The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two Repressors Regulate the Core Promoter of the Human Pro-α3(V) Collagen Gene (COL5A3)

Abstract

To elucidate the mechanisms underlining α3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the –129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to –122 to –117, the –101 to –96, the –83 to –78, and the –68 to –57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity. To elucidate the mechanisms underlining α3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the –129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to –122 to –117, the –101 to –96, the –83 to –78, and the –68 to –57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity. Vertebrate collagens, a large family of extracellular proteins, are critically important for the formation and function of virtually every organ system (1Vuorio E. de Crombregghe B. Annu. Rev. Biochem. 1990; 59: 837-872Crossref PubMed Scopus (392) Google Scholar). Among them, fibrillar collagen, which includes five different molecular types I, II, III, V, and XI, participates in the formation of fibrils with molecules packed in quarter-staggered arrays (2van der Rest M. Garrone R. FASEB J. 1991; 5: 2814-2823Crossref PubMed Scopus (994) Google Scholar, 3Brown J.C. Timpl R. Int. Arch. Allergy Immunol. 1995; 107: 484-490Crossref PubMed Scopus (125) Google Scholar). The fibrillar collagens are divided into major types (I–III) and minor types (V and XI) based on their relative expression levels. Minor fibrillar collagen types V and XI are incorporated into the fibrils of the much more abundant collagen types I and II, respectively, and act as regulators of the sizes and shapes of the resultant heterotypic fibrils (4Adachi E. Hayashi T. Connect. Tissue Res. 1986; 14: 257-266Crossref PubMed Scopus (143) Google Scholar, 5Mendler M. Eich-Bender S.G. Vaughn L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (405) Google Scholar, 6Linsenmayer T.F. Gibney E. Igoe F. Gordon M.K. Fitch J.M. Fessler L.I. Birk D.E. J. Cell Biol. 1993; 121: 1181-1189Crossref PubMed Scopus (247) Google Scholar, 7Andrikopoulos K. Liu X. Keene D.R. Jaenisch R. Ramirez F. Nat. Genet. 1995; 9: 31-39Crossref PubMed Scopus (189) Google Scholar). The collagen molecules are either homotrimers with α chains or heterotrimers with two or three different α chains. The predominant molecular form of type V is the heterotrimer [α1(V)]2α2(V) and is expressed in most tissues (8Burgeson R.E. El Adli F.A. Kaitila I.I. Hollister D.W. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2579-2583Crossref PubMed Scopus (306) Google Scholar). Other forms of type V collagen include the [α1(V)]3 homotrimer that is synthesized in cultures of hamster lung cells (9Haralson M.A. Michell W.M. Rhodes R.K. Kresina T.F. Gay R. Miller E.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5206-5210Crossref PubMed Scopus (65) Google Scholar) and the α1(V)α2(V)α3(V) heterotrimer that can be extracted from the placenta (10Sage H. Bornstein P. Biochemistry. 1979; 18: 3815-3822Crossref PubMed Scopus (151) Google Scholar, 11Brown R.A. Shuttleworth C.A. Weiss J.B. Biochem. Biophys. Res. Commun. 1978; 71: 1167-1174Google Scholar, 12Rhodes R.K. Miller E.J. Collagen Relat. Res. 1981; 1: 337-343Crossref PubMed Scopus (45) Google Scholar). Cross-type heterotrimer composed of α2(V) and α1(XI) chains is present in the rhabdomyosarcoma cell line A204 and bovine vitreous (13Kleman J.-P. Hartmann D.J. Ramirez F. van der Rest M. Eur. J. Biochem. 1992; 210: 329-335Crossref PubMed Scopus (67) Google Scholar, 14Mayne R. Brewton R.G. Mayne P.M. Baker J.R. J. Biol. Chem. 1993; 268: 9381-9386Abstract Full Text PDF PubMed Google Scholar). These findings suggest that type V and type XI chains constitute a single collagen type in which different combinations of chains associate in a tissue-specific manner. Recently, a fourth chain, α4(V), expressed in rat Schwann cells was reported (15Chernousov M.A. Rothblum K. Tyler W.A. Stahl R.C. Carey D.J. J. Biol. Chem. 2000; 275: 28208-28215Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This α chain, which can form molecules with α1(V) and α2(V) chains, seems to be the counterpart of the mouse and human α3(V) chain (16Imamura Y. Scott I.C. Greenspan D.S. J. Biol. Chem. 2000; 275: 8749-8759Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Type V collagen, which is widely expressed spatially and temporally, is expressed in the mouse embryo as early as 11 days post-coitum (17Wu Y.-L. Sumiyoshi H. Khaleduzzaman M. Ninomiya Y. Yoshioka H. Biochim. Biophys. Acta. 1998; 1397: 275-284Crossref PubMed Scopus (10) Google Scholar). Altered production of type V collagen is associated with some connective tissue pathology, such as inflammation, some forms of cancer, and atherosclerosis (18Narayanan A. Engel S. Page R.C. Collagen Relat. Res. 1983; 3: 323-334Crossref PubMed Scopus (36) Google Scholar, 19Barsky S.H. Rao C.N. Grotendorst G.R. Liotta L.A. Am. J. Pathol. 1982; 108: 276-283PubMed Google Scholar, 20Ooshima A. Science. 1981; 213: 666-668Crossref PubMed Scopus (113) Google Scholar). Characterization of the cis-acting elements and trans-acting nuclear factors that modulate correct patterns of gene expression is necessary for understanding physiological and pathological conditions. Among the collagen genes, the transcriptional regulation of type I collagen has been most extensively studied, showing common features of the proximal promoter and tissue-specific enhancer (21Ramirez F. Di Liberto M. FASEB J. 1990; 4: 16116-16123Crossref Scopus (88) Google Scholar, 22Slack J.L. Liska D.J. Bornstein P. Am. J. Med. Genet. 1993; 45: 140-151Crossref PubMed Scopus (110) Google Scholar). With regard to minor collagen, Penkov et al. (23Penkov D. Tanaka S. Rocco G.D. Berthelsen J. Blasi F. Ramirez F. J. Biol. Chem. 2000; 275: 16681-16689Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) identified the nuclear factors to the proximal promoter of that of the complex and of and in regulation of a of the gene repressed the gene promoter by with the that to the D.J. Biol. 18: PubMed Scopus Google Scholar). and Greenspan S. Greenspan D.S. Biochem. J. 1995; PubMed Scopus Google Scholar) that the in the promoter and the of the gene the the proximal promoter of the Sumiyoshi H. K. H. K. M. Yoshioka H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and K. Sumiyoshi H. T. Yoshioka H. Biol. PubMed Scopus Google Scholar). In we that the bound to the motif in the proximal promoter and is in of the Recently, human and mouse (16Imamura Y. Scott I.C. Greenspan D.S. J. Biol. Chem. 2000; 275: 8749-8759Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These to be to the In of mouse embryo its expression in of and within to and in forms molecules with α1(V) and In the present we the human promoter and the core promoter of the α3(V) collagen gene. DNase I footprinting and assays four nuclear protein in the promoter Cell transfection showed that as an and two as analysis with oligonucleotide competition and supershift assays showed that CBF/NF-Y as a transcriptional of and Cell of the cell in this from the Type and the of cell A204 cells rhabdomyosarcoma cell cells cell cells cell cells cell and cells cell in bovine in a cells cell and a cell line of human cells by Y. of in bovine in the of was from cells using to the was by and of electrophoretic mobility shift chromatin immunoprecipitation expression of COL5A3 in cell was by of was by using The was by using and The are in was for using a of for for for by for The by on a The was from the and into the for and of human was to the COL5A3 using the by the The specific for the COL5A3 in the are in The from the was and the by a using and specific for the The with in the To a that the promoter of the was with this site was upstream of the The into the The was by of was to the by et al. M.A. M.K. G.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of from A204 or cells was using the or was to the of the using in the of The was with using a the and a or which was upstream of or was for using a of for for for by for The by on a and into the for In and assays to the transcription A transcription from the of the was by This was into by with and into the site of with the was synthesized using the in transcription to the This was by and the was from the analysis was performed using a the of from A204 or cells and of in of and with the on a a was the the the was and The was by using a of to the from to bp of the COL5A3 was with and into the site of To the was performed using of oligonucleotide that are and specific for the COL5A3 and the as a These into the by with and and into the site of and by using the as a The in the are in The with by with and and into the site of of the CBF-B/NF-YA was by as Sumiyoshi H. K. H. K. M. Yoshioka H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. U. van D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and transfection using A204 and The cells a of transient of was into cells by using the system was as an for transfection an for the cells to the and to as in a using the to the The with a mutant type of CBF-B/NF-YA expression performed as Sumiyoshi H. K. H. K. M. Yoshioka H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Sumiyoshi H. T. Yoshioka H. Biol. PubMed Scopus Google Scholar). in and the expressed as the of three experiments. of from A204 and cells to the of et al. R.G. 1983; PubMed Scopus Google Scholar). in for and with The in on for and by for by in and by for for the and The protein of the nuclear was by using bovine as a DNase I for DNase I footprinting by using of oligonucleotides that are and specific for and into the These with and with using to in the with by a and from the DNase I footprinting was performed to the by and D.J. A. Res. 1978; 5: PubMed Scopus Google Scholar) with some the was for with of nuclear bovine as in a of bovine and of the of of DNase I with bovine as to the the was for The was by of a of and extracted with and with a the was and to a Electrophoretic and mutant for by using of and into the with and the with using to in the The was for in of of of and of nuclear the competition oligonucleotides a consensus and site by The consensus are in the supershift assay, and antibodies and of or of was to the for the of The DNA-protein complexes on a in a of the was and The was by using a assays performed using a chromatin immunoprecipitation to the A204 and cells a of The cells in a of for with cells from in and by for The cell in for and four for bp in the was in with and with the antibodies on the and with high and and with The by for with of To the of the with and for by with for The extracted with and with for using a of for for for by for The by on a by using the of COL5A3 in we the expression of the COL5A3 in cells using the in the of bp was in and A204 The by to the of the COL5A3 the was in and we to A204 and cell for the analysis of the COL5A3 of the of the COL5A3 we a human by and with the of of the with of two and in of the this which was in the promoter and and of COL5A3 To a the promoter region, the was with and into the The was by and the of the was using or a a the of COL5A3 was To the transcription of and assays The was using from A204 and from A204 cells and from and in the of the COL5A3 to bp upstream from the and of the from the which that the major transcription site is bp upstream from the the of we performed assays to the transcription In this we a the transcription and two specific in A204 and cells The and in and the to the of the we that COL5A3 two transcription and that the major transcription site is bp upstream from the The COL5A3 promoter lacks a TATA and has a high GC Functional of the COL5A3 the proximal in the COL5A3 promoter, a of and to the gene into A204 and cells and assays The from a of the of transcriptional activity. The of was with this in from to –129 bp on to bp in a with that of and to the bp showed of in A204 and In the mutant of which is –129 and showed a in These that the the –129 and bp is important for transcriptional of the COL5A3 of in the of the transcription on the proximal promoter of we performed DNase I different which the from the to bp and from the to bp in this in A and B, two in the to bp, and in the to bp in the COL5A3 To the of nuclear proteins to the proximal COL5A3 promoter, we experiments. the footprinting we three oligonucleotides the and bp The the to region, which the bind nuclear protein extracted from A204 and cells in a The was inhibited by oligonucleotide two and The was the site and the a site to in the form a DNA-protein whereas the in A204 and Furthermore, this DNA-protein complex A in the of of in the of analysis of the COL5A3 A, the and with nuclear from A204 and The DNA-protein complex is by A with an and performed nuclear B, competition analysis using an of The was with nuclear from A204 and cells in the of and and and and assays performed nuclear and with nuclear and C, competition analysis using an of The was with nuclear from A204 and cells in the of and of oligonucleotide and and and as The specific DNA-protein complexes are by B and C with and complex is by an D, using The and with nuclear from A204 and cells competition analysis using an of was with nuclear from A204 and cells in the of and of and and and as The specific DNA-protein complex is by D with an and the complex is by an using The and with nuclear from A204 and cells a was in DNase I footprinting and bp, the bind nuclear proteins extracted from A204 and and three and two DNA-protein complexes in A204 cells and and To complexes specific or we using and as The three DNA-protein complexes in A204 cells in the of the in a whereas the DNA-protein complex was in the of a The and complexes in the of that the and DNA-protein complexes are specific the complex is in A204 the the two DNA-protein complexes of cells in the of These showed two nuclear protein the and bp of the COL5A3 To nuclear protein we and and experiments. in the and form the DNA-protein complexes B and C, respectively. The from to bp the in the bind nuclear proteins extracted from A204 and cells and and two and DNA-protein complexes in A204 cells and respectively. the competition the DNA-protein complex in A204 cells was in the of a of the complex was in the of that the DNA-protein complex D, the and in form the DNA-protein complex D, and These with the of the DNase I footprinting of in the of the nuclear proteins in the core promoter of the we performed a analysis The a protein to form complex A, has the motif that is by transcription factors such as and and The site for DNA-protein complex B transcription for and and that of complex C was with and the a protein to form complex D, a be in an of the consensus oligonucleotides of inhibited the of the whereas and oligonucleotides the activity. the and and consensus oligonucleotides complexes B and C, To the of CBF/NF-Y to the we performed an using specific in DNA-protein complex A was by and by specific antibodies and and To CBF/NF-Y directly to we performed a DNA-protein complexes with the and by using for the to bp of the COL5A3 gene. in with antibodies CBF/NF-Y be by using the as as the whereas with or These that CBF/NF-Y to the motif in the COL5A3 core promoter, in immunoprecipitation A was performed to the of CBF/NF-Y to the COL5A3 promoter, in vivo. immunoprecipitation immunoprecipitation with immunoprecipitation with antibodies and by with the The CBF/NF-Y and the of the DNase I footprinting and four nuclear protein identified and To nuclear protein a of of a into A204 and cells and assays The of was with that of in the of the was whereas of and in A204 and the the of the and with that of the These that the nuclear protein of DNA-protein complex A, the COL5A3 promoter and of complex B and C with overexpression of the CBF-B/NF-YA subunit, which with the CBF-A/NF-YB and CBF-C/NF-YC to form CBF/NF-Y K. Sumiyoshi H. T. Yoshioka H. Biol. PubMed Scopus Google the promoter in A204 and cells by of overexpression of CBF-B/NF-YA on the COL5A3 promoter activity. with of and of mutant CBF/NF-Y expression or the of and the are the of three experiments. The from the of COL5A3 transcriptional activity. The promoter has transcriptional start lacks the TATA bp from the major transcriptional start and has a high GC These features are for the promoter of To the proximal in the COL5A3 promoter, a of and to the gene into A204 and The from –129 to bp was necessary for the transcriptional of With the combination analysis using footprinting and we identified four nuclear protein the and Among them, we that CBF/NF-Y bound to the motif in the –122 to and as an and the factors in the –101 to bp and in the –83 to act as of collagen is by a of complex of and transcription A of elements for and been identified in the promoter of type I collagen (21Ramirez F. Di Liberto M. FASEB J. 1990; 4: 16116-16123Crossref Scopus (88) Google Scholar, 22Slack J.L. Liska D.J. Bornstein P. Am. J. Med. Genet. 1993; 45: 140-151Crossref PubMed Scopus (110) Google Scholar). The identified the and and CBF/NF-Y factors for of Y. S. Ramirez F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. K. M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, H. K. T. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, de Crombregghe B. Science. PubMed Scopus Google Scholar, A. van E. de Crombregghe B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, F. de Crombregghe B. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Other transcription factors such as and are in the transcriptional regulation of type I collagen P. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. de Crombregghe B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, de Crombregghe B. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). In we that CBF/NF-Y and two are in the regulation of the core CBF/NF-Y was identified as the proteins bound to the major complex in and the A. J. A. D. Full Text PDF PubMed Scopus Google Scholar, S. L. Science. Scopus Google Scholar). was using the the collagen and the A. van E. de Crombregghe B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, D. H. D. J. 1990; 9: PubMed Scopus Google Scholar). CBF/NF-Y is a heterotrimer composed of three subunits, and necessary for S. J. de Crombregghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). CBF/NF-Y the in a R. Res. 1998; PubMed Scopus Google Scholar). CBF/NF-Y to bind to the three and five to be important These and are in COL5A3 for the of the bind to the –83 to bp, in the core promoter of The to the as an such as T. A. D. H. 1991; Full Text PDF PubMed Scopus Google Scholar). of the factors for the specific expression of mouse in to the and P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in the is in the of transcription of R.A. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The a of the has a and The which is a bound to to bp in the promoter of rat gene, and repressed its transcription D. P. R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). et al. K. T. P. A. T. Y. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported in the and the type protein the promoter of and that four to the bp from the transcription start site with the Other bound to –101 to bp with the competition using the oligonucleotide of The showed which factors from a the gene by bp, whereas which has a P. P. J. M. P. J. M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). repressed the transcription of type I collagen D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and D.J. Biol. 18: PubMed Scopus Google Scholar). The family a in the regulation of α1(V)α2(V)α3(V) molecules extracted from human placenta (10Sage H. Bornstein P. Biochemistry. 1979; 18: 3815-3822Crossref PubMed Scopus (151) Google Scholar, 11Brown R.A. Shuttleworth C.A. Weiss J.B. Biochem. Biophys. Res. Commun. 1978; 71: 1167-1174Google Scholar, 12Rhodes R.K. Miller E.J. Collagen Relat. Res. 1981; 1: 337-343Crossref PubMed Scopus (45) Google Scholar). The α3(V) chain was from R.A. Shuttleworth C.A. Weiss J.B. Biochem. Biophys. Res. Commun. 1978; 71: 1167-1174Google and S. Weiss J.B. 1982; PubMed Scopus Google Scholar). In of mouse embryo α3(V) expression in the of and within to and in (16Imamura Y. Scott I.C. Greenspan D.S. J. Biol. Chem. 2000; 275: 8749-8759Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). et al. M.A. Stahl R.C. Carey D.J. J. PubMed Google Scholar) reported the of which is expressed by Schwann cells in named the rat chain that to human α3(V) chain (15Chernousov M.A. Rothblum K. Tyler W.A. Stahl R.C. Carey D.J. J. Biol. Chem. 2000; 275: 28208-28215Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In human α3(V) chain is expressed in the Schwann cell The expression of α3(V) chain is abundant with α1(V) chain, in more tissues The [α1(V)]2α2(V) form is the tissue and has been extensively is with type I collagen and forms type heterotypic The of the α1(V)α2(V)α3(V) form has that CBF/NF-Y is for the expression of the α1(V) gene and type I collagen Sumiyoshi H. K. H. K. M. Yoshioka H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this we showed that to the COL5A3 Penkov et al. (23Penkov D. Tanaka S. Rocco G.D. Berthelsen J. Blasi F. Ramirez F. J. Biol. Chem. 2000; 275: 16681-16689Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) identified and proteins as an complex in the proximal a cis-acting that in some of the In fibrillar collagen genes, the expression of the type collagen gene is different from of type I collagen The promoter of the gene has a and the tissue-specific expression is by the enhancer in the the family de Crombregghe B. Biol. PubMed Google Scholar). type collagen is expressed with type I collagen in tissues and the of regulation seems to be different from of type I collagen of which is a protein of bind in the proximal promoter of the gene de Crombregghe B. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). CBF/NF-Y is a be for the expression in tissue such as type I and V collagen is to the in promoter from –129 to bp by the of CBF/NF-Y the mutant of CBF-B/NF-YA was Other that be identified the of system bind in this the we showed two in this of The of factors CBF/NF-Y on the type V collagen expression, by of different for the of type V Y. for the of K. for this and the of the of and and the of of we performed some experiments. with