Michael W. Starbuck

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1−/− mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1−/− pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1−/− animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1−/− mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1−/− mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

Biologically active molecular clones of avian sarcoma virus 17 (ASV 17) contain a replication-defective proviral genome of 3.5 kilobases (kb). The genome retains partial gag and env sequences, which flank a cell-derived putative oncogene of 0.93 kb, termed jun. The jun gene lacks preserved coding domains of tyrosine-specific protein kinases. It also shows no significant nucleic acid homology with other known oncogenes. The probable transformation-specific protein in ASV 17-transformed cells is a 55-kDa gag-jun fusion product.

Type I collagen is composed of two chains, α1(I) and α2(I), encoded by two distinct genes, the α1(I) and α2(I) collagen genes, that are highly expressed in osteoblasts. In most physiological situations, α1(I) and α2(I) collagen expression is coregulated, suggesting that identical transcription factors control their expression. Here, we studied the role of Cbfa1, an osteoblast-specific transcription factor, in the control of α1(I) and α2(I) collagen expression in osteoblasts. A consensus Cbfa1-binding site, termed OSE2, is present at the same location in the α1(I) collagen promoter at approximately −1347 base pairs (bp) of the rat, mouse, and human genes. Cbfa1 can bind to this site, as demonstrated by electrophoretic mobility shift assay (EMSA) and supershift experiments using an anti-Cbfa1 antibody. Mutagenesis of the α1(I) collagenOSE2 at −1347 bp reduced the activity of a α1(I) collagen promoter fragment 2- to 3-fold. Moreover, multimers of this OSE2 at −1347bp confer osteoblast-specific activity to a minimum α1(I) collagen promoter fragment in DNA transfection experiments as well as in transgenic mice. An additional Cbfa1-binding element is present in the α1(I) collagen promoter of mouse, rat, and human at approximately position −372. This site binds Cbfa1 only weakly and does not act as a cis-acting activator of transcription when tested in DNA transfection experiments. Similar to α1(I) collagen, the mouse α2(I) collagen gene contains multiple OSE2 sites, of which one is conserved across multiple species. In EMSA, Cbfa1 binds to this site and multimers of this α2(I) OSE2 element confer osteoblast-specific activity to the minimum α1(I) collagen promoter in DNA transfection experiments. Thus, our results suggest that Cbfa1 is one of the positive regulators of the osteoblast-specific expression of both type I collagengenes. Type I collagen is composed of two chains, α1(I) and α2(I), encoded by two distinct genes, the α1(I) and α2(I) collagen genes, that are highly expressed in osteoblasts. In most physiological situations, α1(I) and α2(I) collagen expression is coregulated, suggesting that identical transcription factors control their expression. Here, we studied the role of Cbfa1, an osteoblast-specific transcription factor, in the control of α1(I) and α2(I) collagen expression in osteoblasts. A consensus Cbfa1-binding site, termed OSE2, is present at the same location in the α1(I) collagen promoter at approximately −1347 base pairs (bp) of the rat, mouse, and human genes. Cbfa1 can bind to this site, as demonstrated by electrophoretic mobility shift assay (EMSA) and supershift experiments using an anti-Cbfa1 antibody. Mutagenesis of the α1(I) collagenOSE2 at −1347 bp reduced the activity of a α1(I) collagen promoter fragment 2- to 3-fold. Moreover, multimers of this OSE2 at −1347bp confer osteoblast-specific activity to a minimum α1(I) collagen promoter fragment in DNA transfection experiments as well as in transgenic mice. An additional Cbfa1-binding element is present in the α1(I) collagen promoter of mouse, rat, and human at approximately position −372. This site binds Cbfa1 only weakly and does not act as a cis-acting activator of transcription when tested in DNA transfection experiments. Similar to α1(I) collagen, the mouse α2(I) collagen gene contains multiple OSE2 sites, of which one is conserved across multiple species. In EMSA, Cbfa1 binds to this site and multimers of this α2(I) OSE2 element confer osteoblast-specific activity to the minimum α1(I) collagen promoter in DNA transfection experiments. Thus, our results suggest that Cbfa1 is one of the positive regulators of the osteoblast-specific expression of both type I collagengenes. kilobase(s) dithiothreitol electrophoretic mobility shift assay base pair(s) Type I collagen is the most abundant protein of the bone extracellular matrix, accounting for 90% of the matrix protein content (1Gehron-Robey P. Principles of Bone Biology. Academic Press, San Diego, CA1996: 155-166Google Scholar). It is a heterotrimer made of two α1(I) chains and one α2(I) chain (2van der Rest M. Garrone R. FASEB. J. 1991; 5: 2814-2823Crossref PubMed Scopus (1015) Google Scholar). The α1(I) and α2(I) chains are encoded by two distinct genes that are expressed most highly in two cell types: the fibroblast and the osteoblast. Moreover, the expression of these two genes is often regulated by identical transcription factors (3Maity S.N. Golumbek P.T. Karsenty G. de Crombrugghe B. Science. 1988; 241: 582-585Crossref PubMed Scopus (171) Google Scholar, 4Karsenty G. de Crombrugghe B. J. Biol. Chem. 1990; 265: 9934-9942Abstract Full Text PDF PubMed Google Scholar, 5Nehls M.C. Rippe R.A. Veloz L. Brenner D.A. Mol. Cell. Biol. 1991; 11: 4065-4073Crossref PubMed Google Scholar, 6Karsenty G. de Crombrugghe B. Biochem. Biophys. Res. Commun. 1991; 177: 538-544Crossref PubMed Scopus (66) Google Scholar, 7Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Thetype I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life (8Sandberg M. Autio-Harmainen H. Vuorio E. Dev. Biol. 1988; 130: 324-334Crossref PubMed Scopus (87) Google Scholar), suggesting that the factor(s) controlling their expression in these cells could also control osteoblast differentiation and function. Another possibility, which is not exclusive of the previous one, is that different transcription factors may control their expression in osteoblasts at various stages of development and of postnatal life. Thus, the elucidation of the molecular mechanisms controlling α1(I)and α2(I) collagen gene expression in osteoblasts is of critical importance in understanding how osteoblast differentiation and, thereby, bone matrix deposition by differentiated osteoblasts is regulated. Ultimately, these studies may shed light on the pathogenesis of genetically acquired bone diseases and help design appropriate therapies for some of these diseases. The critical role that Cbfa1, a Runt-related osteoblast-specific transcription factor, plays in osteoblast differentiation and function has been demonstrated in mouse and in human using both molecular and genetic approaches (9Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3708) Google Scholar, 10Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3731) Google Scholar, 11Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2456) Google Scholar, 12Mundlos S. Otto F. Mundlos C. Mulliken J.B. Aylsworth A.S. Albright S. Lindhout D. Cole W.G. Henn W. Knoll J.H. Owen M.J. Mertelsmann R. Zabel B.U. Olsen B.R. Cell. 1997; 89: 773-779Abstract Full Text Full Text PDF PubMed Scopus (1294) Google Scholar, 13Lee B. Thirunavukkarasu K. Zhou L. Pastore L. Baldini A. Hecht J. Geoffroy V. Ducy P. Karsenty G. Nat. Genet. 1997; 16: 307-310Crossref PubMed Scopus (500) Google Scholar, 14Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (715) Google Scholar). Cbfa1 was identified as a key regulator of osteoblast-specific gene expression through its binding to the OSE2 element of the mouse Osteocalcin genes 1 and 2 (OG1 and OG2) (9Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3708) Google Scholar) and other genes expressed in osteoblasts. The early and cell-specific expression of this gene together with its biological role in vivo as a factor required for osteoblast differentiation (9Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3708) Google Scholar, 10Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3731) Google Scholar, 11Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2456) Google Scholar), indicate that Cbfa1 must control the expression of multiple target genes that are expressed earlier than Osteocalcin. Conceivably, these target genes could include the α1(I) and α2(I) collagengenes that are expressed early during development. This hypothesis was confirmed indirectly by the observation that expression of a dominant negative form of Cbfa1 in differentiated osteoblasts leads to a decrease in expression of the type I collagen genes in vivo (14Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (715) Google Scholar). To date, no osteoblast-specific cis-acting elements to which Cbfa1 may bind have been identified in these genes. Two groups have extensively studied the regulation of expression of the α1(I) collagen gene in osteoblasts and have identified a region in the promoter of the rat and mouse α1(I) collagengene that plays an important role in this regulation of expression (15Bogdanovic Z. Bedalov A. Krebsbach P.H. Pavlin D. Woody C.O. Clark S.H. Thomas H.F. Rowe D.W. Kream B.E. Lichtler A.C. J. Bone Miner. Res. 1994; 9: 285-292Crossref PubMed Scopus (95) Google Scholar,16Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar). The sequence of this region bears no homology to a Cbfa1-binding site, and several homeobox-containing proteins can bind to this sequence and affect α1(I) collagen expression. However, a cell-specific transcription factor binding to this region has not yet been identified. Moreover, no osteoblast-specific cis-acting element has yet been identified in the α2(I) collagenpromoter. Given the large size of these genes and their expression at multiple stages of osteoblast differentiation, it is likely that several distinct osteoblast-specific cis-acting elements, besides those already described (15Bogdanovic Z. Bedalov A. Krebsbach P.H. Pavlin D. Woody C.O. Clark S.H. Thomas H.F. Rowe D.W. Kream B.E. Lichtler A.C. J. Bone Miner. Res. 1994; 9: 285-292Crossref PubMed Scopus (95) Google Scholar, 16Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar), contribute to the expression of the type I collagen genes in osteoblast progenitors and/or in fully differentiated osteoblasts. Consistent with this hypothesis, we noticed the existence of two Cbfa1-binding sites (OSE2s) in the mouse α1(I) collagen promoter and one OSE2 in the mouse α2(I) collagen gene that are conserved among multiple species. The functional importance of these sites has never been studied before. Here we present evidence suggesting that Cbfa1 is one of the factors controlling osteoblast-specific expression of bothtype I collagen genes. For DNA transfection and generation of transgenic mice, multimers of double-stranded oligonucleotides (see Table I) were cloned into the SmaI site upstream of a chimeric pK1-luc reporter plasmid containing the α1(I) collagen −86 minimal promoter (4Karsenty G. de Crombrugghe B. J. Biol. Chem. 1990; 265: 9934-9942Abstract Full Text PDF PubMed Google Scholar) fused to a luciferase gene. The expression plasmid was pCMV-Osf2 (9Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3708) Google Scholar).Table IOligonucleotides used in this studyOligonucleotide nameSequenceα1AB5′-GATCCCTTCCCACACCACCCACACAGA-3′α1wtAmutB5′-GATCCCTTCGAACACCACCCACACAGA-3′α1mutAwtB5′-GATCCCTTCCCACACCACGAACACAGA-3′α1mutAmutB5′-GATCCCTTCGAACACCACGAACACAGA-3′α1C5′-GATCTACTGAGGGCCCAGCCACACTCCA-3′α1mutC5′-GATCTACTGAGGGCCCAGGAACACTCCA-3′α2A5′-GATCCCTTTGTGGATACGCGGACTTTGA-3′α2mutA5′-GATCCCTTTGTTCATACGCGGACTTTGA-3′Boldface letters indicate mutated nucleotides. Open table in a new tab Boldface letters indicate mutated nucleotides. Site-specific mutations were created in the intact promoter by using polymerase chain reaction-directed mutagenesis (17Ausubel F.M. Brent R. Kingson R.E. Moore D.D. Seidman J.G. K. in Biology. Scholar) on a containing of the α1(I) collagen promoter upstream of a luciferase reporter gene. of the mutations was by and cells were in minimal cells were in minimal cells were at a of and to For we used of Cbfa1 expression of reporter plasmid and 2 of using the (17Ausubel F.M. Brent R. Kingson R.E. Moore D.D. Seidman J.G. K. in Biology. Scholar). were identical to those used in the that of reporter plasmid were cells were in and in an additional cells were to containing and to for were by into and by and luciferase were as described R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). assay results were used to the luciferase assay results for transfection DNA transfection experiments were at in and other were as described P. Karsenty G. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar) and at was using as described (17Ausubel F.M. Brent R. Kingson R.E. Moore D.D. Seidman J.G. K. in Biology. Scholar). oligonucleotides (see Table I) were and as described P. Karsenty G. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). of was with of of Cbfa1 α2(I) collagen experiments used the of The for binding of binding 2 S. A. 1995; PubMed Scopus Google Scholar), 2 of and of at for experiments were as described that the was for at with an Cbfa1 in binding to their with the for at For protein binding the of binding and 1 of at for by the of 1 of The were on 2 for at V. The were and to at The described containing multimers of the oligonucleotides were and the was by two of DNA were into the of mouse which were in the of were identified by of The transgenic the were as were and on in a containing and 1 dithiothreitol were and were for luciferase activity to P. Karsenty G. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). were using the protein luciferase were expressed as luciferase light of protein expressed as a of the activity in DNA sequence identified OSE2 sites in the promoter of the α1(I) collagen gene. Two are at −1347 and in the mouse gene and a site is at in the mouse gene 1 OSE2 sites were termed and The of and not of at approximately the same location in the α1(I) collagen promoter of rat and human 1 a biological role for these sites and to these two To Cbfa1 could bind to the in the mouse α1(I) collagen we double-stranded oligonucleotides to used in DNA binding of contains the and sites and their A one, termed contains the site and a mutated A a mutated sequence and a A contains mutations in both the and the Two other termed and were to the binding activity of the The mutations into all the oligonucleotides have been to binding of Cbfa1 to the OSE2 sequence present in the Osteocalcin promoter P. Karsenty G. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). double-stranded oligonucleotides were used as in electrophoretic mobility shift (EMSA) using Cbfa1 as a of The of with at the same location as the of with the 1 it was of In no was when using as a To which of the two upstream OSE2 sites was binding to this factor, we used as in of with a that the same mobility as the one when using as a and was of In when using as a we only a binding of to the DNA 2 To this osteoblast-specific factor binding to the element was Cbfa1, we of experiments. we the factor present in and binding to the site was expressed only in osteoblasts. For that we osteoblasts and several other and used in in 2 the factor binding to the was present only in osteoblast and not in of other we supershift experiments using anti-Cbfa1 a of the with an Cbfa1 to of to the of a of mobility 2 a no that the of the with contains Cbfa1, this is for Cbfa1 (14Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (715) Google Scholar). we Cbfa1 could bind to not to 2 of with Cbfa1 in the of a 2 using not 2 these results indicate that the site, a site conserved in multiple is the binding site for Cbfa1 in this region of the α1(I) collagen the functional of the and sites using two additional to the of the site on activity of a promoter a was in the site polymerase chain and into a α1(I) chimeric gene. mutations in a decrease in promoter activity when tested in DNA transfection experiments in cells The same mutations not the activity of this α1(I) collagen chimeric gene in other cell of of and This that this cis-acting element is only in osteoblasts. in the site not affect the activity of the fragment of the α1(I) used in this in DNA in a cell that does not Cbfa1 M. T. K. S. K. Y. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), Cbfa1 a containing of the fused to a minimal α1(I) collagen chimeric of with a in a an expression no This of is to we when using a containing of the as a reporter The minimal α1(I) fragment has no on its (4Karsenty G. de Crombrugghe B. J. Biol. Chem. 1990; 265: 9934-9942Abstract Full Text PDF PubMed Google Scholar). using a containing multimers of oligonucleotides cloned upstream of the collagen promoter we a in luciferase that the site is the to the function of this region of the α1(I) collagen This is with the observation that only the site is to bind Cbfa1 in The decrease in activity with the of the site may indicate a of these two sites in this type of The activity of containing of of upstream of the minimal α1(I) collagen chimeric gene could not with the the of the In this of we used multimers of the α1(I) collagen OSE2 sites, Cbfa1 does not the α1(I) collagen promoter fragment in this type of This is a of Cbfa1 we of the promoter when with Cbfa1 (9Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3708) Google Scholar), with the of Cbfa1 when using (9Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3708) Google Scholar). we these OSE2 sites could confer expression to a reporter gene in For that we transgenic containing two of the used in the and In transgenic luciferase activity could in bone in other type I collagen, in which not type I collagen their the expression of was than that of α1(I) collagen not The was not expressed in bone other together with the results of the mutagenesis of the site and of the site, these results indicate that Cbfa1 to the expression of α1(I) osteoblasts through the at the of is a OSE2 sequence in collagen at in mouse 1 This site is also conserved across and we this sequence could by Cbfa1 in oligonucleotides were with as described to the of a that at the same location as that of with 1 However, the was of with that we when using oligonucleotides as 1 and of this size was of with To that Cbfa1 was of this supershift experiments were using an anti-Cbfa1 a and as a of The of with an Cbfa1 to the of to the of a mobility This was it was not when using a and experiments using Cbfa1 evidence that this site could Cbfa1, Cbfa1 was to bind to not to 1 and results indicate that Cbfa1 is to bind only weakly to the site in the mouse α1(I) collagen suggesting that this OSE2 site may not a critical To this hypothesis, we cloned of mutated oligonucleotides upstream of the minimal α1(I) collagen promoter chimeric gene used in A. in in DNA transfection experiments in could the activity of this reporter gene with a Cbfa1 expression This of an role for the site is with the binding of Cbfa1 to this these results indicate that the site is not a critical cis-acting element in controlling the osteoblast-specific expression of the collagen gene. α1(I) collagen genes are often coregulated, we Cbfa1 was also the expression of collagen promoter the existence of several OSE2 one of in the of the is present at the same location in collagen gene of multiple 1 For this this site was studied DNA binding was was using a double-stranded containing the OSE2 element as a and as a of of with in the generation of a at the same location as the of with and 1 This was it not form of with an containing a in this OSE2 sequence the binding of to was using a of we also used Cbfa1 protein in The of Cbfa1 with using a of Cbfa1 with that used to binding of Cbfa1 to in the of a This not form of Cbfa1 with that the conserved OSE2 sequence present in collagen gene can bind Cbfa1, weakly than the To the function of the site, were in In this Cbfa1 a containing a of oligonucleotides fused to a minimal α1(I) collagen chimeric an in luciferase This was the containing a of oligonucleotides no The in luciferase activity with the with multimers of the is with the binding of Cbfa1 to the our evidence that Cbfa1 is one of the transcription factors to the expression of the two Type I collagen genes in osteoblasts in Moreover, with (3Maity S.N. Golumbek P.T. Karsenty G. de Crombrugghe B. Science. 1988; 241: 582-585Crossref PubMed Scopus (171) Google Scholar, 4Karsenty G. de Crombrugghe B. J. Biol. Chem. 1990; 265: 9934-9942Abstract Full Text PDF PubMed Google Scholar, R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and M.C. Rippe R.A. Veloz L. Brenner D.A. Mol. Cell. Biol. 1991; 11: 4065-4073Crossref PubMed Google Scholar, 7Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), Cbfa1 to a of transcription factors accounting for the regulation of both genes. The that Cbfa1 type I collagen expression in osteoblasts is with the of bone extracellular matrix in and with the decrease of Type I collagen expression in transgenic a dominant negative form of Cbfa1 in osteoblasts T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3731) Google Scholar, 11Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2456) Google Scholar, 14Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (715) Google Scholar). results the of transcription factors to type I collagen gene expression at various stages of osteoblast differentiation and our understanding of type I collagen The indicate that is a functional the different Cbfa1-binding sites, OSE2 sites, present in the α1(I) collagen the element is the most activator of expression of all the OSE2 elements we studied in this not the that OSE2 sites present upstream in the promoter and/or in the gene may also contribute to the osteoblast-specific expression of the α1(I) collagen gene. Conceivably, one of these as of yet OSE2 sites may bind Cbfa1 with a and act as a osteoblast-specific cis-acting not conserved consensus OSE2 sites by the DNA sequence of the region and that has been to required for osteoblast expression Eberspaecher H. de Crombrugghe B. S. A. PubMed Scopus (95) Google Scholar). This the hypothesis that other cell-specific transcription factors must contribute to osteoblastic expression of the type I collagen genes. Cbfa1 can bind to a site present in collagen gene and the expression of this gene is in transgenic a dominant negative form of Cbfa1, the of in experiments with the was than that when using the on the α1(I) collagen two could for this and most the binding of to the site was than its binding to the site of α1(I) collagen, that this site has a to the OSE2 sites present in the α2(I) collagen it is likely for this gene and for collagen gene as some of the other OSE2 sites act in with the conserved OSE2 site to control its expression in osteoblasts in Cbfa1 is one positive regulator of type I in it is the that it is not the only Cbfa1 expression is in osteoblast progenitors type I collagen expression can noticed in Moreover, at one element has been to in osteoblast-specific expression of the α1(I) collagen gene in mouse and rat (15Bogdanovic Z. Bedalov A. Krebsbach P.H. Pavlin D. Woody C.O. Clark S.H. Thomas H.F. Rowe D.W. Kream B.E. Lichtler A.C. J. Bone Miner. Res. 1994; 9: 285-292Crossref PubMed Scopus (95) Google Scholar, 16Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar). of the of proteins are to bind to this sequence and to transcription T. M. M. R.A. D.A. Lichtler A.C. Scholar). Another can bind to this sequence and expression of the α1(I) collagen gene M. Bedalov A. Kream G. Clark S.H. K. Y. R. Rowe D.W. Lichtler A.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and genetic evidence has demonstrated that is upstream of Cbfa1 L. H. M. K. T. T. M. S. H. Z. R. R. Nat. Genet. PubMed Scopus Google Scholar). proteins are likely to expressed earlier than Cbfa1 and may control its It is to that these with other act early during the of cells to the osteoblast and that Cbfa1 is required for osteoblast differentiation and for the of the osteoblast This hypothesis when for several proteins are the observation that Cbfa1 binds to and the activity of both type I collagen genes in osteoblasts how important Cbfa1 is in osteoblast P. Ducy and T. for the