Identification of CCAAT/Enhancer-binding Protein α as a Transactivator of the Mouse Amelogenin Gene

Abstract

Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels. In a previous transgenic mouse analysis, the expression pattern of the endogenous amelogenin gene was recapitulated by a reporter gene driven by a 2.2-kilobase mouse amelogenin proximal promoter. To understand the molecular mechanisms underlying the spatiotemporal expression of the amelogenin gene during odontogenesis, the mouse amelogenin promoter was systematically analyzed in mouse ameloblast-like LS8 cells. Deletion analysis identified a minimal promoter (−70/+52) containing a CCAAT/enhancer-binding protein (C/EBP)-binding site upstream of the TATA box. In transient transfection assays, C/EBPα up-regulated the promoter activity in a dose-dependent manner. The C/EBP-binding site was necessary for both C/EBPα-mediated transactivation and basal promoter activity. Electrophoresis mobility shift assays demonstrated that C/EBPα bound to its cognate site in the amelogenin promoter and that the binding was specific. Endogenous C/EBPα was detected in LS8 cells, and overexpression of exogenous C/EBPα in LS8 cells was able to increase the expression level of the endogenous amelogenin protein. The activity of the amelogenin promoter in rat parotid Pa-4 cells and Madin-Darby canine kidney cells was minimal, ranging from 20 to 30% of the activity in ameloblast-like cells. Transient transfection experiments showed that C/EBPα transactivated the mouse amelogenin reporter gene in Pa-4 cells, but not in Madin-Darby canine kidney cells. Taken together, these data indicate that C/EBPα is a bona fide transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels. In a previous transgenic mouse analysis, the expression pattern of the endogenous amelogenin gene was recapitulated by a reporter gene driven by a 2.2-kilobase mouse amelogenin proximal promoter. To understand the molecular mechanisms underlying the spatiotemporal expression of the amelogenin gene during odontogenesis, the mouse amelogenin promoter was systematically analyzed in mouse ameloblast-like LS8 cells. Deletion analysis identified a minimal promoter (−70/+52) containing a CCAAT/enhancer-binding protein (C/EBP)-binding site upstream of the TATA box. In transient transfection assays, C/EBPα up-regulated the promoter activity in a dose-dependent manner. The C/EBP-binding site was necessary for both C/EBPα-mediated transactivation and basal promoter activity. Electrophoresis mobility shift assays demonstrated that C/EBPα bound to its cognate site in the amelogenin promoter and that the binding was specific. Endogenous C/EBPα was detected in LS8 cells, and overexpression of exogenous C/EBPα in LS8 cells was able to increase the expression level of the endogenous amelogenin protein. The activity of the amelogenin promoter in rat parotid Pa-4 cells and Madin-Darby canine kidney cells was minimal, ranging from 20 to 30% of the activity in ameloblast-like cells. Transient transfection experiments showed that C/EBPα transactivated the mouse amelogenin reporter gene in Pa-4 cells, but not in Madin-Darby canine kidney cells. Taken together, these data indicate that C/EBPα is a bona fide transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. CCAAT/enhancer-binding protein(s) Madin-Darby canine kidney electrophoresis mobility shift assay nuclear factor Y CAAT-binding factor One unique characteristic of tooth development is the formation of mineralizing extracellular matrices. Enamel, the only epithelially derived calcified tissue in vertebrates, is synthesized by ameloblasts. Amelogenins are essential to the proper regulation of enamel mineralization. Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels (1.Snead M.L. Bringas Jr., P. Bessem C. Slavkin H.C. Dev. Biol. 1984; 104: 255-258Crossref PubMed Scopus (35) Google Scholar, 2.Snead M.L. Lau E. Zeichner-David M. Nanci T. Bendayan N. Bringas P. Bessem C. Slavkin H.C. Firtel A. Davidson E. Molecular Approaches to Developmental Biology. Alan Liss, Inc., New York1987: 641-652Google Scholar, 3.Snead M.L. Luo W. Lau E.C. Slavkin H.C. Development (Camb.). 1988; 104: 77-85Crossref PubMed Google Scholar, 4.Couwenhoven R.I. Schwartz S.A. Snead M.L. J. Craniofacial Genet. Dev. Biol. 1993; 13: 259-269PubMed Google Scholar, 5.Snead M.L. Paine M.L. Chen L.S. Luo B.Y. Zhou D.H. Lei Y.P. Liu Y.H. Maxson Jr., R.E. Connect. Tissue Res. 1996; 35: 41-47Crossref PubMed Scopus (36) Google Scholar, 6.Diekwisch T.G. Ware J. Fincham A.G. Zeichner-David M. J. Histochem. Cytochem. 1997; 45: 859-866Crossref PubMed Scopus (39) Google Scholar, 7.Zeichner-David M. Vo H. Tan H. Diekwisch T. Berman B. Thiemann F. Alcocer M.D. Hsu P. Wang T. Eyna J. Caton J. Slavkin H.C. MacDougall M. Int. J. Dev. Biol. 1997; 41: 27-38PubMed Google Scholar, 8.Chen E. Piddington R. Decker S. Park J. Yuan Z.A. Abrams W.R. Rosenbloom J. Feldman G. Gibson C.W. Dev. Dyn. 1994; 199: 189-198Crossref PubMed Scopus (45) Google Scholar). A 2263-nucleotide proximal promoter element from the mouse X-chromosomal amelogenin gene has been demonstrated by transgenic mouse analysis to direct the expression of a reporter gene in a temporal and spatial pattern that is essentially identical to that of the endogenous amelogenin gene (5.Snead M.L. Paine M.L. Chen L.S. Luo B.Y. Zhou D.H. Lei Y.P. Liu Y.H. Maxson Jr., R.E. Connect. Tissue Res. 1996; 35: 41-47Crossref PubMed Scopus (36) Google Scholar). During organogenesis, a programmed differentiation of embryonic epithelial cells is often characterized by the highly regulated expression of tissue-specific genes activated in response to inductive interactions with embryonic mesenchyme. The biochemical identities of the inductive signals that direct mammalian epithelial determination and differentiation have been elusive; however, studies of the regulated expression of tissue-specific gene products in developing epithelia may facilitate the molecular dissection of these interactions. The regulated expression of the ameloblast-specific amelogenin gene in the developing mouse tooth organ is an excellent model for studying developmentally regulated gene expression. We hypothesize that the regulated transcription of amelogenin by specific activator(s) and repressor(s) in ameloblast cell lineage results in the spatiotemporal expression of amelogenin required for proper enamel formation. Gene expression is regulated at several levels, including activation of gene structure, transcription initiation, termination of transcription, nuclear RNA processing, mRNA translation, and mRNA stability. Unlike several other developmentally regulated genes (9.Cedar H. Cell. 1988; 53: 3-4Abstract Full Text PDF PubMed Scopus (730) Google Scholar), the methylation pattern of CpG islands in the promoter region is not associated with the regulated transcription of the amelogenin gene (10.Couwenhoven R.I. Snead M.L. Dev. Biol. 1994; 164: 290-299Crossref PubMed Scopus (44) Google Scholar). Extensive homologies exist in the promoter regions of the bovine, human, and murine X-chromosomal amelogenin genes. There is a 70% identity within the 300-base pair region upstream of the transcription initiation site, suggesting that transcriptional regulation is likely to play an important role in the spatiotemporal expression of amelogenin. For a TATA box-containing promoter, the preinitiation complex is assembled in a highly regulated and defined order (11.Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar). The assembly of the preinitiation complex on a core promoter is sufficient to initiate transcription at a minimal level. However, the rate of transcription can be increased by an activator or turned off by a repressor. The CCAAT/enhancer-binding proteins (C/EBPs)1 are a family of related basic region leucine zipper transcription factors involved in the regulation of various aspects of cellular differentiation and function in multiple tissues. Six different members of the family (C/EBPα, -β, -γ, -δ, -ε, and -ζ) have been isolated and characterized. The expression of C/EBPs is tissue- and stage-specific during development. C/EBPs have been shown to play a key role in regulating cellular differentiation, terminal function, and response to inflammatory insults (12.Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar, 13.Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 14.Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 15.Diehl A.M. J. Biol. Chem. 1998; 273: 30843-30846Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 16.Croniger C. Leahy P. Reshef L. Hanson R.W. J. Biol. Chem. 1998; 273: 31629-31632Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). To investigate the role of C/EBPα in the regulation of amelogenin gene expression, the 2.2-kilobase mouse amelogenin promoter has been systematically analyzed in mouse ameloblast-like LS8 cells. Our experimental strategy includes the following four analyses. First, the potential of C/EBPα to serve as a transcriptional activator of the amelogenin gene is tested in LS8, Pa-4, and MDCK cells the C/EBP-binding site on the amelogenin promoter is and the specific binding to the site by C/EBPα is by electrophoresis mobility shift assay the of C/EBPα overexpression on the endogenous amelogenin gene in LS8 cells is by To reporter the to from containing of the mouse amelogenin promoter (5.Snead M.L. Paine M.L. Chen L.S. Luo B.Y. Zhou D.H. Lei Y.P. Liu Y.H. Maxson Jr., R.E. Connect. Tissue Res. 1996; 35: 41-47Crossref PubMed Scopus (36) Google was the to site of To was and with by and For and the promoter regions by with as the a site is and and the are and the site is in with the to site of and by was by the site is in as the and as the and by A mouse ameloblast cell by of enamel organ with was in with and The rat parotid epithelial cell Pa-4, as the parotid cell In Cell. Dev. Biol. 1998; PubMed Scopus Google Scholar), was by of and as J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The MDCK cells by C. of and in minimal essential with and was for transient transfection of in To transfection of was as an the of the cells was with and in and The was with of in a of was and at for of was of in a The of these and at for The complex was with of and on of the cells. The cells at and for of the cells in of for an and to assay with a to the cells with and in of a and by at for activity was by of with of A and of a at activity was by of in a was the in to the The was synthesized at the of To the the analyzed by and LS8 cells in a with and off in of 20 for with a A on to a and at for at The at in nuclear and with a of and on a at for was by at for at in and at The protein was a protein assay with by an to a of and in with and The was for the binding was as by the The on a in the The was and the by The of the as and are cell from LS8 cells, LS8 cells with and LS8 cells with C/EBPα expression in by the protein assay with a of protein to The proteins on the and the was with a or and the to the bound To the required for the promoter activity of the mouse amelogenin a of reporter tested in LS8 cells with transient transfection with the transgenic analysis (5.Snead M.L. Paine M.L. Chen L.S. Luo B.Y. Zhou D.H. Lei Y.P. Liu Y.H. Maxson Jr., R.E. Connect. Tissue Res. 1996; 35: 41-47Crossref PubMed Scopus (36) Google Scholar), the pair region from the mouse amelogenin promoter in the reporter was sufficient to direct the expression of the reporter gene in LS8 cells, a mouse ameloblast-like cell of the region and in the mouse amelogenin promoter to a increase in promoter activity. However, of a to in a of reporter gene activity in LS8 cells, with a level of activity to the the region of the mouse amelogenin promoter in the as a minimal promoter in LS8 cells, the region was to promoter activity. In ameloblast-like LS8 cells, the region to have a on the promoter activity of the mouse amelogenin To a for tissue of the amelogenin promoter is in the the of reporter was cells. parotid Pa-4 and MDCK cells terminal and epithelial cells, The reporter gene activity of in Pa-4 and MDCK cells was that of the in with in LS8 cells. to only to a increase in reporter gene in Pa-4 cells and in MDCK cells. However, the reporter gene of in Pa-4, and LS8 cells The transfection studies that the mouse amelogenin promoter is ameloblast-specific as demonstrated in a previous transgenic analysis (5.Snead M.L. Paine M.L. Chen L.S. Luo B.Y. Zhou D.H. Lei Y.P. Liu Y.H. Maxson Jr., R.E. Connect. Tissue Res. 1996; 35: 41-47Crossref PubMed Scopus (36) Google Scholar). the that the amelogenin reporter promoter in Pa-4 and MDCK cells that 20 and 30% of the activity in LS8 cells, the region to to the tissue of the mouse amelogenin promoter. However, to that in LS8 cells, the region was necessary for promoter activity in both Pa-4 and MDCK cells. A transcription site was in the region of the mouse amelogenin promoter. To C/EBPα function as a transcriptional activator of the mouse amelogenin promoter, a C/EBPα expression was LS8 cells with the amelogenin or In response to of the reporter gene activity was increased to for and to for data indicate that C/EBPα the mouse amelogenin promoter in a dose-dependent in LS8 cells. To C/EBPα is sufficient to the mouse amelogenin promoter in cells, studies in Pa-4 and MDCK cells. of C/EBPα on the promoter activity of both and in MDCK cells. However, in Pa-4 cells, the reporter gene activity was increased to for and to for in response to of In the of exogenous the reporter gene activity of in Pa-4 cells was 30% of the basal level in LS8 of C/EBPα was able to increase the level to A was for In the of exogenous the reporter gene activity of in Pa-4 cells was of the basal level in LS8 of C/EBPα was able to increase the level to Taken together, these data indicate that C/EBPα as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. To understand the function of the C/EBP-binding site, a was the core of the site in the of and the to C/EBPα was in LS8 cells with transient or of the site the basal promoter activity of these reporter and C/EBPα-mediated transactivation of these was to the to that of the reporter gene activity of the was increased an order of by with C/EBPα Taken together, these data indicate that the C/EBP-binding site in the region of the mouse amelogenin promoter is required not only for C/EBPα-mediated but for basal promoter activity in LS8 cells. the C/EBP-binding site identified in the region of the mouse amelogenin promoter is as shown in the of bovine, and X-chromosomal amelogenin promoter The results of a role for C/EBPα as a of mouse amelogenin gene expression. To C/EBPα can to the mouse amelogenin promoter, a mobility shift assay was the mouse amelogenin site is in A complex was nuclear from LS8 cells as by the The of the was increased nuclear from LS8 cells was In an specific to C/EBPα was able to the complex the not to the The of a or of the binding of C/EBPα to the However, was with a of an a C/EBP-binding site, site is in data that C/EBPα is able to to the mouse amelogenin promoter and that the binding is specific. shown in the within the mouse amelogenin promoter not a box. containing a C/EBP-binding site was to a the In the a complex in a dose-dependent data indicate that the C/EBP-binding site, of the is required for the binding of C/EBPα to the mouse amelogenin promoter. To that C/EBPα is a bona of mouse amelogenin gene expression, First, is the expression of C/EBPα in is C/EBPα able to the expression of the endogenous amelogenin gene in the of The expression of C/EBPα was analyzed in an ameloblast-like cell by specific to C/EBPα a be off with a to the that C/EBPα is in LS8 cells. the overexpression of exogenous C/EBPα in LS8 cells was by of C/EBPα expression C/EBPα was the endogenous protein required a for with The expression level of the endogenous amelogenin protein in LS8 cells was A increase in the amelogenin protein level was in LS8 cells C/EBPα the level of amelogenin expression in LS8 cells essentially the as that in LS8 cells a transfection in transient transfection the increase in the amelogenin protein level C/EBPα activation a increase in the cells that exogenous the of C/EBPα and its of the endogenous amelogenin gene in the ameloblast-like LS8 cells indicate that C/EBPα is a of amelogenin gene expression. a to other family members play a role in mouse amelogenin gene expression, was The expression of was analyzed in LS8 cells by specific to a be off with a to the that is in LS8 cells. To can function as a transcriptional activator of the mouse amelogenin promoter, a expression was LS8 cells with amelogenin or was C/EBPα in amelogenin reporter gene for for C/EBPα The transfection data indicate that the of in the mouse amelogenin gene is To endogenous in LS8 cells can to the amelogenin promoter, a mobility shift analysis was the as a A complex was nuclear from LS8 cells as by the However, an specific to was not able to or to the complex and the was not able to to the data that was level of in the Taken together, the results indicate that has only a on the transcriptional regulation of the mouse amelogenin The proximal region of the mouse amelogenin promoter is the region upstream of the TATA box. region includes to with the TATA and of the promoter activity in ameloblast-like LS8 cells to the 2.2-kilobase promoter tested in transgenic (5.Snead M.L. Paine M.L. Chen L.S. Luo B.Y. Zhou D.H. Lei Y.P. Liu Y.H. Maxson Jr., R.E. Connect. Tissue Res. 1996; 35: 41-47Crossref PubMed Scopus (36) Google Scholar). The increase in promoter activity that a may be in the region and is with a previous in an upstream from the amelogenin promoter the activity of a minimal promoter by in cells E. Yuan Z.A. Abrams W.R. Gibson C.W. Gene 1998; PubMed Scopus Google Scholar). In the mouse amelogenin promoter, the region the binding site for Deletion of the C/EBP-binding site results in the of promoter activity. of the ameloblast-like LS8 cells, have identified the mouse amelogenin minimal promoter, of the in the C/EBP-binding region is essential to promoter activity. is highly of in the promoter regions of human, bovine, and murine X-chromosomal amelogenin genes the core for the C/EBP-binding site is identical in suggesting that region a basic role in We tested the that C/EBPα is involved in the regulation of mouse amelogenin gene expression. Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels. experiments (1.Snead M.L. Bringas Jr., P. Bessem C. Slavkin H.C. Dev. Biol. 1984; 104: 255-258Crossref PubMed Scopus (35) Google Scholar, 2.Snead M.L. Lau E. Zeichner-David M. Nanci T. Bendayan N. Bringas P. Bessem C. Slavkin H.C. Firtel A. Davidson E. Molecular Approaches to Developmental Biology. Alan Liss, Inc., New York1987: 641-652Google Scholar, 3.Snead M.L. Luo W. Lau E.C. Slavkin H.C. Development (Camb.). 1988; 104: 77-85Crossref PubMed Google demonstrated that the transcription of amelogenin is to enamel epithelial cells that terminal differentiation to the ameloblast is characterized by from the cell and the promoter region as that in the is able to the spatiotemporal expression pattern of the endogenous amelogenin gene in transgenic mouse (5.Snead M.L. Paine M.L. Chen L.S. Luo B.Y. Zhou D.H. Lei Y.P. Liu Y.H. Maxson Jr., R.E. Connect. Tissue Res. 1996; 35: 41-47Crossref PubMed Scopus (36) Google Scholar). In the the promoter of the mouse amelogenin gene was systematically analyzed in ameloblast-like LS8 cells, rat parotid Pa-4 cells, and MDCK cells. The minimal promoter was in a containing the transcription site was required for basal promoter activity. C/EBPs are of a family of basic region leucine zipper transcription factors that are of cell differentiation (12.Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar, 13.Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 14.Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 15.Diehl A.M. J. Biol. Chem. 1998; 273: 30843-30846Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 16.Croniger C. Leahy P. Reshef L. Hanson R.W. J. Biol. Chem. 1998; 273: 31629-31632Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). C/EBPα has been demonstrated to cell cellular and transcriptional regulation of tissue-specific genes in and M. M. G.J. Dev. 1996; PubMed Scopus Google Scholar, M. G.J. Cell. Biol. 1997; PubMed Google Scholar, PubMed Scopus Google Scholar, Cell. Biol. 1996; PubMed Google Scholar, A. M.D. G.J. PubMed Scopus Google Scholar, P. C. H. L. Xanthopoulos K.G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. E. H. J. Google Scholar, 1997; PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar, S. L. 1993; PubMed Scopus Google Scholar, J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, F. M. J. Biol. Chem. 1997; PubMed Scopus Google Scholar, Dev. 1994; PubMed Scopus Google Scholar). In and levels of C/EBPα mRNA are detected only in B. S. J. Dev. PubMed Scopus Google Scholar, P. Xanthopoulos K.G. Biochem. Res. PubMed Scopus Google Scholar). C/EBPα as a transcriptional activator in and the of C/EBPα in differentiation is with the expression of differentiation O.A. M.D. Biochem. PubMed Scopus Google Scholar, M. Dev. PubMed Scopus Google Scholar, M.D. MacDougald O.A. M. Int. J. 1996; 20 Google Scholar). that C/EBPα as a transcriptional activator of the mouse amelogenin gene by the following The overexpression of C/EBPα in mouse ameloblast-like LS8 cells can the promoter activity of the mouse amelogenin gene in a dose-dependent manner. The of C/EBPα to the mouse amelogenin gene is cell The C/EBP-binding site in the region in the mouse amelogenin promoter is necessary for both C/EBPα-mediated transactivation and basal promoter activity in LS8 cells. C/EBPα is of binding to the site in the promoter. C/EBPα is in ameloblast-like LS8 cells, and overexpression of C/EBPα can increase the expression level of the endogenous amelogenin protein. with the that a is in the region of the mouse amelogenin promoter, the transactivation of the reporter by C/EBPα is that of that the repressor(s) can with the function of interactions. The may be to direct interactions with C/EBPα of the interactions C/EBPα and basal transcriptional The mouse amelogenin promoter is cell In cell reporter tested have promoter and of the region only to a increase in reporter gene activity. There are several for The tissue is in the The region that to tissue-specific The region a tissue-specific however, of region is not sufficient to increase promoter activity to the of transcriptional activator(s) in cells. the of C/EBPα to the mouse amelogenin gene is cell The of repressor(s) the of may for the of C/EBPα to the mouse amelogenin promoter in MDCK cells. The formation of a complex detected in LS8 nuclear likely from endogenous C/EBPα in LS8 cells. is by the that exogenous C/EBPα was able to increase the of the complex The complex in not be by a be to the following First, the by the is C/EBPα to as a the binding of to the C/EBPα is that to the as in the C/EBPα and its is by other proteins in the complex as transcription C/EBPα was identified as a binding the for that the is not an essential of a C/EBP-binding In the of the mouse amelogenin promoter, a is in the region on the of the core of the site is that proteins other C/EBPα are of binding to the in One of the is is a transcription factor of and S. J. B. Sci. S. A. PubMed Scopus Google Scholar). The in both and a and with S. B. Cell. Biol. 1996; PubMed Scopus Google Scholar). The by is in the promoter and regions of genes R. Res. 1998; PubMed Scopus Google Scholar). binding site has been W. L. F. B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google to the for binding as in or in the of the binding core have been shown to only binding W. L. F. B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The in both the site and the However, of the detected in as of a of and First, complex was detected in shift assay in the site was in only the in the binding site and is to that the binding of C/EBPα and to the amelogenin promoter is to the of binding the that is a transcription is that a role in the spatiotemporal expression of the amelogenin gene during tooth development. The may be necessary for amelogenin promoter however, in the of a site, the is not sufficient to reporter gene transcription in LS8 cells, as by the and A is of or the complex There are First, is protein in the the to protein in the the that has a on amelogenin reporter genes is to play a direct role in mouse amelogenin gene expression. Our results that the C/EBP-binding site in the proximal region of the mouse amelogenin promoter is necessary not only for C/EBPα-mediated but for basal promoter activity in ameloblast-like cells, and that overexpression of C/EBPα is sufficient to increase the protein level of the endogenous amelogenin that C/EBPα a key role in the developmentally regulated expression of the amelogenin the of enamel has been demonstrated to play an important role in proper enamel mineralization. Amelogenins of of the enamel in the X-chromosomal amelogenin gene have been identified in with the enamel E. Genet. PubMed Scopus Google Scholar, M. N. B. PubMed Scopus Google Scholar, M. M. B. E. L. PubMed Scopus Google Scholar). of amelogenin during tooth development with or results in enamel T. S. Bringas Jr., P. V. Slavkin H.C. Development (Camb.). 1993; PubMed Google Scholar, S. J. PubMed Scopus Google Scholar). the developmentally regulated expression pattern of the gene during tooth facilitate the of the molecular involved in enamel formation. C/EBPα is the transcription factor identified to to function as a of amelogenin expression. the C/EBPα and amelogenin expression the of the underlying the tissue-specific expression of the amelogenin In the the expression of C/EBPα and amelogenin are with other during ameloblast However, is likely that C/EBPα in with transcription to the spatiotemporal expression of the amelogenin gene during tooth development. and activation are required for the regulation of the amelogenin can function cognate on the amelogenin promoter the of The of C/EBPα as a transcriptional activator of amelogenin not only the direct that are involved in amelogenin gene expression, but the of potential We and Paine for of the for Pa-4 and for MDCK cells. We and Chen for