Negative Regulatory Role of Sp1 in Metal Responsive Element-mediated Transcriptional Activation

Abstract

Transcription of mammalian metallothionein (MT) genes is activated by heavy metals via multiple copies of acis-acting DNA element, the metal-responsive element (MRE). Our previous studies have shown that certain MREs of the human MT-IIA gene (MREb, MREc, MREd, and MREf) are less active than the others (MREa, MREe, and MREg). Gel shift analysis of HeLa cell nuclear proteins revealed that whereas the active MREs strongly bind the transcription factor MTF-1 essential for metal regulation, the less active MREs bind another distinct protein, MREb-BF. This protein recognizes the GC-rich region of MREb rather than the MRE core required for MTF-1 binding. All the MREs recognized by MREb-BF contain the CGCCC and/or CACCC motif, suggesting that the MREb-BF·MRE complex contains Sp1 or related proteins. Supershift analysis using antibodies against Sp1 family proteins as well as gel shift analysis using the recombinant Sp1 demonstrated that Sp1 represents the majority of MREb-BF activity. An MREb mutant with reduced affinity to Sp1 mediated zinc-inducible transcription much more actively than the wild-type MREb. Furthermore, when placed in the native promoter, this mutant MREb raised the overall promoter activity. These results strongly suggest that Sp1 acts as a negative regulator of transcription mediated by specific MREs. Transcription of mammalian metallothionein (MT) genes is activated by heavy metals via multiple copies of acis-acting DNA element, the metal-responsive element (MRE). Our previous studies have shown that certain MREs of the human MT-IIA gene (MREb, MREc, MREd, and MREf) are less active than the others (MREa, MREe, and MREg). Gel shift analysis of HeLa cell nuclear proteins revealed that whereas the active MREs strongly bind the transcription factor MTF-1 essential for metal regulation, the less active MREs bind another distinct protein, MREb-BF. This protein recognizes the GC-rich region of MREb rather than the MRE core required for MTF-1 binding. All the MREs recognized by MREb-BF contain the CGCCC and/or CACCC motif, suggesting that the MREb-BF·MRE complex contains Sp1 or related proteins. Supershift analysis using antibodies against Sp1 family proteins as well as gel shift analysis using the recombinant Sp1 demonstrated that Sp1 represents the majority of MREb-BF activity. An MREb mutant with reduced affinity to Sp1 mediated zinc-inducible transcription much more actively than the wild-type MREb. Furthermore, when placed in the native promoter, this mutant MREb raised the overall promoter activity. These results strongly suggest that Sp1 acts as a negative regulator of transcription mediated by specific MREs. metallothionein metal-responsive element MRE-binding transcription factor-1 mouse MT-I human MT-IIA nuclear extract electrophoretic mobility shift assay chloramphenicol acetyltransferase herpes simplex virus thymidine kinase Heavy metal-induced transcriptional activation of the genes coding for metallothioneins (MTs)1is mediated by multiple copies of a cis-acting DNA element, the metal-responsive element (MRE; Refs. 1Karin M. Haslinger A. Holtgreve H. Richards R.I. Krauter P. Westphal H.M. Beato M. Nature. 1984; 308: 513-519Crossref PubMed Scopus (568) Google Scholar, 2Carter A.D. Felber B.K. Walling M. Jubier M.-F. Schmidt C.J. Hamer D.H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7392-7396Crossref PubMed Scopus (104) Google Scholar, 3Stuart G.W. Searle P.F. Palmiter R.D. Nature. 1985; 317: 828-831Crossref PubMed Scopus (264) Google Scholar). It has been shown that the mouse MRE-binding transcription factor-1 (MTF-1) is essential for MRE-mediated transcription from gene knockout (4Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar) and antisense RNA expression studies (5Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1219-1223Crossref PubMed Scopus (327) Google Scholar). Nevertheless, it remains possible that metal regulation of MT genes involves additional transcriptional regulators. A number of MRE-binding proteins have been reported (reviewed in Ref.6Koizumi S. Otsuka F. Suzuki K.T. Imura N. Kimura M. Metallothionein III. Birkhäuser, Basel1993: 457-474Google Scholar), and cDNAs encoding MRE-binding proteins distinct from MTF-1 have been cloned (7Xu C. DNA Cell Biol. 1993; 12: 517-525Crossref PubMed Scopus (16) Google Scholar, 8Inouye C. Remondelli P. Karin M. Elledge S. DNA Cell Biol. 1994; 13: 731-742Crossref PubMed Scopus (71) Google Scholar). However, no protein except MTF-1 has been demonstrated to be functionally relevant to metal regulation.MREs were first identified by deletion analysis in duplicated sites upstream of the mouse MT-I (mMT-I) and human MT-IIA (hMT-IIA) genes (1Karin M. Haslinger A. Holtgreve H. Richards R.I. Krauter P. Westphal H.M. Beato M. Nature. 1984; 308: 513-519Crossref PubMed Scopus (568) Google Scholar, 2Carter A.D. Felber B.K. Walling M. Jubier M.-F. Schmidt C.J. Hamer D.H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7392-7396Crossref PubMed Scopus (104) Google Scholar, 9Stuart G.W. Searle P.F. Chen H.Y. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7318-7322Crossref PubMed Scopus (243) Google Scholar). After that, a search for MRE-related sequences revealed that there are multiple MRE homologs in the upstream region of all the mammalian MT genes reported so far (3Stuart G.W. Searle P.F. Palmiter R.D. Nature. 1985; 317: 828-831Crossref PubMed Scopus (264) Google Scholar,10Koizumi S. Otsuka F. Sarkar B. Genetic Response to Metals. Marcel Dekker, New York1995: 397-410Google Scholar). These facts imply that such a redundancy is probably essential for the high metal inducibility of MT gene promoters. However, MREs are not always functionally equivalent. In transfection assays using reporter genes driven by synthetic MRE-containing promoters, striking differences in zinc-induced transcriptional activity were observed among MREs of the mMT-I (3Stuart G.W. Searle P.F. Palmiter R.D. Nature. 1985; 317: 828-831Crossref PubMed Scopus (264) Google Scholar) and hMT-IIA (11Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar) genes, despite the observation that all these sequences share the highly conserved MRE core (whose functional importance has been demonstrated; Refs. 12Searle P.F. Stuart G.W. Palmiter R.D. Experientia Suppl. 1987; 52: 407-414Crossref PubMed Scopus (50) Google Scholar, 13Culotta V.C. Hamer D.H. Mol. Cell. Biol. 1989; 9: 1376-1380Crossref PubMed Scopus (159) Google Scholar) and the flanking semi-conserved GC-rich sequence. In addition, we have observed that the transcriptional activity of MRE does not always reflect its binding affinity to MTF-1; certain MREs of the hMT-IIA gene are able to bind the purified human MTF-1 in vitro, although they show no or very low transcriptional activity in vivo (11Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar). We had assumed that additional transcriptional regulators might be responsible for such a discrepancy and explored HeLa cell crude nuclear extracts for another MRE binding activity. Here we show that a nuclear protein distinct from MTF-1, which was finally proven to be the transcription factor Sp1 (SV40 protein 1), binds to the less active MREs and probably acts as a negative regulator of MRE-mediated transcription. Heavy metal-induced transcriptional activation of the genes coding for metallothioneins (MTs)1is mediated by multiple copies of a cis-acting DNA element, the metal-responsive element (MRE; Refs. 1Karin M. Haslinger A. Holtgreve H. Richards R.I. Krauter P. Westphal H.M. Beato M. Nature. 1984; 308: 513-519Crossref PubMed Scopus (568) Google Scholar, 2Carter A.D. Felber B.K. Walling M. Jubier M.-F. Schmidt C.J. Hamer D.H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7392-7396Crossref PubMed Scopus (104) Google Scholar, 3Stuart G.W. Searle P.F. Palmiter R.D. Nature. 1985; 317: 828-831Crossref PubMed Scopus (264) Google Scholar). It has been shown that the mouse MRE-binding transcription factor-1 (MTF-1) is essential for MRE-mediated transcription from gene knockout (4Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar) and antisense RNA expression studies (5Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1219-1223Crossref PubMed Scopus (327) Google Scholar). Nevertheless, it remains possible that metal regulation of MT genes involves additional transcriptional regulators. A number of MRE-binding proteins have been reported (reviewed in Ref.6Koizumi S. Otsuka F. Suzuki K.T. Imura N. Kimura M. Metallothionein III. Birkhäuser, Basel1993: 457-474Google Scholar), and cDNAs encoding MRE-binding proteins distinct from MTF-1 have been cloned (7Xu C. DNA Cell Biol. 1993; 12: 517-525Crossref PubMed Scopus (16) Google Scholar, 8Inouye C. Remondelli P. Karin M. Elledge S. DNA Cell Biol. 1994; 13: 731-742Crossref PubMed Scopus (71) Google Scholar). However, no protein except MTF-1 has been demonstrated to be functionally relevant to metal regulation. MREs were first identified by deletion analysis in duplicated sites upstream of the mouse MT-I (mMT-I) and human MT-IIA (hMT-IIA) genes (1Karin M. Haslinger A. Holtgreve H. Richards R.I. Krauter P. Westphal H.M. Beato M. Nature. 1984; 308: 513-519Crossref PubMed Scopus (568) Google Scholar, 2Carter A.D. Felber B.K. Walling M. Jubier M.-F. Schmidt C.J. Hamer D.H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7392-7396Crossref PubMed Scopus (104) Google Scholar, 9Stuart G.W. Searle P.F. Chen H.Y. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7318-7322Crossref PubMed Scopus (243) Google Scholar). After that, a search for MRE-related sequences revealed that there are multiple MRE homologs in the upstream region of all the mammalian MT genes reported so far (3Stuart G.W. Searle P.F. Palmiter R.D. Nature. 1985; 317: 828-831Crossref PubMed Scopus (264) Google Scholar,10Koizumi S. Otsuka F. Sarkar B. Genetic Response to Metals. Marcel Dekker, New York1995: 397-410Google Scholar). These facts imply that such a redundancy is probably essential for the high metal inducibility of MT gene promoters. However, MREs are not always functionally equivalent. In transfection assays using reporter genes driven by synthetic MRE-containing promoters, striking differences in zinc-induced transcriptional activity were observed among MREs of the mMT-I (3Stuart G.W. Searle P.F. Palmiter R.D. Nature. 1985; 317: 828-831Crossref PubMed Scopus (264) Google Scholar) and hMT-IIA (11Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar) genes, despite the observation that all these sequences share the highly conserved MRE core (whose functional importance has been demonstrated; Refs. 12Searle P.F. Stuart G.W. Palmiter R.D. Experientia Suppl. 1987; 52: 407-414Crossref PubMed Scopus (50) Google Scholar, 13Culotta V.C. Hamer D.H. Mol. Cell. Biol. 1989; 9: 1376-1380Crossref PubMed Scopus (159) Google Scholar) and the flanking semi-conserved GC-rich sequence. In addition, we have observed that the transcriptional activity of MRE does not always reflect its binding affinity to MTF-1; certain MREs of the hMT-IIA gene are able to bind the purified human MTF-1 in vitro, although they show no or very low transcriptional activity in vivo (11Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar). We had assumed that additional transcriptional regulators might be responsible for such a discrepancy and explored HeLa cell crude nuclear extracts for another MRE binding activity. Here we show that a nuclear protein distinct from MTF-1, which was finally proven to be the transcription factor Sp1 (SV40 protein 1), binds to the less active MREs and probably acts as a negative regulator of MRE-mediated transcription.