashi A. Sogawa K. Imataka H. Fujii-Kuriyama Y. J. Biochem. (Tokyo). 1995; 117: 91-95Crossref PubMed Scopus (32) Google Scholar) and TIEG2 (10Cook T. Gebelein B. Mesa K. Mladek A. Urrutia R. J. Biol. Chem. 1998; 273: 25929-25936Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Cook T. Gebelein B. Belal M. Mesa K. Urrutia R. J. Biol. Chem. 1999; 274: 29500-29504Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 49Asano H. Li S.X. Stamatoyannopoulos G. Mol. Cell. Biol. 1999; 19: 3571-3579Crossref PubMed Google Scholar) display both activator and repressor function depending on the promoter context. Indeed, the activation previously reported for BTEB3 was observed in a different promoter context and includes the SV40 (41Martin K. Cooper W. Metcalfe J. Kemp P. Biochem. J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar), β-globin (40Asano H. Li X.S. Stamatoyannopoulos G. Blood. 2000; 95: 3578-3584Crossref PubMed Google Scholar), and RANTES (42Song A. Chen Y. Thamatrakoln K. Storm T. Krensky A. Immunity. 1999; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) promoters. In addition, the cell lines used for these studies may also express other factors that have an effect on both the DNA binding and transcriptional regulatory activity of BTEB3. These differences in the systems need to be taken into consideration when evaluating the data and will require further investigation to better understand BTEB3-mediated transcriptional control.In summary, the fact that BTEB3 can repress transcription through at least two mechanisms makes it a good candidate for a repressor that can counterbalance the potent activator function of Sp1 on GC promoters. More importantly, we show that BTEB3 represses transcription by a mechanism different from that of BKLF proteins, suggesting that Sp1-like proteins can be classified into subgroups based on the mode of repression. However, it remains possible that these proteins interact with different co-repressors under different conditions and future experiments may better clarify these data. Thus, our results support the hypothesis that Sp1-like proteins with similar DNA binding domains but different transcription regulatory properties have evolved to provide fine-tune gene expression regulation on certain GC-rich sequences present in mammalian promoters. Here, we have characterized the transcriptional repressor and DNA binding activities of BTEB3, a novel member of the BTEB subfamily of Sp1-like proteins. We demonstrated that the BTEB3 N-terminal repressor activity was TSA-sensitive and associated with mSin3A and HDAC-1in vivo (Fig. 2). However, TSA did not completely reduce BTEB3-mediated repression, a phenomenon also observed for the well characterized mSin3A-interacting protein Mad1 (46Laherty C.D. Yang W.-M. Sun J.-M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Interestingly, mSin3A lacking the HDAC-1 interacting domain can still repress (46Laherty C.D. Yang W.-M. Sun J.-M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar,47Ayer D.E. Trends Cell Biol. 1999; 9: 193-198Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar), which indicates that other mechanisms participate in mSin3a-mediated repression. Thus, in a similar manner, other histone deacetylase-independent co-repressors or chromatin remodeling complexes may contribute to BTEB3 repression activity. The N terminus of BTEB3 contains three separable repressor domains (R1, R2, and R3) (Fig. 3). R1 is separated from R2 by a linker and exhibits homology to the R1 domains of TIEG proteins, which bind to mSin3A (50Zhang J.-S. Moncrieffe M.C. Kaczynski J. Ellenrieder V. Prendergast F.G. Urrutia R. Mol. Cell. Biol. 2001; 21: 5041-5049Crossref PubMed Scopus (155) Google Scholar), suggesting that it may function as a distinct repressor domain in vivo. In contrast, since a linker does not separate R2 and R3, these may comprise a single repression domain. Fig. 4 shows that each domain silences transcription of a BTE-containing promoter via the native DNA binding domain to the same extent as the full-length protein. This is different from the data of the GAL4 assay (Fig. 3), which show that each domain is less potent than the entire N terminus. This discrepancy may be due to different basal promoters present in the reporter constructs (promoter context). In addition, different DNA binding domains and associated proteins may influence the function of these domains. Currently, it is difficult to define which of these possibilities is operating in vivo. Fig. 4Ddemonstrates that the repression domains of BTEB3 bind mSin3A, but not HDAC-1, supporting a model in which BTEB3 directly interacts with mSin3A, to recruit HDAC-1. These data are consistent with results showing that mSin3A and HDAC-1 co-precipitate with BTEB3 (Fig.2C). Thus, BTEB3 represses transcription by a mechanism involving the recruitment of the HDAC-1·mSin3A complex via three N-terminal repression domains. Since Sp1 is ubiquitously expressed, displacing it from promoters by competition may be a significant mechanism of repression. Fig. 5 shows that BTEB3 binds the BTE element with an affinity comparable to Sp1, suggesting that competition between the two proteins is possible. Fig.6 shows that BTEB3 does compete with Sp1 to antagonize its activation function. These data support a model in which competition with Sp1 via the zinc finger domain can contribute to the repressor function of BTEB3. To evaluate the contribution of each mechanism to the overall repression of BTEB3, we created a BTEB3 mutant containing the zinc fingers and evaluated its ability to compete with and antagonize Sp1. Fig. 6A shows that this domain can compete with Sp1 similar to the full-length protein. It also retains its ability to repress a BTE-containing promoter, although to a lesser degree than the full-length protein (Fig. 6C). Since the BTEB3 zinc finger domain alone is not able to interact with co-repressor proteins (Fig.2C) but retains partial repression activity, this further supports the idea that BTEB3 represses transcription by competing with Sp1. Thus, it is likely that, in vivo, the competition for DNA binding as well as direct repressor function are acting together to suppress transcription at promoters activated by Sp1. However, the contribution of these mechanisms to the overall repression activity may vary according to promoter context, co-repressor availability, or signaling. This model is attractive since a potent repression mechanism of this type may be required to counterbalance Sp1-mediated activation, the major GC binding activity detected in most mammalian cells. Indeed, several Sp1-like proteins may function according to this model, including BTEB1 (26Sogawa K. Kikuchi Y. Imataka H. Fujii-Kuriyama Y. J. Biochem. 1993; 114: 605-609Crossref PubMed Scopus (49) Google Scholar), Sp3 (25Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 270: 20-28Abstract Full Text Full Text PDF Scopus (96) Google Scholar), GKLF (8Zhang W. Shields J.M. Kazuhiro S. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and TIEG2. 2T. Cook and R. Urrutia, unpublished results. In addition, like BTEB3, some of these proteins also contain an N-terminal repressor activity. Thus, it is likely that both of these mechanisms of regulation may be a common motif among a subset of Sp1-like proteins. Recently, BTEB3 has been reported to display activator function (41Martin K. Cooper W. Metcalfe J. Kemp P. Biochem. J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar,42Song A. Chen Y. Thamatrakoln K. Storm T. Krensky A. Immunity. 1999; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this regard, although we do not rule that this protein may have activator function, the detailed characterization reported here strongly supports a repressor role for this gene. Interestingly, other Sp1-like proteins including BTEB1 (45Imataka H. Sogawa K. Yasumoto K. Kikuchi Y. Sasano K. Kobayashi A. Hayami M. Fujii-Kuriyama Y. EMBO J. 1992; 11: 3663-3671Crossref PubMed Scopus (307) Google Scholar, 48Kobayashi A. Sogawa K. Imataka H. Fujii-Kuriyama Y. J. Biochem. (Tokyo). 1995; 117: 91-95Crossref PubMed Scopus (32) Google Scholar) and TIEG2 (10Cook T. Gebelein B. Mesa K. Mladek A. Urrutia R. J. Biol. Chem. 1998; 273: 25929-25936Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Cook T. Gebelein B. Belal M. Mesa K. Urrutia R. J. Biol. Chem. 1999; 274: 29500-29504Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 49Asano H. Li S.X. Stamatoyannopoulos G. Mol. Cell. Biol. 1999; 19: 3571-3579Crossref PubMed Google Scholar) display both activator and repressor function depending on the promoter context. Indeed, the activation previously reported for BTEB3 was observed in a different promoter context and includes the SV40 (41Martin K. Cooper W. Metcalfe J. Kemp P. Biochem. J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar), β-globin (40Asano H. Li X.S. Stamatoyannopoulos G. Blood. 2000; 95: 3578-3584Crossref PubMed Google Scholar), and RANTES (42Song A. Chen Y. Thamatrakoln K. Storm T. Krensky A. Immunity. 1999; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) promoters. In addition, the cell lines used for these studies may also express other factors that have an effect on both the DNA binding and transcriptional regulatory activity of BTEB3. These differences in the systems need to be taken into consideration when evaluating the data and will require further investigation to better understand BTEB3-mediated transcriptional control. In summary, the fact that BTEB3 can repress transcription through at least two mechanisms makes it a good candidate for a repressor that can counterbalance the potent activator function of Sp1 on GC promoters. More importantly, we show that BTEB3 represses transcription by a mechanism different from that of BKLF proteins, suggesting that Sp1-like proteins can be classified into subgroups based on the mode of repression. However, it remains possible that these proteins interact with different co-repressors under different conditions and future experiments may better clarify these data. Thus, our results support the hypothesis that Sp1-like proteins with similar DNA binding domains but different transcription regulatory properties have evolved to provide fine-tune gene expression regulation on certain GC-rich sequences present in mammalian promoters. We thank the members of the Mayo Optical Morphology and Flow Cytometry and Molecular Core Facilities for the use of these facilities. We kindly thank Drs. C. Seiser and R. Eisenman for providing the HDAC-1 and mSin3A constructs, respectively. We also thank Drs. Karen Hedin, Tiffany Cook, and Brian Gebelein for critically reviewing the manuscript." @default.
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W1991293300 title "The Sp1-like Protein BTEB3 Inhibits Transcription via the Basic Transcription Element Box by Interacting with mSin3A and HDAC-1 Co-repressors and Competing with Sp1" @default.
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