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W2102013368 abstract "The transcription rates of glycolytic enzyme genes are coordinately induced when cells are exposed to low oxygen tension. This effect has been described in many cell types and is not restricted to species or phyla. In mammalian cells, there are 11 distinct glycolytic enzymes, at least 9 of which are induced by hypoxia. Recent reports described a role for the hypoxia-inducible factor-1 (HIF-1) in the transcriptional activation of lactate dehydrogenase A, aldolase-A, phosphoglycerate kinase, and enolase-1 genes. It is not known whether the HIF-1 factor acts exclusively to regulate these genes during hypoxia, or how the other genes of the pathway are regulated. In this paper, we describe analyses of the muscle-specific pyruvate kinase-M and β-enolase promoters that implicate additional mechanisms for the regulation of glycolytic enzyme gene transcription by hypoxia. Transient transcription of a reporter gene directed by either promoter was activated when transfected muscle cells were exposed to hypoxia. Neither of these promoters contain HIF-1 binding sites. Instead, the hypoxia response was localized to a conserved GC-rich element positioned immediately upstream of a GATAA site in the proximal promoter regions of both genes. The GC element was essential for both basal and hypoxia-induced expression and bound the transcription factors Sp1 and Sp3. Hypoxia caused the progressive depletion of Sp3 determined by DNA binding studies and Western analyses, whereas Sp1 protein levels remained unchanged. Overexpression of Sp3 repressed expression of β-enolase promoters. It is concluded that hypoxia activates these glycolytic enzyme gene promoters by down-regulating Sp3, thereby removing the associated transcriptional repression. The transcription rates of glycolytic enzyme genes are coordinately induced when cells are exposed to low oxygen tension. This effect has been described in many cell types and is not restricted to species or phyla. In mammalian cells, there are 11 distinct glycolytic enzymes, at least 9 of which are induced by hypoxia. Recent reports described a role for the hypoxia-inducible factor-1 (HIF-1) in the transcriptional activation of lactate dehydrogenase A, aldolase-A, phosphoglycerate kinase, and enolase-1 genes. It is not known whether the HIF-1 factor acts exclusively to regulate these genes during hypoxia, or how the other genes of the pathway are regulated. In this paper, we describe analyses of the muscle-specific pyruvate kinase-M and β-enolase promoters that implicate additional mechanisms for the regulation of glycolytic enzyme gene transcription by hypoxia. Transient transcription of a reporter gene directed by either promoter was activated when transfected muscle cells were exposed to hypoxia. Neither of these promoters contain HIF-1 binding sites. Instead, the hypoxia response was localized to a conserved GC-rich element positioned immediately upstream of a GATAA site in the proximal promoter regions of both genes. The GC element was essential for both basal and hypoxia-induced expression and bound the transcription factors Sp1 and Sp3. Hypoxia caused the progressive depletion of Sp3 determined by DNA binding studies and Western analyses, whereas Sp1 protein levels remained unchanged. Overexpression of Sp3 repressed expression of β-enolase promoters. It is concluded that hypoxia activates these glycolytic enzyme gene promoters by down-regulating Sp3, thereby removing the associated transcriptional repression. hypoxia-inducible factor-1 lactate dehydrogenase polymerase chain reaction wild type base pair(s) pyruvate kinase serum response factor SRF recognition site. Glycolysis is induced in most cell types by anaerobic or hypoxic conditions when oxidative metabolism is repressed. In mammalian cells, the glycolytic pathway has 11 separate enzymes, some with multiple tissue-specific isoforms, each encoded by separate genes and mostly situated on unlinked chromosomal loci (reviewed in Ref. 1Webster K.A. Murphy B.J. Can. J. Zool. 1988; 66: 1046-1058Crossref Google Scholar). We reported previously that the transcription rates of glycolytic enzyme genes are coordinately induced in muscle cells subjected to hypoxia (2Webster K.A. Mol. Cell. Biochem. 1987; 77: 19-28Crossref PubMed Scopus (97) Google Scholar, 3Webster K.A. Gunning P.W. Hardeman E. Wallace D.C. Kedes L.H. J. Cell. Physiol. 1990; 142: 566-573Crossref PubMed Scopus (89) Google Scholar). Consistent with the appearance of elevated steady state mRNA levels, the transcription rates increased gradually over 1–2 days (2Webster K.A. Mol. Cell. Biochem. 1987; 77: 19-28Crossref PubMed Scopus (97) Google Scholar). Similar effects have been described in endothelial cells exposed to hypoxia (4Aaronson R.M. Graven K.K. Tucci M. McDonald R.J. Farber H.W. J. Biol. Chem. 1995; 270: 27752-27757Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The signaling pathways and genetic elements that control this response are probably complex. In recent reports, the hypoxia-inducible factor 1 (HIF-1),1 originally identified as a factor in the regulation of the erythropoietin gene (reviewed in Ref. 5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar), has been shown to play a role in the transcriptional activation of a number of genes by hypoxia (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 7Semenza G.L. Roth P.H. Fang H-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 8Shanna Y.L. Cox R. Morita T. Kourembanas S. Circ. Res. 1995; 77: 638-643Crossref PubMed Scopus (802) Google Scholar, 9Taylor J.M. Davies J.D. Peterson C.A. J. Biol. Chem. 1995; 270: 2535-2540Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2023) Google Scholar). HIF-1 binds to an enhancer element containing the core sequence ACGTGC, which is an obligatory and minimal component of HIF-1-responsive genes.DNA sequence and functional analyses have revealed the presence of active HIF-1-binding sites in the non-coding regions of mammalian lactate dehydrogenase A (LDH-A), aldolase-A, enolase-1, and phosphoglycerate kinase (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 7Semenza G.L. Roth P.H. Fang H-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 9Taylor J.M. Davies J.D. Peterson C.A. J. Biol. Chem. 1995; 270: 2535-2540Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). They have not been described in a number of the other mammalian glycolytic enzyme genes including hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-phosphate isomerase, β-enolase, or some of the other tissue-specific isogenes. The absence of appropriately located sites indicates that there may be other mechanisms for regulating these genes. An additional consideration is that hypoxia response elements distinct from HIF-1 binding sites have been described in plant alcohol dehydrogenase, LDH, and aldolase gene promoters (11Good A.G. Paetkau D.H. Plant Mol. Biol. 1992; 19: 693-697Crossref PubMed Scopus (17) Google Scholar, 12Walker J.C. Howard E.A. Dennis E.S. Peacock W.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6624-6628Crossref PubMed Google Scholar, 13Olive M.R. Peacock W.J. Dennis E.S. Nucleic Acids Res. 1991; 19: 7053-7060Crossref PubMed Scopus (68) Google Scholar). Therefore, there is a precedent and perhaps a requirement for additional hypoxia-regulated mechanisms for mammalian glycolytic enzyme gene expression.Promoter elements with the core sequence GGGC/T/AGG bind a number of transcription factors including members of the Sp family (14Boulikas T. Crit. Rev. Eukaryotic Gene Exp. 1994; 4: 117-321Crossref PubMed Scopus (64) Google Scholar, 15Hagen G. Muller S. Beato M. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (522) Google Scholar, 16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar). The factors Sp1 and Sp3 appear to be ubiquitous in mammalian cells where they compete with similar binding affinities for common target sequences (18Birnbaum 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). Sp1 is typically a positive-acting transcription factor and may interact directly with the basal TFIID transcriptional complex (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar). In contrast, Sp3 appears to possess both transcriptional repressor and activating properties, the relative strengths of which depend on the promoter context as well as the cellular background (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 18Birnbaum 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, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar).Here, we present evidence that changes in the relative abundances of Sp1 and Sp3 contribute to the positive regulation of the glycolytic enzyme genes PK-M and β-enolase by hypoxia. Exposure of C2C12 myocytes to hypoxia caused depletion of Sp3 from nuclear extracts without affecting Sp1. The loss of Sp3 binding to the conserved GC element in these promoters correlated with transcriptional activation. Overexpression of Sp3 caused transcriptional repression of cotransfected β-enolase promoters in support of a regulatory mechanism involving the hypoxia-mediated alleviation of Sp3 repression.DISCUSSIONWe show in this article that the region between −80 and −28 of the human β-enolase gene confers positive regulation by hypoxia. The region contains a GC-rich, Sp-family binding site and a 3′ proximal GATAA element. A similar sequence combination is present in the same location of the hypoxia-responsive PK-M promoter in the rat (27Takenaka M. Noguchi T. Inoue H. Yamada K. Matsuda T. Tanaka T. J. Biol. Chem. 1989; 264: 2363-2367Abstract Full Text PDF PubMed Google Scholar) and is a common combination in erythroid-specific promoters, many of which are directly or indirectly regulated by oxygen availability (31Max-Audit I. Eleouet J.F. Romeo P.H. J. Biol. Chem. 1993; 268: 5431-5437Abstract Full Text PDF PubMed Google Scholar, 32Youssoufian H. Blood. 1994; 83: 1428-1435Crossref PubMed Google Scholar). When the β-enolase GC element was mutated or deleted, basal (aerobic) expression was reduced and the response to hypoxia was lost. This indicates that the GC element is required alone or in combination with downstream element(s) to promote transcriptional activation by hypoxia.Gel mobility shift binding studies demonstrated that mutation of the GC element eliminated the binding of Sp proteins coincident with loss in function of the promoter. We reported previously that this site was also responsible for hyperoxic regulation of β-enolase expression through the reversible oxidation of Sp1 cysteine sulfhydryl groups (29Wu X. Bishopric N.H. Discher D.J. Murphy B.J. Webster K.A. Mol. Cell. Biol. 1996; 16: 1035-1046Crossref PubMed Scopus (181) Google Scholar). These observations implicate the GC element as a critical redox control site in the β-enolase, PK-M, and possibly other promoters with a similar arrangement of proximal elements.Protein binding studies identified Sp1 and Sp3 as the major components of the complexes formed between nuclear extracts and the PK-M/β-enolase GC element. Although we cannot preclude involvement of other factors, the combination of Sp1 and Sp3 antibodies blocked or supershifted each of the specific complexes. Therefore, these proteins are implicated in the regulation. Protein binding as well as Western blots indicate that Sp1 levels did not change markedly in response to hypoxia whereas Sp3 levels fell dramatically.The loss of Sp3 binding could account for the transcriptional activation if Sp3 functions as a repressor when bound to these promoter elements. The effects of overexpressing Sp3 indicates that Sp3 can indeed repress transcription from the β-enolase promoters. Previous studies, typically using Schneider SL2 cells that lacks endogenous Sp factors, have shown that Sp3 is a strong repressor of some GC-dependent promoters, and it can antagonize multiple classes of positive-acting factors (17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 18Birnbaum 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, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar). Our observation that pCMV-Sp3 caused >60% repression of −101β-Eno in hypoxic C2C12 cells is substantial because these cells already have a high Sp background. The observation that overexpression of Sp3 countered the promoter induction by hypoxia provides strong evidence that Sp3 depletion is associated with gene activation. We propose that the hypoxia-mediated depletion of Sp3 alleviates a repressor activity, thereby activating transcription from the β-enolase promoters.It is not clear whether Sp1 has an active or passive role in this regulation. Previous studies indicated that Sp3 may repress transcription independently of Sp1 activity, probably by interacting directly with other transcription factors (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Sp3 may also compete with Sp1 for the common DNA binding site, thereby reducing transcriptional activation by Sp1 (17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar). The observation that overexpressing Sp1 did not augment the activity of −101β-Eno suggests either that endogenous Sp1 is saturating, or that Sp3 repression is independent of Sp1. Indeed, we have no direct evidence to indicate that Sp3 binding is replaced by Sp1 during hypoxia. The apparent increase of Sp1 p106 during hypoxia (Fig. 5) indicates an increased proportion of phosphorylated Sp1 in hypoxic nuclear extracts, and this in turn may reflect an increased binding of Sp1 because DNA-bound Sp1 is phosphorylated by a DNA-dependent kinase (33Jackson S.P. MacDonald J. Lees-Miller S. Tjian R. Cell. 1990; 63: 155-165Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 34Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1020) Google Scholar). Alternatively displaced Sp3 may be substituted by the new GC-binding factor that appears to be induced by hypoxia (Fig. 4 C). We have no information on this new complex, except that it is a GC-binding factor with a similar migration rate to Sp3, it accumulates under hypoxia, and it is not recognized by Sp1 or Sp3 antibodies.Sp3 down-regulation by hypoxia appears to be by a post-transcriptional pathway since Sp3 transcript levels did not change. Hypoxia-mediated degradation, post-translational modifications, or translational mechanisms may be involved. Interestingly, a post-translational mechanism involving ubiquitin-mediated degradation has recently been described for the regulation of HIF-1 by hypoxia (35Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1392) Google Scholar). Previous studies suggest that the repressor function of Sp3 is stronger when there are multiple Sp1/Sp3 binding sites (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Therefore, positive responses to hypoxia and the regulation of gene expression by this pathway may be selective for a subset of genes with multiple GC-rich promoter elements.Results presented here suggest that mechanisms other than, or in addition to, HIF-1 regulate the expression of glycolytic enzyme genes by hypoxia. Functional HIF-1 binding sites have been described in LDH-A, phosphoglycerate kinase, enolase-1, and aldolase-A genes, and mutational analyses as well as studies of cells from HIF-1 knockout mice support the involvement of HIF-1 in the regulation of these genes (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2023) Google Scholar, 36Semenza G.L. Jiang B.-H. Leung S.W. Passantino R. Concordet J.-P. Maire P. Giallongo A. J. Biol. Chem. 1996; 271: 32529-32537Abstract Full Text Full Text PDF PubMed Scopus (1333) Google Scholar). Our GenBank data base analyses revealed 2 HIF-1 binding sites in the first intron of the PK-M gene but no sites in 7194 bp of the human β-enolase gene. HIF-1-regulated genes are induced by both hypoxia and transition metals (5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar), although these functions may be controlled by separate regulatory elements in other genes. GenBank screens also revealed multiple metal response element binding sequences (TGCACT) in both PK-M and β-enolase gene promoters (not shown). These elements bind the metal response factor MTF-1 that mediates positive transcriptional responses to metals (37Dalton T. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (250) Google Scholar, 38Smith A. Alam J. Escriba P.V. Morgan W.T. J. Biol. Chem. 1993; 268: 7365-7371Abstract Full Text PDF PubMed Google Scholar, 39Koizumi S. Yamada H. Suzuki K. Otsuka F. Eur. J. Biochem. 1992; 210: 555-560Crossref PubMed Scopus (40) Google Scholar, 40Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar). For genes that do not contain HIF-1 sites, separate MREs and HREs could account for the dual regulation by hypoxia and metals (41Ho V.T. Bunn H.F. Biochem. Biophys. Res. Commun. 1996; 223: 175-180Crossref PubMed Scopus (117) Google Scholar).Finally, although contributions of Sp-binding sites to the hypoxia response have not been reported previously in mammalian cells, anaerobic response elements have been described in the maize alcohol dehydrogenase, LDH, and aldolase gene promoters (11Good A.G. Paetkau D.H. Plant Mol. Biol. 1992; 19: 693-697Crossref PubMed Scopus (17) Google Scholar, 42Ferl R.J. Nick H.S. J. Biol. Chem. 1987; 262: 7947-7950Abstract Full Text PDF PubMed Google Scholar, 43Dennis E.S. Gerlach W.L. Walker J.C. Lavin M. Peacock W.J. Mol. Biol. 1988; 202: 759-767Crossref Scopus (50) Google Scholar). The anaerobic response element in the alcohol dehydrogenase promoter contains a GC-rich element that constitutively binds Sp1 and is essential for activation by hypoxia (12Walker J.C. Howard E.A. Dennis E.S. Peacock W.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6624-6628Crossref PubMed Google Scholar, 13Olive M.R. Peacock W.J. Dennis E.S. Nucleic Acids Res. 1991; 19: 7053-7060Crossref PubMed Scopus (68) Google Scholar). Therefore, hypoxia-mediated regulation of gene expression through the modulation of GC-binding factors may be an ancient pathway that has been conserved in plant and mammalian glycolytic enzyme genes. Glycolysis is induced in most cell types by anaerobic or hypoxic conditions when oxidative metabolism is repressed. In mammalian cells, the glycolytic pathway has 11 separate enzymes, some with multiple tissue-specific isoforms, each encoded by separate genes and mostly situated on unlinked chromosomal loci (reviewed in Ref. 1Webster K.A. Murphy B.J. Can. J. Zool. 1988; 66: 1046-1058Crossref Google Scholar). We reported previously that the transcription rates of glycolytic enzyme genes are coordinately induced in muscle cells subjected to hypoxia (2Webster K.A. Mol. Cell. Biochem. 1987; 77: 19-28Crossref PubMed Scopus (97) Google Scholar, 3Webster K.A. Gunning P.W. Hardeman E. Wallace D.C. Kedes L.H. J. Cell. Physiol. 1990; 142: 566-573Crossref PubMed Scopus (89) Google Scholar). Consistent with the appearance of elevated steady state mRNA levels, the transcription rates increased gradually over 1–2 days (2Webster K.A. Mol. Cell. Biochem. 1987; 77: 19-28Crossref PubMed Scopus (97) Google Scholar). Similar effects have been described in endothelial cells exposed to hypoxia (4Aaronson R.M. Graven K.K. Tucci M. McDonald R.J. Farber H.W. J. Biol. Chem. 1995; 270: 27752-27757Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The signaling pathways and genetic elements that control this response are probably complex. In recent reports, the hypoxia-inducible factor 1 (HIF-1),1 originally identified as a factor in the regulation of the erythropoietin gene (reviewed in Ref. 5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar), has been shown to play a role in the transcriptional activation of a number of genes by hypoxia (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 7Semenza G.L. Roth P.H. Fang H-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 8Shanna Y.L. Cox R. Morita T. Kourembanas S. Circ. Res. 1995; 77: 638-643Crossref PubMed Scopus (802) Google Scholar, 9Taylor J.M. Davies J.D. Peterson C.A. J. Biol. Chem. 1995; 270: 2535-2540Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2023) Google Scholar). HIF-1 binds to an enhancer element containing the core sequence ACGTGC, which is an obligatory and minimal component of HIF-1-responsive genes. DNA sequence and functional analyses have revealed the presence of active HIF-1-binding sites in the non-coding regions of mammalian lactate dehydrogenase A (LDH-A), aldolase-A, enolase-1, and phosphoglycerate kinase (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 7Semenza G.L. Roth P.H. Fang H-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 9Taylor J.M. Davies J.D. Peterson C.A. J. Biol. Chem. 1995; 270: 2535-2540Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). They have not been described in a number of the other mammalian glycolytic enzyme genes including hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-phosphate isomerase, β-enolase, or some of the other tissue-specific isogenes. The absence of appropriately located sites indicates that there may be other mechanisms for regulating these genes. An additional consideration is that hypoxia response elements distinct from HIF-1 binding sites have been described in plant alcohol dehydrogenase, LDH, and aldolase gene promoters (11Good A.G. Paetkau D.H. Plant Mol. Biol. 1992; 19: 693-697Crossref PubMed Scopus (17) Google Scholar, 12Walker J.C. Howard E.A. Dennis E.S. Peacock W.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6624-6628Crossref PubMed Google Scholar, 13Olive M.R. Peacock W.J. Dennis E.S. Nucleic Acids Res. 1991; 19: 7053-7060Crossref PubMed Scopus (68) Google Scholar). Therefore, there is a precedent and perhaps a requirement for additional hypoxia-regulated mechanisms for mammalian glycolytic enzyme gene expression. Promoter elements with the core sequence GGGC/T/AGG bind a number of transcription factors including members of the Sp family (14Boulikas T. Crit. Rev. Eukaryotic Gene Exp. 1994; 4: 117-321Crossref PubMed Scopus (64) Google Scholar, 15Hagen G. Muller S. Beato M. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (522) Google Scholar, 16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar). The factors Sp1 and Sp3 appear to be ubiquitous in mammalian cells where they compete with similar binding affinities for common target sequences (18Birnbaum 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). Sp1 is typically a positive-acting transcription factor and may interact directly with the basal TFIID transcriptional complex (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar). In contrast, Sp3 appears to possess both transcriptional repressor and activating properties, the relative strengths of which depend on the promoter context as well as the cellular background (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 18Birnbaum 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, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar). Here, we present evidence that changes in the relative abundances of Sp1 and Sp3 contribute to the positive regulation of the glycolytic enzyme genes PK-M and β-enolase by hypoxia. Exposure of C2C12 myocytes to hypoxia caused depletion of Sp3 from nuclear extracts without affecting Sp1. The loss of Sp3 binding to the conserved GC element in these promoters correlated with transcriptional activation. Overexpression of Sp3 caused transcriptional repression of cotransfected β-enolase promoters in support of a regulatory mechanism involving the hypoxia-mediated alleviation of Sp3 repression. DISCUSSIONWe show in this article that the region between −80 and −28 of the human β-enolase gene confers positive regulation by hypoxia. The region contains a GC-rich, Sp-family binding site and a 3′ proximal GATAA element. A similar sequence combination is present in the same location of the hypoxia-responsive PK-M promoter in the rat (27Takenaka M. Noguchi T. Inoue H. Yamada K. Matsuda T. Tanaka T. J. Biol. Chem. 1989; 264: 2363-2367Abstract Full Text PDF PubMed Google Scholar) and is a common combination in erythroid-specific promoters, many of which are directly or indirectly regulated by oxygen availability (31Max-Audit I. Eleouet J.F. Romeo P.H. J. Biol. Chem. 1993; 268: 5431-5437Abstract Full Text PDF PubMed Google Scholar, 32Youssoufian H. Blood. 1994; 83: 1428-1435Crossref PubMed Google Scholar). When the β-enolase GC element was mutated or deleted, basal (aerobic) expression was reduced and the response to hypoxia was lost. This indicates that the GC element is required alone or in combination with downstream element(s) to promote transcriptional activation by hypoxia.Gel mobility shift binding studies demonstrated that mutation of the GC element eliminated the binding of Sp proteins coincident with loss in function of the promoter. We reported previously that this site was also responsible for hyperoxic regulation of β-enolase expression through the reversible oxidation of Sp1 cysteine sulfhydryl groups (29Wu X. Bishopric N.H. Discher D.J. Murphy B.J. Webster K.A. Mol. Cell. Biol. 1996; 16: 1035-1046Crossref PubMed Scopus (181) Google Scholar). These observations implicate the GC element as a critical redox control site in the β-enolase, PK-M, and possibly other promoters with a similar arrangement of proximal elements.Protein binding studies identified Sp1 and Sp3 as the major components of the complexes formed between nuclear extracts and the PK-M/β-enolase GC element. Although we cannot preclude involvement of other factors, the combination of Sp1 and Sp3 antibodies blocked or supershifted each of the specific complexes. Therefore, these proteins are implicated in the regulation. Protein binding as well as Western blots indicate that Sp1 levels did not change markedly in response to hypoxia whereas Sp3 levels fell dramatically.The loss of Sp3 binding could account for the transcriptional activation if Sp3 functions as a repressor when bound to these promoter elements. The effects of overexpressing Sp3 indicates that Sp3 can indeed repress transcription from the β-enolase promoters. Previous studies, typically using Schneider SL2 cells that lacks endogenous Sp factors, have shown that Sp3 is a strong repressor of some GC-dependent promoters, and it can antagonize multiple classes of positive-acting factors (17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 18Birnbaum 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, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar). Our observation that pCMV-Sp3 caused >60% repression of −101β-Eno in hypoxic C2C12 cells is substantial because these cells already have a high Sp background. The observation that overexpression of Sp3 countered the promoter induction by hypoxia provides strong evidence that Sp3 depletion is associated with gene activation. We propose that the hypoxia-mediated depletion of Sp3 alleviates a repressor activity, thereby activating transcription from the β-enolase promoters.It is not clear whether Sp1 has an active or passive role in this regulation. Previous studies indicated that Sp3 may repress transcription independently of Sp1 activity, probably by interacting directly with other transcription factors (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Sp3 may also compete with Sp1 for the common DNA binding site, thereby reducing transcriptional activation by Sp1 (17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar). The observation that overexpressing Sp1 did not augment the activity of −101β-Eno suggests either that endogenous Sp1 is saturating, or that Sp3 repression is independent of Sp1. Indeed, we have no direct evidence to indicate that Sp3 binding is replaced by Sp1 during hypoxia. The apparent increase of Sp1 p106 during hypoxia (Fig. 5) indicates an increased proportion of phosphorylated Sp1 in hypoxic nuclear extracts, and this in turn may reflect an increased binding of Sp1 because DNA-bound Sp1 is phosphorylated by a DNA-dependent kinase (33Jackson S.P. MacDonald J. Lees-Miller S. Tjian R. Cell. 1990; 63: 155-165Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 34Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1020) Google Scholar). Alternatively displaced Sp3 may be substituted by the new GC-binding factor that appears to be induced by hypoxia (Fig. 4 C). We have no information on this new complex, except that it is a GC-binding factor with a similar migration rate to Sp3, it accumulates under hypoxia, and it is not recognized by Sp1 or Sp3 antibodies.Sp3 down-regulation by hypoxia appears to be by a post-transcriptional pathway since Sp3 transcript levels did not change. Hypoxia-mediated degradation, post-translational modifications, or translational mechanisms may be involved. Interestingly, a post-translational mechanism involving ubiquitin-mediated degradation has recently been described for the regulation of HIF-1 by hypoxia (35Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1392) Google Scholar). Previous studies suggest that the repressor function of Sp3 is stronger when there are multiple Sp1/Sp3 binding sites (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Therefore, positive responses to hypoxia and the regulation of gene expression by this pathway may be selective for a subset of genes with multiple GC-rich promoter elements.Results presented here suggest that mechanisms other than, or in addition to, HIF-1 regulate the expression of glycolytic enzyme genes by hypoxia. Functional HIF-1 binding sites have been described in LDH-A, phosphoglycerate kinase, enolase-1, and aldolase-A genes, and mutational analyses as well as studies of cells from HIF-1 knockout mice support the involvement of HIF-1 in the regulation of these genes (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2023) Google Scholar, 36Semenza G.L. Jiang B.-H. Leung S.W. Passantino R. Concordet J.-P. Maire P. Giallongo A. J. Biol. Chem. 1996; 271: 32529-32537Abstract Full Text Full Text PDF PubMed Scopus (1333) Google Scholar). Our GenBank data base analyses revealed 2 HIF-1 binding sites in the first intron of the PK-M gene but no sites in 7194 bp of the human β-enolase gene. HIF-1-regulated genes are induced by both hypoxia and transition metals (5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar), although these functions may be controlled by separate regulatory elements in other genes. GenBank screens also revealed multiple metal response element binding sequences (TGCACT) in both PK-M and β-enolase gene promoters (not shown). These elements bind the metal response factor MTF-1 that mediates positive transcriptional responses to metals (37Dalton T. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (250) Google Scholar, 38Smith A. Alam J. Escriba P.V. Morgan W.T. J. Biol. Chem. 1993; 268: 7365-7371Abstract Full Text PDF PubMed Google Scholar, 39Koizumi S. Yamada H. Suzuki K. Otsuka F. Eur. J. Biochem. 1992; 210: 555-560Crossref PubMed Scopus (40) Google Scholar, 40Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar). For genes that do not contain HIF-1 sites, separate MREs and HREs could account for the dual regulation by hypoxia and metals (41Ho V.T. Bunn H.F. Biochem. Biophys. Res. Commun. 1996; 223: 175-180Crossref PubMed Scopus (117) Google Scholar).Finally, although contributions of Sp-binding sites to the hypoxia response have not been reported previously in mammalian cells, anaerobic response elements have been described in the maize alcohol dehydrogenase, LDH, and aldolase gene promoters (11Good A.G. Paetkau D.H. Plant Mol. Biol. 1992; 19: 693-697Crossref PubMed Scopus (17) Google Scholar, 42Ferl R.J. Nick H.S. J. Biol. Chem. 1987; 262: 7947-7950Abstract Full Text PDF PubMed Google Scholar, 43Dennis E.S. Gerlach W.L. Walker J.C. Lavin M. Peacock W.J. Mol. Biol. 1988; 202: 759-767Crossref Scopus (50) Google Scholar). The anaerobic response element in the alcohol dehydrogenase promoter contains a GC-rich element that constitutively binds Sp1 and is essential for activation by hypoxia (12Walker J.C. Howard E.A. Dennis E.S. Peacock W.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6624-6628Crossref PubMed Google Scholar, 13Olive M.R. Peacock W.J. Dennis E.S. Nucleic Acids Res. 1991; 19: 7053-7060Crossref PubMed Scopus (68) Google Scholar). Therefore, hypoxia-mediated regulation of gene expression through the modulation of GC-binding factors may be an ancient pathway that has been conserved in plant and mammalian glycolytic enzyme genes. We show in this article that the region between −80 and −28 of the human β-enolase gene confers positive regulation by hypoxia. The region contains a GC-rich, Sp-family binding site and a 3′ proximal GATAA element. A similar sequence combination is present in the same location of the hypoxia-responsive PK-M promoter in the rat (27Takenaka M. Noguchi T. Inoue H. Yamada K. Matsuda T. Tanaka T. J. Biol. Chem. 1989; 264: 2363-2367Abstract Full Text PDF PubMed Google Scholar) and is a common combination in erythroid-specific promoters, many of which are directly or indirectly regulated by oxygen availability (31Max-Audit I. Eleouet J.F. Romeo P.H. J. Biol. Chem. 1993; 268: 5431-5437Abstract Full Text PDF PubMed Google Scholar, 32Youssoufian H. Blood. 1994; 83: 1428-1435Crossref PubMed Google Scholar). When the β-enolase GC element was mutated or deleted, basal (aerobic) expression was reduced and the response to hypoxia was lost. This indicates that the GC element is required alone or in combination with downstream element(s) to promote transcriptional activation by hypoxia. Gel mobility shift binding studies demonstrated that mutation of the GC element eliminated the binding of Sp proteins coincident with loss in function of the promoter. We reported previously that this site was also responsible for hyperoxic regulation of β-enolase expression through the reversible oxidation of Sp1 cysteine sulfhydryl groups (29Wu X. Bishopric N.H. Discher D.J. Murphy B.J. Webster K.A. Mol. Cell. Biol. 1996; 16: 1035-1046Crossref PubMed Scopus (181) Google Scholar). These observations implicate the GC element as a critical redox control site in the β-enolase, PK-M, and possibly other promoters with a similar arrangement of proximal elements. Protein binding studies identified Sp1 and Sp3 as the major components of the complexes formed between nuclear extracts and the PK-M/β-enolase GC element. Although we cannot preclude involvement of other factors, the combination of Sp1 and Sp3 antibodies blocked or supershifted each of the specific complexes. Therefore, these proteins are implicated in the regulation. Protein binding as well as Western blots indicate that Sp1 levels did not change markedly in response to hypoxia whereas Sp3 levels fell dramatically. The loss of Sp3 binding could account for the transcriptional activation if Sp3 functions as a repressor when bound to these promoter elements. The effects of overexpressing Sp3 indicates that Sp3 can indeed repress transcription from the β-enolase promoters. Previous studies, typically using Schneider SL2 cells that lacks endogenous Sp factors, have shown that Sp3 is a strong repressor of some GC-dependent promoters, and it can antagonize multiple classes of positive-acting factors (17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 18Birnbaum 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, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar). Our observation that pCMV-Sp3 caused >60% repression of −101β-Eno in hypoxic C2C12 cells is substantial because these cells already have a high Sp background. The observation that overexpression of Sp3 countered the promoter induction by hypoxia provides strong evidence that Sp3 depletion is associated with gene activation. We propose that the hypoxia-mediated depletion of Sp3 alleviates a repressor activity, thereby activating transcription from the β-enolase promoters. It is not clear whether Sp1 has an active or passive role in this regulation. Previous studies indicated that Sp3 may repress transcription independently of Sp1 activity, probably by interacting directly with other transcription factors (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 20De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Sp3 may also compete with Sp1 for the common DNA binding site, thereby reducing transcriptional activation by Sp1 (17Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 21Kumar A.P. Butler A.P. Nucleic Acids Res. 1997; 25: 2012-2019Crossref PubMed Scopus (81) Google Scholar). The observation that overexpressing Sp1 did not augment the activity of −101β-Eno suggests either that endogenous Sp1 is saturating, or that Sp3 repression is independent of Sp1. Indeed, we have no direct evidence to indicate that Sp3 binding is replaced by Sp1 during hypoxia. The apparent increase of Sp1 p106 during hypoxia (Fig. 5) indicates an increased proportion of phosphorylated Sp1 in hypoxic nuclear extracts, and this in turn may reflect an increased binding of Sp1 because DNA-bound Sp1 is phosphorylated by a DNA-dependent kinase (33Jackson S.P. MacDonald J. Lees-Miller S. Tjian R. Cell. 1990; 63: 155-165Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 34Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1020) Google Scholar). Alternatively displaced Sp3 may be substituted by the new GC-binding factor that appears to be induced by hypoxia (Fig. 4 C). We have no information on this new complex, except that it is a GC-binding factor with a similar migration rate to Sp3, it accumulates under hypoxia, and it is not recognized by Sp1 or Sp3 antibodies. Sp3 down-regulation by hypoxia appears to be by a post-transcriptional pathway since Sp3 transcript levels did not change. Hypoxia-mediated degradation, post-translational modifications, or translational mechanisms may be involved. Interestingly, a post-translational mechanism involving ubiquitin-mediated degradation has recently been described for the regulation of HIF-1 by hypoxia (35Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1392) Google Scholar). Previous studies suggest that the repressor function of Sp3 is stronger when there are multiple Sp1/Sp3 binding sites (16Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 19Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Therefore, positive responses to hypoxia and the regulation of gene expression by this pathway may be selective for a subset of genes with multiple GC-rich promoter elements. Results presented here suggest that mechanisms other than, or in addition to, HIF-1 regulate the expression of glycolytic enzyme genes by hypoxia. Functional HIF-1 binding sites have been described in LDH-A, phosphoglycerate kinase, enolase-1, and aldolase-A genes, and mutational analyses as well as studies of cells from HIF-1 knockout mice support the involvement of HIF-1 in the regulation of these genes (6Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2023) Google Scholar, 36Semenza G.L. Jiang B.-H. Leung S.W. Passantino R. Concordet J.-P. Maire P. Giallongo A. J. Biol. Chem. 1996; 271: 32529-32537Abstract Full Text Full Text PDF PubMed Scopus (1333) Google Scholar). Our GenBank data base analyses revealed 2 HIF-1 binding sites in the first intron of the PK-M gene but no sites in 7194 bp of the human β-enolase gene. HIF-1-regulated genes are induced by both hypoxia and transition metals (5Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1041) Google Scholar), although these functions may be controlled by separate regulatory elements in other genes. GenBank screens also revealed multiple metal response element binding sequences (TGCACT) in both PK-M and β-enolase gene promoters (not shown). These elements bind the metal response factor MTF-1 that mediates positive transcriptional responses to metals (37Dalton T. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (250) Google Scholar, 38Smith A. Alam J. Escriba P.V. Morgan W.T. J. Biol. Chem. 1993; 268: 7365-7371Abstract Full Text PDF PubMed Google Scholar, 39Koizumi S. Yamada H. Suzuki K. Otsuka F. Eur. J. Biochem. 1992; 210: 555-560Crossref PubMed Scopus (40) Google Scholar, 40Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar). For genes that do not contain HIF-1 sites, separate MREs and HREs could account for the dual regulation by hypoxia and metals (41Ho V.T. Bunn H.F. Biochem. Biophys. Res. Commun. 1996; 223: 175-180Crossref PubMed Scopus (117) Google Scholar). Finally, although contributions of Sp-binding sites to the hypoxia response have not been reported previously in mammalian cells, anaerobic response elements have been described in the maize alcohol dehydrogenase, LDH, and aldolase gene promoters (11Good A.G. Paetkau D.H. Plant Mol. Biol. 1992; 19: 693-697Crossref PubMed Scopus (17) Google Scholar, 42Ferl R.J. Nick H.S. J. Biol. Chem. 1987; 262: 7947-7950Abstract Full Text PDF PubMed Google Scholar, 43Dennis E.S. Gerlach W.L. Walker J.C. Lavin M. Peacock W.J. Mol. Biol. 1988; 202: 759-767Crossref Scopus (50) Google Scholar). The anaerobic response element in the alcohol dehydrogenase promoter contains a GC-rich element that constitutively binds Sp1 and is essential for activation by hypoxia (12Walker J.C. Howard E.A. Dennis E.S. Peacock W.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6624-6628Crossref PubMed Google Scholar, 13Olive M.R. Peacock W.J. Dennis E.S. Nucleic Acids Res. 1991; 19: 7053-7060Crossref PubMed Scopus (68) Google Scholar). Therefore, hypoxia-mediated regulation of gene expression through the modulation of GC-binding factors may be an ancient pathway that has been conserved in plant and mammalian glycolytic enzyme genes." @default.
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W2102013368 title "Hypoxia Regulates β-Enolase and Pyruvate Kinase-M Promoters by Modulating Sp1/Sp3 Binding to a Conserved GC Element" @default.
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