Regulation of Murine Cytochrome Oxidase Vb Gene Expression in Different Tissues and during Myogenesis

The mouse cytochrome oxidase (COX) Vb promoter contains three sequence motifs with partial or full consensus for YY-1 and GTG factor binding and a CArG box, located between positions −480 and −390. Individually, all three motifs stimulated transcription of the TKCAT promoter, and bound distinctly different proteins from the liver and differentiated C2C12 nuclear extracts. Collectively, these motifs, together with the downstream flanking sequence, −378 to −320, suppressed the transcription activity of heterologous promoters, thymidine kinase-chloramphenicol acetyltransferase (TKCAT) and COXIV/CAT. The transcription activities of both TKCAT and COXIV/CAT constructs were induced 3-4-fold during induced myogenesis of C2C12 cells. The downstream CArG-like motif binds transcription factor YY-1, while the upstream YY-1-like motif binds to a yet unidentified factor. Co-expression with intact YY-1, but not that lacking the DNA binding domain suppressed the transcriptional activity. Mutations targeted to the CArG-like motif abolished the suppressive effect of the negative enhancer and the inducibility of the promoter during myogenic differentiation. Our results suggest that the activity of the negative enhancer may determine the level of expression of the COX Vb gene in different tissues. The mouse cytochrome oxidase (COX) Vb promoter contains three sequence motifs with partial or full consensus for YY-1 and GTG factor binding and a CArG box, located between positions −480 and −390. Individually, all three motifs stimulated transcription of the TKCAT promoter, and bound distinctly different proteins from the liver and differentiated C2C12 nuclear extracts. Collectively, these motifs, together with the downstream flanking sequence, −378 to −320, suppressed the transcription activity of heterologous promoters, thymidine kinase-chloramphenicol acetyltransferase (TKCAT) and COXIV/CAT. The transcription activities of both TKCAT and COXIV/CAT constructs were induced 3-4-fold during induced myogenesis of C2C12 cells. The downstream CArG-like motif binds transcription factor YY-1, while the upstream YY-1-like motif binds to a yet unidentified factor. Co-expression with intact YY-1, but not that lacking the DNA binding domain suppressed the transcriptional activity. Mutations targeted to the CArG-like motif abolished the suppressive effect of the negative enhancer and the inducibility of the promoter during myogenic differentiation. Our results suggest that the activity of the negative enhancer may determine the level of expression of the COX Vb gene in different tissues. INTRODUCTIONCytochrome c oxidase (COX), 1The abbreviations used are:COXcytochrome oxidaseCATchloramphenicol acetyltransferaseTKCATthymidine kinase-CATbpbase pair(s)PCRpolymerase chain reactionCMVcytomegalovirus. the terminal oxidase of the mitochondrial electron transport chain, is a major site of regulation of oxidative metabolism (1Calhoun M.W. Thomas J.W. Gennis R.B. Trends Biochem. Sci. 1994; 15: 325-330Abstract Full Text PDF Scopus (162) Google Scholar, 2Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (512) Google Scholar). The enzyme from mammalian tissues contains 13 subunits, 10 of which are encoded by nuclear genes and the 3 largest catalytic subunits by the mitochondrial genes (3Cao X. Hengst L. Schlerf A. Droste M. Mengel T. Kadenbach B. Ann. N. Y. Acad. Sci. 1988; 550: 337-347Crossref PubMed Scopus (20) Google Scholar, 4Hart F.-U. Pfanner N. Nicholson D.W. Neupert W. Biochim Biophys. Acta. 1989; 988: 1-45Crossref PubMed Scopus (545) Google Scholar). We have recently characterized the mouse genes for nuclear encoded subunits IV, Vb, and VIII (H) of the COX complex (5Basu A. Avadhani N.G. J. Biol. Chem. 1991; 266: 15450-15456Abstract Full Text PDF PubMed Google Scholar, 6Carter R.S. Avadhani N.G. Arch. Biochem. Biophys. 1991; 288: 97-106Crossref PubMed Scopus (30) Google Scholar, 7Carter R.S. Bhat N.K. Basu A. Avadhani N.G. J. Biol. Chem. 1992; 267: 23418-23426Abstract Full Text PDF PubMed Google Scholar, 8Carter R.S. Avadhani N.G. J. Biol. Chem. 1994; 269: 4381-4387Abstract Full Text PDF PubMed Google Scholar, 9Lenka N. Basu A. Mullick J. Avadhani N.G. J. Biol. Chem. 1996; 271: 30281-30289Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The expression of the COX Vb gene is particularly interesting, since it appears to involve a large combinatorial array of DNA elements and protein transactivators located at both the distal, as well as immediate upstream regions of the transcription initiation sites (7Carter R.S. Bhat N.K. Basu A. Avadhani N.G. J. Biol. Chem. 1992; 267: 23418-23426Abstract Full Text PDF PubMed Google Scholar, 9Lenka N. Basu A. Mullick J. Avadhani N.G. J. Biol. Chem. 1996; 271: 30281-30289Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Virbasius J.V. Virbasius C.A. Scarpulla R.C. Genes Dev. 1993; 7: 380-392Crossref PubMed Scopus (237) Google Scholar, 12Basu A. Park K. Atchison M.L. Carter R.S. Avadhani N.G. J. Biol. Chem. 1993; 268: 4188-4196Abstract Full Text PDF PubMed Google Scholar). Additionally, the COX Vb gene encodes multiple mRNAs that exhibit 5′ heterogeneity due to transcription initiation at multiple initiator sites (5Basu A. Avadhani N.G. J. Biol. Chem. 1991; 266: 15450-15456Abstract Full Text PDF PubMed Google Scholar, 10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar).The immediate upstream region of the mouse COX Vb gene consists of a TATA-less GC-rich sequence characteristic of a number of “housekeeping” genes. The basal promoter region has been mapped to the −8 to +40 region of the gene, which contains several protein-binding motifs. At least five of these protein-binding motifs correspond to discrete transcription initiation sites (10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar). Initiation of RNA mapping to the +1 position of the promoter has been shown previously to depend upon a YY-1-binding initiator motif (12Basu A. Park K. Atchison M.L. Carter R.S. Avadhani N.G. J. Biol. Chem. 1993; 268: 4188-4196Abstract Full Text PDF PubMed Google Scholar). Recently we have found that the GA-binding protein factor-binding Ets repeat sequence GTTCCCGGAAG at the +17 to +26 position also functions as an independent initiator for transcription of two clusters of RNAs mapping to the +18/19 and +23/24 regions (10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar).Although the COX Vb gene is constitutively expressed, its level of expression varies in different tissues. C0X Vb mRNA is 10-20-fold more abundant in the heart and kidney as compared to the liver. Furthermore, the COX Vb mRNA level increases markedly during induced differentiation of C2C12 myoblasts into myotubes. In the present study we have investigated the contributions of various protein-binding motifs from the 5′ distal region of the promoter in the overall transcriptional rates of the promoter. We have identified a negative regulatory region, which encompasses a YY-1-like (YY-1′) and CArG-like (CArG′) consensus motifs flanking a GTG element (13Zhao X.-A. Hung M.-C. Mol. Cell Biol. 1992; 12: 2739-2748Crossref PubMed Scopus (24) Google Scholar), a novel enhancer found in the neu oncogene promoter. Results presented in this paper show that factor YY-1 binding to the downstream CArG′ motif is important for the activity of the negative enhancer. Our results therefore suggest that in addition to the multiple positive cis acting elements (7Carter R.S. Bhat N.K. Basu A. Avadhani N.G. J. Biol. Chem. 1992; 267: 23418-23426Abstract Full Text PDF PubMed Google Scholar, 8Carter R.S. Avadhani N.G. J. Biol. Chem. 1994; 269: 4381-4387Abstract Full Text PDF PubMed Google Scholar, 9Lenka N. Basu A. Mullick J. Avadhani N.G. J. Biol. Chem. 1996; 271: 30281-30289Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar, 11Virbasius J.V. Virbasius C.A. Scarpulla R.C. Genes Dev. 1993; 7: 380-392Crossref PubMed Scopus (237) Google Scholar), a negative enhancer region is involved in the complex regulatory circuit of the COX Vb gene expression.DISCUSSIONSubunit Vb of the mammalian COX is classified as a ubiquitously expressed subunit of the enzyme complex (2Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (512) Google Scholar). Results presented in this study, along with previous results (5Basu A. Avadhani N.G. J. Biol. Chem. 1991; 266: 15450-15456Abstract Full Text PDF PubMed Google Scholar, 10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar), demonstrate that the relative abundance of the COX Vb mRNA in different tissues varies markedly; the mouse kidney and heart contain about 10-20-fold higher level of the mRNA than the liver (Fig. 1A). Furthermore, a 4-6-fold higher level of the mRNA is detected during induced differentiation of C2C12 myocytes. Our results suggest that widely varying levels of Cox Vb mRNA transcription in different tissues are due to the presence of an unusual negative regulatory element at the 5′ distal region of the promoter, whose suppressor activity appears to vary in different cell types and during induced myogenesis. The activity of this negative enhancer is regulated by transcription factor YY-1 in conjunction with other protein factors that bind to this region in a sequence- and tissue-specific manner.Results of deletion analyses (Fig. 3, Fig. 4) show that the negative regulatory region spans sequences −481 to −321, and consists of two distinct domains. A region between −481 and −390 contains three different sequence motifs showing partial consensus to the YY-1-binding site (NF-E1′), a GTG enhancer element, and a CArG-like element (CArG′). The second region between −390 and −321 contains an Ets sequence motif, which binds to an unknown factor from 3T3, liver, and C2C12 nuclear extracts (results not shown). All three upstream region sequence motifs, YY-1′, GTG, and CArG′, stimulated transcription of the TKCAT promoter in 3T3, Hep 3B, and C2C12 cells at different levels (Fig. 5). All three DNA motifs exhibited distinctly different patterns of protein binding with nuclear extracts from liver and C2C12 myotubes (Fig. 9), suggesting that the cell- or tissue-specific binding patterns may reflect upon the differing levels of transcription activation with these activator motifs in different cells. The three motifs placed together in a tandem array, as they exist in the COX Vb promoter, nearly completely reversed the transcriptional activity obtained with the individual sequence motifs. The transcriptional down-regulation of the heterologous promoter was more pronounced when further downstream sequences up to −320 were linked to the TKCAT promoter or the mouse COXIV promoter (Fig. 5). It is therefore likely that the 90-bp region, containing the three protein-binding motifs, together with the flanking downstream sequence is involved in conferring the negative regulatory activity.An interesting observation of this study relates to the unusual nature of YY-1 binding to the negative regulatory region. Gel mobility shift patterns in Fig. 6 show that the YY-1′ and CArG′ sequences fail to bind to bacterially expressed purified YY-1 factor under conditions that detect only complexes with slow off rates. Under conditions that detect rapidly dissociating complexes, however, the CArG′ sequence motif, but not the YY-1′ motif binds to the YY-1 factor (Fig. 6). The 90-bp DNA probe containing all three sequence motifs, on the other hand, binds to the YY-1 factor under slow off rate conditions and the bound complex yields a single-site methylation footprint, which maps to the CArG′ sequence region. Thus, although the CArG′ sequence region appears to provide the site for binding, the overall binding efficiency appears to be modulated by a still unknown sequence or structure within the 90-bp DNA sequence. YY-1 is a multifunctional factor, which functions both as a transcriptional activator and a repressor (18Bushmeyer S. Park K. Atchison M.L. J. Biol. Chem. 1995; 270: 30213-30220Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 27Flanagan J.R. Becker K.G. Ennist D.L. Gleason S.L. Driggers P.H. Levi B.-Z. Appella E. Ozato K. Mol. Cell Biol. 1992; 12: 38-44Crossref PubMed Scopus (216) Google Scholar, 28Shi Y. Seto Chang L.-S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar, 29Meier V.S. Groner B. Mol. Cell Biol. 1994; 14: 128-137Crossref PubMed Google Scholar, 30Bauknecht T. Jundt F. Herr I. Oehler J. Delius H. Shi Y. Angel P. zur Hausen H. J. Virol. 1995; 69: 1-12Crossref PubMed Google Scholar, 31Riggs K.J. Saleque S. Wong K.-K. Merrell K.T. Lee J.-S. Shi Y. Calame K. Mol. Cell Biol. 1993; 13: 7487-7495Crossref PubMed Scopus (162) Google Scholar, 32Hariharan N. Kelley D.E. Perry R.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9799-9803Crossref PubMed Scopus (245) Google Scholar, 33Satyamoorthy K. Park K. Atchison M.L. Howe C.C. Mol. Cell Biol. 1993; 13: 6621-6628Crossref PubMed Scopus (49) Google Scholar, 34Shrivastava A. Calame K. Nucleic Acids Res. 1994; 22: 5151-5155Crossref PubMed Scopus (279) Google Scholar, 35Natesan S. Gilman M.Z. Genes Dev. 1993; 7: 2497-2509Crossref PubMed Scopus (210) Google Scholar) of different promoters under different cellular conditions and in different cell systems. Additionally, YY-1 binding is known to induce DNA bending in some cases (35Natesan S. Gilman M.Z. Genes Dev. 1993; 7: 2497-2509Crossref PubMed Scopus (210) Google Scholar). In the present case, YY-1 binding may induce similar DNA bending, facilitating specific interactions between protein-bound complexes at both upstream and downstream regions of the COX Vb negative enhancer.Experiments in Fig. 10 (A and D), demonstrating a 60% suppression of transcription in 3T3 and C2C12 myoblasts by co-expression with YY-1 cDNA, provide evidence for its involvement in the negative regulation of the COX Vb gene under transient transfection conditions. Several lines of evidence suggest that the YY-1-mediated inhibition is specific. First, as shown in Fig. 10, the YY-1-mediated inhibition is dose-dependent. Results of cotransfection with various deletion constructs of YY-1 (expressed as Gal-4 fusion proteins) in Fig. 11 demonstrate that the DNA binding and repressor domains of YY-1 are essential for transcription suppression of the COX Vb promoter, while the transcription activation domain is not necessary. Third, mutations targeted to the CArG′ region of the promoter resulted in vastly reduced YY-1 binding and reduced effectiveness of the negative enhancer (Fig. 10, C and D). It is therefore likely that the relative levels of YY-1 in a given cell type may determine the level of expression of the COX Vb gene. It is also likely that other tissue-specific factors binding to the CArG′ or other flanking motifs might affect the binding or the activity of the YY-1 factor. It was recently reported that although nuclear extract from differentiated chick embryonic myocytes contained YY-1 protein, its DNA binding efficiency was reduced, possibly due to interference from another muscle-specific factor (36Lee T.-C. Shi Y. Schwartz R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9814-9818Crossref PubMed Scopus (197) Google Scholar). It is known that a number of different nuclear protein factors, including Sp1 (37Lee J.S. Galvin K.M. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6145-6149Crossref PubMed Scopus (276) Google Scholar), E1A (38Lewis B.A. Tullis G. Seto E. Horihoshi N. Weinmann R. Shenk T. J. Virol. 1995; 69: 1628-1636Crossref PubMed Google Scholar), FKBP12 (39Yang W.-M. Inouye C.J. Seto E. J. Biol. Chem. 1995; 270: 15187-15193Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and c-Myc (40Shrivastava A. Saleque S. Kalpana G.V. Calame K. Science. 1993; 262: 1889-1892Crossref PubMed Scopus (231) Google Scholar), interact with YY-1 and modulate its functional properties. Our results showing a reduced transcription activity of the −481/CArG′M1 and −481/CArG′M2 constructs in differentiated myotube suggests that in the context of the COX Vb promoter, the CArG′ sequence functions as a transcription activator in differentiated myocytes, and mutations in this region may affect the binding of myotube-specific transcriptional activator protein(s).A number of different negative enhancer mechanisms have been reported (41Levine M. Manley J.L. Cell. 1989; 59: 405-408Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 42Johnson A.D. Cell. 1995; 81: 655-658Abstract Full Text PDF PubMed Scopus (203) Google Scholar), including the repression due to competition for DNA-binding sites, the quenching mechanism, where the repressor interferes with the function of activator(s) without inhibiting its binding ability, and the direct repression where the negative regulatory factor blocks the activity of the basal transcription complex. It appears likely that the COX Vb negative regulatory region represents a mechanism similar to the quenching phenomenon. The activity of the negative enhancer may require specific interactions between proteins bound to the upstream 90-bp DNA sequence with those bound to sequences downstream of the −390 region, although the details of this interaction currently remain unknown. We also postulate that positive acting sequence motifs like YY-1′, GTG, and CArG′, when correctly juxtaposed to the downstream regulatory region, alter the activity of various transactivator proteins, with the net effect being the transcriptional repression of the promoter. Recent studies have shown that in a number of different promoters, regulatory regions consist of alternatively positioned positive and negative acting regulatory elements. In the case of the low density lipoprotein receptor gene, the sterol-dependent binding of a protein to a regulatory sequence inhibits or alters the activity of an adjacent Sp1 factor-binding site (43Dawson P.A. Hoffman S.L. van der Westhuyzen D.R. Sudhof T.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 1988; 263: 3372-3379Abstract Full Text PDF PubMed Google Scholar). In several other cases, including the mouse IgH T. P. Nucleic Acids Res. 14: PubMed Scopus Google Scholar), the cell receptor gene A. Cell. 1989; 59: Full Text PDF PubMed Scopus Google Scholar), gene P. T. Y. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), and regulatory gene R. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar), transcriptional regulation interaction between both positive and negative elements. the nature of proteins binding to the individual sequence motifs of the COX Vb negative regulatory region, the nature of interaction between the various bound complexes, and the of the YY-1 in these interactions remain unknown. are under to these INTRODUCTIONCytochrome c oxidase (COX), 1The abbreviations used are:COXcytochrome oxidaseCATchloramphenicol acetyltransferaseTKCATthymidine kinase-CATbpbase pair(s)PCRpolymerase chain reactionCMVcytomegalovirus. the terminal oxidase of the mitochondrial electron transport chain, is a major site of regulation of oxidative metabolism (1Calhoun M.W. Thomas J.W. Gennis R.B. Trends Biochem. Sci. 1994; 15: 325-330Abstract Full Text PDF Scopus (162) Google Scholar, 2Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (512) Google Scholar). The enzyme from mammalian tissues contains 13 subunits, 10 of which are encoded by nuclear genes and the 3 largest catalytic subunits by the mitochondrial genes (3Cao X. Hengst L. Schlerf A. Droste M. Mengel T. Kadenbach B. Ann. N. Y. Acad. Sci. 1988; 550: 337-347Crossref PubMed Scopus (20) Google Scholar, 4Hart F.-U. Pfanner N. Nicholson D.W. Neupert W. Biochim Biophys. Acta. 1989; 988: 1-45Crossref PubMed Scopus (545) Google Scholar). We have recently characterized the mouse genes for nuclear encoded subunits IV, Vb, and VIII (H) of the COX complex (5Basu A. Avadhani N.G. J. Biol. Chem. 1991; 266: 15450-15456Abstract Full Text PDF PubMed Google Scholar, 6Carter R.S. Avadhani N.G. Arch. Biochem. Biophys. 1991; 288: 97-106Crossref PubMed Scopus (30) Google Scholar, 7Carter R.S. Bhat N.K. Basu A. Avadhani N.G. J. Biol. Chem. 1992; 267: 23418-23426Abstract Full Text PDF PubMed Google Scholar, 8Carter R.S. Avadhani N.G. J. Biol. Chem. 1994; 269: 4381-4387Abstract Full Text PDF PubMed Google Scholar, 9Lenka N. Basu A. Mullick J. Avadhani N.G. J. Biol. Chem. 1996; 271: 30281-30289Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The expression of the COX Vb gene is particularly interesting, since it appears to involve a large combinatorial array of DNA elements and protein transactivators located at both the distal, as well as immediate upstream regions of the transcription initiation sites (7Carter R.S. Bhat N.K. Basu A. Avadhani N.G. J. Biol. Chem. 1992; 267: 23418-23426Abstract Full Text PDF PubMed Google Scholar, 9Lenka N. Basu A. Mullick J. Avadhani N.G. J. Biol. Chem. 1996; 271: 30281-30289Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Virbasius J.V. Virbasius C.A. Scarpulla R.C. Genes Dev. 1993; 7: 380-392Crossref PubMed Scopus (237) Google Scholar, 12Basu A. Park K. Atchison M.L. Carter R.S. Avadhani N.G. J. Biol. Chem. 1993; 268: 4188-4196Abstract Full Text PDF PubMed Google Scholar). Additionally, the COX Vb gene encodes multiple mRNAs that exhibit 5′ heterogeneity due to transcription initiation at multiple initiator sites (5Basu A. Avadhani N.G. J. Biol. Chem. 1991; 266: 15450-15456Abstract Full Text PDF PubMed Google Scholar, 10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar).The immediate upstream region of the mouse COX Vb gene consists of a TATA-less GC-rich sequence characteristic of a number of “housekeeping” genes. The basal promoter region has been mapped to the −8 to +40 region of the gene, which contains several protein-binding motifs. At least five of these protein-binding motifs correspond to discrete transcription initiation sites (10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar). Initiation of RNA mapping to the +1 position of the promoter has been shown previously to depend upon a YY-1-binding initiator motif (12Basu A. Park K. Atchison M.L. Carter R.S. Avadhani N.G. J. Biol. Chem. 1993; 268: 4188-4196Abstract Full Text PDF PubMed Google Scholar). Recently we have found that the GA-binding protein factor-binding Ets repeat sequence GTTCCCGGAAG at the +17 to +26 position also functions as an independent initiator for transcription of two clusters of RNAs mapping to the +18/19 and +23/24 regions (10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar).Although the COX Vb gene is constitutively expressed, its level of expression varies in different tissues. C0X Vb mRNA is 10-20-fold more abundant in the heart and kidney as compared to the liver. Furthermore, the COX Vb mRNA level increases markedly during induced differentiation of C2C12 myoblasts into myotubes. In the present study we have investigated the contributions of various protein-binding motifs from the 5′ distal region of the promoter in the overall transcriptional rates of the promoter. We have identified a negative regulatory region, which encompasses a YY-1-like (YY-1′) and CArG-like (CArG′) consensus motifs flanking a GTG element (13Zhao X.-A. Hung M.-C. Mol. Cell Biol. 1992; 12: 2739-2748Crossref PubMed Scopus (24) Google Scholar), a novel enhancer found in the neu oncogene promoter. Results presented in this paper show that factor YY-1 binding to the downstream CArG′ motif is important for the activity of the negative enhancer. Our results therefore suggest that in addition to the multiple positive cis acting elements (7Carter R.S. Bhat N.K. Basu A. Avadhani N.G. J. Biol. Chem. 1992; 267: 23418-23426Abstract Full Text PDF PubMed Google Scholar, 8Carter R.S. Avadhani N.G. J. Biol. Chem. 1994; 269: 4381-4387Abstract Full Text PDF PubMed Google Scholar, 9Lenka N. Basu A. Mullick J. Avadhani N.G. J. Biol. Chem. 1996; 271: 30281-30289Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 10Sucharov C. Basu A. Avadhani N.G. Gene Exp. 1996; 5: 93-111Google Scholar, 11Virbasius J.V. Virbasius C.A. Scarpulla R.C. Genes Dev. 1993; 7: 380-392Crossref PubMed Scopus (237) Google Scholar), a negative enhancer region is involved in the complex regulatory circuit of the COX Vb gene