Keratin 75 (K75) was recently discovered in ameloblasts and enamel organic matrix. Carriers of A161T substitution in K75 present with the skin condition Pseudofollicullitis barbae . This mutation is also associated with high prevalence of caries and compromised structural and mechanical properties of enamel. Krt75 tm1Der knock-in mouse (KI) with deletion of Asn159, located two amino acids away from KRT75 A161T , can be a potential model for studying the role of K75 in enamel and the causes of the higher caries susceptibility associated with KRT75 A161T mutation. To test the hypotheses that KI enamel is more susceptible to a simulated acid attack (SAA), and has altered structural and mechanical properties, we conducted in vitro SAA experiments, microCT, and microhardness analyses on 1st molars of one-month-old WT and KI mice. KI and WT hemimandibles were subjected to SAA and contralateral hemimandibles were used as controls. Changes in enamel porosity were assessed by immersion of the hemimandibles in rhodamine, followed by fluorescent microscopy analysis. Fluorescence intensity of KI enamel after SSA was significantly higher than in WT, indicating that KI enamel is more susceptible to acid attack. MicroCT analysis of 1st molars revealed that while enamel volumes were not significantly different, enamel mineral density was significantly lower in KI, suggesting a potential defect of enamel maturation. Microhardness tests revealed that in KI enamel is softer than in WT, and potentially less resilient to damages. These results suggest that the KI enamel can be used as a model to study the role of K75 in enamel.
Background EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells. Methodology/Principal Findings To gain insights into EpCAM function in vivo, we generated EpCAM −/− mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to βgeo. EpCAM +/− mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/− embryos revealed that βgeo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM −/− mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM −/− embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM −/− placentas. Conclusion EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs.
The Distal-less Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells, and there is evidence to support an essential role as a transcriptional regulator of the terminal differentiation process in these cells. In an attempt to determine the factors that induce Dlx3 gene expression, we have cloned the 1.2-kilobase pair proximal region of murine gene and analyzed its cis-regulatory elements and potential trans-acting factors. The proximal region of the Dlx3 gene has a canonical TATA box and CCAAT box, and the transcription start site was located 205 base pairs upstream from the initiation of translation site. Serial deletion analysis showed that the region between −84 and −34 confers the maximal promoter activity both in undifferentiated and differentiated primary mouse keratinocytes. Gel retardation assays and mutational analysis demonstrated that the transcriptional regulator NF-Y (also referred to as CBF) binds to a CCAAT box motif within this region and is responsible for the majority of the Dlx3 promoter activity. In addition, an Sp1-binding site was located immediately upstream of transcription start site that acts as a positive regulatory element of the Dlx3 promoter, independent of the CCAAT box motif. Importantly, elements residing between +30 to +60 of the Dlx3 gene are responsible for the Ca2+-dependent induction of Dlx3 during keratinocyte differentiation. The Distal-less Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells, and there is evidence to support an essential role as a transcriptional regulator of the terminal differentiation process in these cells. In an attempt to determine the factors that induce Dlx3 gene expression, we have cloned the 1.2-kilobase pair proximal region of murine gene and analyzed its cis-regulatory elements and potential trans-acting factors. The proximal region of the Dlx3 gene has a canonical TATA box and CCAAT box, and the transcription start site was located 205 base pairs upstream from the initiation of translation site. Serial deletion analysis showed that the region between −84 and −34 confers the maximal promoter activity both in undifferentiated and differentiated primary mouse keratinocytes. Gel retardation assays and mutational analysis demonstrated that the transcriptional regulator NF-Y (also referred to as CBF) binds to a CCAAT box motif within this region and is responsible for the majority of the Dlx3 promoter activity. In addition, an Sp1-binding site was located immediately upstream of transcription start site that acts as a positive regulatory element of the Dlx3 promoter, independent of the CCAAT box motif. Importantly, elements residing between +30 to +60 of the Dlx3 gene are responsible for the Ca2+-dependent induction of Dlx3 during keratinocyte differentiation. protein kinase C chloramphenicol acetyltransferase polymerase chain reaction base pair During epidermal differentiation, mitotically active basal keratinocytes cease to proliferate, detach from the basement membrane, and migrate through the spinous and granular layers to the outermost terminally differentiated cornified layer of the skin. This cornification process is tightly associated with a stepwise program of transcriptional regulation and is concurrent with the sequential induction and repression of structural and enzymatic differentiation-specific markers (1Fuchs E. Byrne C. Curr. Opin. Genet. & Dev. 1994; 4: 725-736Crossref PubMed Scopus (222) Google Scholar). This process can be achieved in mouse keratinocytes cultivated in vitro by increasing the Ca2+ concentration from 0.05 to 0.12 mm in the culture medium (2Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. J. Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (511) Google Scholar), which produces a situation that mimics the endogenous Ca2+ gradient present in the skin (3Menon G.K. Grayson S. Elias P.M. J. Invest. Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (387) Google Scholar). The Ca2+ signaling differentiation pathway is associated with increased phospholipase C activity (4Punnonen K. Denning M. Lee E. Li L. Rhee S.G. Yuspa S.H. J. Invest. Dermatol. 1993; 101: 719-726Abstract Full Text PDF PubMed Google Scholar) and activation of protein kinase C (PKC)1 (5Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar). Previous work has demonstrated an essential role of PKC signaling in the late stages of epidermal differentiation. Activation of PKC has been shown to be necessary for expression of late differentiation markers loricrin and profilaggrin and for the suppression of the spinous-specific markers K1 and K10 (6Dlugosz A.A. Yuspa S.H. J. Cell Biol. 1993; 120: 217-225Crossref PubMed Scopus (218) Google Scholar).Dlx33, a murine ortholog of the Drosophila Distal-less homeodomain protein (7Cohen S.M. Brönner G. Kütter F. Jürgens G. Jäckle H. Nature. 1989; 338: 432-434Crossref PubMed Scopus (323) Google Scholar), is a member of the Dlx vertebrate family. This family comprises to date six genes identified both in mouse and human and found to be organized as three convergently transcribed pairs, each closely linked to one of the four mammalian Hox clusters (8Robinson G.W. Wray S. Mahon K.A. New Biol. 1991; 3: 1183-1194PubMed Google Scholar, 9Porteus M.H. Bulfone A. Ciaranello R.D. Rubenstein J.L.R. Neuron. 1991; 7: 221-229Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 10Simeone A. Acampora D. Pannese M. D'Esposito M. Stornaiuolo A. Gulisano M. Mallamaci A. Kastury K. Druck T. Huebner K. Boncinelli E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2250-2254Crossref PubMed Scopus (260) Google Scholar, 11Nakamura S. Stock D.W. Wynder K.L. Bolekens J.A. Takeshita K. Nagai B.M. Chiba S. Kitamura T. Freeland T.M. Zhao Z. Minowada J. Lawrence J.B. Weiss K.M. Ruddle F.H. Genomics. 1996; 38: 314-324Crossref PubMed Scopus (71) Google Scholar, 12Morasso M.I. Yonescu R. Griffin C.A. Sargent T.D. Mamm. Genome. 1997; 8: 302-303Crossref PubMed Scopus (11) Google Scholar, 13Quinn L.M. Johnson B.V. Nicholl J. Sutherland G.R. Kalionis B. Gene (Amst.). 1997; 187: 55-61Crossref PubMed Scopus (65) Google Scholar). Disruption of the DLX3 coding sequence has been associated with a human disorder, Tricho-Dento-Osseous syndrome. This inherited autosomal dominant disorder is characterized by defects in ectodermal derivatives such as hair and teeth and craniofacial bone abnormalities (14Price J.A. Bowden D.W. Wright J.T. Pettenati M.J. Hart T.C. Hum. Mol. Genet. 1998; 7: 563-569Crossref PubMed Scopus (197) Google Scholar). Dlx3 is expressed in the granular layer of the epidermis and in the hair matrix cells of the hair follicle (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar,16Robinson G.W. Mahon K.A. Mech. Dev. 1994; 48: 199-215Crossref PubMed Scopus (188) Google Scholar), and there is evidence strongly supporting the critical role of the Dlx3 homeoprotein in the regulation of expression of late epidermal differentiation genes (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar). In vitro studies have shown that Dlx3 binds to an AT-rich region and acts as a positive transcriptional regulator (17Feledy J. Morasso M.I. Jang S.-I. Sargent T.D. Nucleic Acids Res. 1999; 27: 764-770Crossref PubMed Scopus (78) Google Scholar), which is activated during the Ca2+ shift in keratinocytes induced to differentiate in culture (18Morasso M. Jamrich M. Sargent T.D. Dev. Biol. 1994; 162: 267-276Crossref PubMed Scopus (27) Google Scholar). In transgenic mice, ectopic expression of Dlx3 in basal cells is accompanied by the cessation of cell proliferation and the up-regulation of expression of late epidermal differentiation structural genes including profilaggrin (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar). A potential binding site for Dlx3 has been identified in the profilaggrin gene, suggesting that the observed up-regulation of this gene in the transgenic mice may result from a direct effect of Dlx3 (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar). Altogether, these data strongly support a role for Dlx3 as a determinant factor in the activation of expression of granular markers during the terminal differentiation of keratinocytes.During the process of terminal epidermal differentiation, many genes expressed in the keratinocyte are regulated at the transcriptional level (1Fuchs E. Byrne C. Curr. Opin. Genet. & Dev. 1994; 4: 725-736Crossref PubMed Scopus (222) Google Scholar). The transcription factors AP1 and AP2 have been characterized as primary regulatory factors of keratinocyte gene expression (19Casatorres J. Navarro J.M. Blessing M. Jorcano J.L. J. Biol. Chem. 1994; 269: 20489-20496Abstract Full Text PDF PubMed Google Scholar, 20Leask A. Byrne C. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7948-7952Crossref PubMed Scopus (201) Google Scholar, 21Lu B. Rothnagel J.A. Longley M.A. Tsai S.Y. Roop D.R. J. Biol. Chem. 1994; 269: 7443-7449Abstract Full Text PDF PubMed Google Scholar, 22DiSepio D. Jones A. Longley M.A. Bundman D. Rothnagel J.A. Roop D.R. J. Biol. Chem. 1995; 270: 10792-10799Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 23Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 24Jang S.-I. Steinert P.M. Markova N.G. J. Biol. Chem. 1996; 271: 24105-24114Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). PKC is an upstream component of the pathway that regulates AP1 in many systems and may play a role in the epidermal differentiation expression of K5, K1, loricrin, profilaggrin, and involucrin (19Casatorres J. Navarro J.M. Blessing M. Jorcano J.L. J. Biol. Chem. 1994; 269: 20489-20496Abstract Full Text PDF PubMed Google Scholar, 21Lu B. Rothnagel J.A. Longley M.A. Tsai S.Y. Roop D.R. J. Biol. Chem. 1994; 269: 7443-7449Abstract Full Text PDF PubMed Google Scholar, 22DiSepio D. Jones A. Longley M.A. Bundman D. Rothnagel J.A. Roop D.R. J. Biol. Chem. 1995; 270: 10792-10799Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 23Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 24Jang S.-I. Steinert P.M. Markova N.G. J. Biol. Chem. 1996; 271: 24105-24114Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Members of the POU family of transcription factors such as Oct1, Oct2, Oct6, Skn1a, and Skn1i have also been implicated as regulators of epidermal genes (25Blumemberg M Darmon M. Blumemberg M. Molecular Biology of the Skin: The Keratinocyte. Academic Press, New York1993: 1-32Google Scholar, 26Faus I. Hsu H.J. Fuchs E. Mol. Cell. Biol. 1994; 14: 3263-3275Crossref PubMed Scopus (86) Google Scholar, 27Andersen B. Schonemann M.D. Flynn S.E. Pearse R.V. Singh H. Rosenfeld M. Science. 1993; 260: 78-82Crossref PubMed Scopus (102) Google Scholar, 28Welter J.F. Gali H. Crish J.F. Eckert R.L. J. Biol. Chem. 1996; 271: 14727-14733Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).In order to elucidate the role of Dlx3 in the cascade of transcriptional events that ultimately leads to terminal differentiation, we have cloned and characterized the mouse Dlx3 promoter. Deletional promoter analysis was utilized to delineate the sequences that regulate the transcription of Dlx3 in differentiated and undifferentiated keratinocytes. In turn, these cis-acting elements were used to identify the transcription factors that regulate the Dlx3 promoter. Importantly, we have identified a region residing between +10 and +60 that responds to the Ca2+ shift used to differentiate the keratinocytes in culture.DISCUSSIONThe cascade of events that leads to terminal differentiation can be triggered in primary mouse keratinocytes by the elevation of extracellular Ca2+. The expression of the Dlx3 homeobox gene is restricted to the suprabasal layer of the epidermis (18Morasso M. Jamrich M. Sargent T.D. Dev. Biol. 1994; 162: 267-276Crossref PubMed Scopus (27) Google Scholar), and this expression is dramatically increased in primary mouse keratinocytes induced to differentiate by Ca2+ in vitro. Evidence obtained in transgenic mice suggests the role of Dlx3 as a positive transcriptional activator of differentiation-specific epidermal structural genes. In an attempt to decipher the different stages of the pathway that culminate in terminal differentiation, we have cloned and characterized the upstream promoter sequence of the murine Dlx3 gene, and we determined the regulatory elements necessary for expression of the gene in keratinocytes. By sequence comparison we have found that the region close to the transcription start site of the mouse Dlx3 gene (−110 to +61) has striking homology with that of the Xenopus and human dlx3 genes. In transgenic mice, 470 bp of the Xenopus Dlx3 promoter conferred an expression pattern to the β-galactosidase reporter that was indistinguishable from that of the endogenous gene, including the Ca2+response (46Morasso M.I. Mahon K.A. Sargent T.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3968-3972Crossref PubMed Scopus (72) Google Scholar). Thus, it could be hypothesized that the cis- and trans-regulatory elements important in the regulation of the Dlx3 orthologs have been conserved through evolution and are contained within the proximal promoter region.To understand the mechanism(s) that control the regulation and expression of the Dlx3 promoter during epidermal differentiation, our studies examined the expression of Dlx3 promoter/CAT constructs in primary mouse keratinocytes to identify putative regulatory elements that are required for Dlx3 expression. The data presented here demonstrate the existence of one positive regulatory region in the Dlx3 promoter located between −84 and −34. We determined that the CCAAT box in this region exerts a significant positive influence on the Dlx3 promoter activity in undifferentiated and differentiated keratinocytes. Competition gel shift analysis and supershift analysis further demonstrated that the transcription factor NF-Y is the protein that binds specifically to the CCAAT box motif. Mutation of this motif dramatically reduced transcription of the Dlx3 promoter. Several genes also contain canonical sites for CCAAT-binding proteins that have been described to be important in early functions of preinitiation complex formation (36Milos P.M. Zaret K.S. Genes Dev. 1992; 6: 991-1004Crossref PubMed Scopus (108) Google Scholar). CCAAT boxes are typically located within 100 bp of the transcription start sites, and CCAAT box binding factors include NF-Y (also known as CBF), C/EBP family members, and NF-1 (35Gronostajski R.M. Nucleic Acids Res. 1986; 14: 9117-9129Crossref PubMed Scopus (76) Google Scholar). NF-Y was originally identified as a ubiquitously expressed protein that binds to the Y box motif, defined as an inverted CCAAT box motif in all major histocompatibility complex class II genes (47Glimcher L.H. Kara C.J. Annu. Rev. Immunol. 1992; 10: 13-49Crossref PubMed Scopus (504) Google Scholar). NF-Y is a heterotrimeric transcription factor composed of three subunits (37Hatamochi A. Golumbek P.T. Van Schaftingen E. de Crombrugghe B. J. Biol. Chem. 1989; 263: 5940-5947Abstract Full Text PDF Google Scholar, 38Osawa H. Robey R.B. Printz R.L. Granner D.K. J. Biol. Chem. 1996; 271: 17296-17303Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 39Dorn A. Bollenkens J. Staub A. Benoist C. Mathis D. Cell. 1987; 50: 863-872Abstract Full Text PDF PubMed Scopus (471) Google Scholar, 40Maity S.N. Vuorio T. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5378-5382Crossref PubMed Scopus (86) Google Scholar, 41van Huijsduijnen R.H. Li X.Y. Black D. Matthes H. Benoist C. Mathis D. EMBO J. 1990; 9: 2127-3119Google Scholar, 42Coustry F. Maity S.N. de Crombrugghe B. J. Biol. Chem. 1995; 270: 468-475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 43Sinha S. Maity S.H. Lu J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (250) Google Scholar). Formation of a complex between the A and C subunits is required to bind the B subunit, and together, the heterotrimeric complex binds DNA (48Coustry F. Maity S.N. Sinha S. de Crombrugghe B. J. Biol. Chem. 1996; 271: 14485-14491Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The activation domains of both B and C subunits, which are rich in glutamine and hydrophobic residues, share protein sequence homology with each other and with the glutamine-rich activation domain of the transcription factor Sp1 (48Coustry F. Maity S.N. Sinha S. de Crombrugghe B. J. Biol. Chem. 1996; 271: 14485-14491Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).NF-Y has been demonstrated to act as both a positive (49Lamb K.A. Johnson L.R. Rizzino A. Mol. Reprod. Dev. 1997; 48: 301-309Crossref PubMed Scopus (16) Google Scholar, 50Osawa H. Robey R.B. Printz R.L. Granner D.K. J. Biol. Chem. 1996; 271: 17296-17303Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 51Katula K.S. Wright K.L. Paul H. Surman D.R. Nuckolls F.J. Smith J.W. Ting J.P-Y. Yates J. Cogswell J.P. Cell Growth Differ. 1997; 8: 811-820PubMed Google Scholar) and a negative (52Moriuchi H. Moriuchi M. Cohen J.I. Virology. 1995; 214: 256-258Crossref PubMed Scopus (15) Google Scholar) regulator of transcription. Several studies suggest that NF-Y acts by stabilizing the binding of additional factors to adjacent regulatory elements, such as RFX in the major histocompatibility complex class II promoter (53Hinkley C. Perry M. Mol. Cell. Biol. 1992; 12: 4400-4411Crossref PubMed Scopus (48) Google Scholar, 54Reith W. Kobr M. Emery P. Durand B. Siegrist C.A. Mach B. J. Biol. Chem. 1994; 269: 20020-20025Abstract Full Text PDF PubMed Google Scholar). NF-Y also interacts with transcription factors binding upstream elements and basal transcription machinery, such as HNF4 and TAF100 (42Coustry F. Maity S.N. de Crombrugghe B. J. Biol. Chem. 1995; 270: 468-475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 55Ueda A. Takeshita F. Yamashiro S. Yoshimura T. J. Biol. Chem. 1998; 273: 19339-19347Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Recently it was shown that the interaction of NF-Y with cAMP-response element-binding protein may be responsible for the gene-specific transcriptional activity of the insulin cAMP-response element (56Eggers A. Siemann G. Blume R. Knepel W. J. Biol. Chem. 1998; 273: 18499-18508Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and similar results have been reported for the albumin promoter (36Milos P.M. Zaret K.S. Genes Dev. 1992; 6: 991-1004Crossref PubMed Scopus (108) Google Scholar). Since the expression of Dlx3 gene is epithelial specific and the activity of the Dlx3 promoter is low in the fibroblast cell line NIH 3T3 (data not shown), NF-Y and some interacting regulatory factors may be involved in conferring tissue- and differentiation-specific expression to the Dlx3 gene.In this study, we also demonstrate that the Sp1-binding site located between −21 and +2 positively regulates, and contributes to, the basal activity of the Dlx3 promoter in undifferentiated and differentiated keratinocytes. The Sp1 consensus motif is highly GC-rich; therefore, certain Sp1-binding sites can overlap with other GC-rich consensus transcription factor binding motifs such as that for Ap-2. The ratio of Sp1/Ap2 might be determinant in the differentiation-specific gene expression (57Chen T.T. Wu R.L. Castro-Munozledo F. Sun T.T. Mol. Cell. Biol. 1997; 17: 3056-3064Crossref PubMed Scopus (104) Google Scholar). Many epidermal specific gene promoters contain consensus sites for ubiquitously expressed regulators such as Sp1 family members. The Sp1 family consists of four members (Sp1–Sp4; see Refs. 58Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4561Crossref PubMed Scopus (487) Google Scholar, 59Hagen G. Muller S. Beato M. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (522) Google Scholar, 60Sogawa K. Imataka H. Yamasaki Y. Kusume H. Abe H. Kuriyama Y.F. Nucleic Acids Res. 1993; 21: 1527-1532Crossref PubMed Scopus (179) Google Scholar). Sp1 is involved with tissue/cell type-specific expression of certain genes in epithelial cells. In most of these cases, Sp1 alone does not affect tissue specificity but requires cooperation with other transcription factors, such as Ets in the promoter of human transglutaminase 3 (61Lee J.H. Jang S.-I. Yang J.M. Markova N.G. Steinert P.M. J. Biol. Chem. 1996; 271: 4561-4568Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), Ap-2 in the K3 keratin gene (57Chen T.T. Wu R.L. Castro-Munozledo F. Sun T.T. Mol. Cell. Biol. 1997; 17: 3056-3064Crossref PubMed Scopus (104) Google Scholar), Egr-1 in the tissue factor gene (62Cui M.-Z. Parry G.C.N. Oeth P. Larson H. Smith M. Huang R.-P. Adamson E.D. Mackman N. J. Biol. Chem. 1996; 271: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), and NF-1 in the mouse vas deferens protein promoter (63Darne C.H. Morel L. Claessens F. Manin M. Fabre S. Veyssiere G. Rombauts W. Jean C.L. Mol. Cell. Endocrinol. 1997; 132: 13-23Crossref PubMed Scopus (33) Google Scholar).From the family of Sp1-related transcription factors, studies have shown that Sp3 acts as an antagonist to Sp1. The human transcobalamin II gene is controlled by the relative ratios of Sp1 and Sp3 (64Li N. Seetharam S. Seetharam B. J. Biol. Chem. 1998; 273: 16104-16111Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and activation of the human papilloma virus type 16 promoter correlates with the ratios of Sp1/Sp3 during epithelial differentiation (65Apt D. Watts R.M. Suske G. Bernard H.U. Virology. 1996; 224: 281-291Crossref PubMed Scopus (133) Google Scholar). We have shown that the Sp1 consensus motif on the Dlx3 promoter can bind specifically Sp1 and Sp3, providing the possibility of antagonistic effects on transcription depending on the specific levels of each of these factors throughout the terminal differentiation process.An essential aspect of the transcriptional regulation of the Dlx3 promoter is the elucidation of the mechanism for inducing its expression by increases in external Ca2+ during differentiation. Until now, the prevailing evidence points to the involvement of PKC isozymes in the induction of keratinocyte differentiation markers by Ca2+, although the exact mechanism is unclear. In this study we show that the region located between +10 and +60 is important for Ca2+ inducibility of the Dlx3 gene. By mutational and gel shift analysis of the +30/+60 sequence, we found that the crucial element responsible for Ca2+ inducibility is located between +42 and +45 (CGAC) and that nuclear factor(s) are involved in the up-regulation of Dlx3 expression by Ca2+. It has been reported that the AP1 and Ets transcription factors are involved in the regulation of the human SPRR1A keratinocyte terminal differentiation marker (45Sark M.W.J. Fisher D.F. de Meijer E. van de Putte P. Backendorf C. J. Biol. Chem. 1998; 273: 24683-24692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, in the case of the Dlx3 gene, the AP1 transcription factor does not bind to the +42/+45 or +46/+52 sequences, which strongly supports that it is not the determinant for the Ca2+ inducibility of Dlx3. Future work will determine the specific nature of the nuclear factor(s) involved in the binding to this regulatory element. The identification of a Ca2+ response element in the Dlx3 gene is the first link between the extracellular signal and the transcriptional control of a regulatory gene involved in keratinocyte differentiation, and represents an important step in the elucidation of the molecular mechanisms underlying this developmental program. During epidermal differentiation, mitotically active basal keratinocytes cease to proliferate, detach from the basement membrane, and migrate through the spinous and granular layers to the outermost terminally differentiated cornified layer of the skin. This cornification process is tightly associated with a stepwise program of transcriptional regulation and is concurrent with the sequential induction and repression of structural and enzymatic differentiation-specific markers (1Fuchs E. Byrne C. Curr. Opin. Genet. & Dev. 1994; 4: 725-736Crossref PubMed Scopus (222) Google Scholar). This process can be achieved in mouse keratinocytes cultivated in vitro by increasing the Ca2+ concentration from 0.05 to 0.12 mm in the culture medium (2Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. J. Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (511) Google Scholar), which produces a situation that mimics the endogenous Ca2+ gradient present in the skin (3Menon G.K. Grayson S. Elias P.M. J. Invest. Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (387) Google Scholar). The Ca2+ signaling differentiation pathway is associated with increased phospholipase C activity (4Punnonen K. Denning M. Lee E. Li L. Rhee S.G. Yuspa S.H. J. Invest. Dermatol. 1993; 101: 719-726Abstract Full Text PDF PubMed Google Scholar) and activation of protein kinase C (PKC)1 (5Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar). Previous work has demonstrated an essential role of PKC signaling in the late stages of epidermal differentiation. Activation of PKC has been shown to be necessary for expression of late differentiation markers loricrin and profilaggrin and for the suppression of the spinous-specific markers K1 and K10 (6Dlugosz A.A. Yuspa S.H. J. Cell Biol. 1993; 120: 217-225Crossref PubMed Scopus (218) Google Scholar). Dlx33, a murine ortholog of the Drosophila Distal-less homeodomain protein (7Cohen S.M. Brönner G. Kütter F. Jürgens G. Jäckle H. Nature. 1989; 338: 432-434Crossref PubMed Scopus (323) Google Scholar), is a member of the Dlx vertebrate family. This family comprises to date six genes identified both in mouse and human and found to be organized as three convergently transcribed pairs, each closely linked to one of the four mammalian Hox clusters (8Robinson G.W. Wray S. Mahon K.A. New Biol. 1991; 3: 1183-1194PubMed Google Scholar, 9Porteus M.H. Bulfone A. Ciaranello R.D. Rubenstein J.L.R. Neuron. 1991; 7: 221-229Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 10Simeone A. Acampora D. Pannese M. D'Esposito M. Stornaiuolo A. Gulisano M. Mallamaci A. Kastury K. Druck T. Huebner K. Boncinelli E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2250-2254Crossref PubMed Scopus (260) Google Scholar, 11Nakamura S. Stock D.W. Wynder K.L. Bolekens J.A. Takeshita K. Nagai B.M. Chiba S. Kitamura T. Freeland T.M. Zhao Z. Minowada J. Lawrence J.B. Weiss K.M. Ruddle F.H. Genomics. 1996; 38: 314-324Crossref PubMed Scopus (71) Google Scholar, 12Morasso M.I. Yonescu R. Griffin C.A. Sargent T.D. Mamm. Genome. 1997; 8: 302-303Crossref PubMed Scopus (11) Google Scholar, 13Quinn L.M. Johnson B.V. Nicholl J. Sutherland G.R. Kalionis B. Gene (Amst.). 1997; 187: 55-61Crossref PubMed Scopus (65) Google Scholar). Disruption of the DLX3 coding sequence has been associated with a human disorder, Tricho-Dento-Osseous syndrome. This inherited autosomal dominant disorder is characterized by defects in ectodermal derivatives such as hair and teeth and craniofacial bone abnormalities (14Price J.A. Bowden D.W. Wright J.T. Pettenati M.J. Hart T.C. Hum. Mol. Genet. 1998; 7: 563-569Crossref PubMed Scopus (197) Google Scholar). Dlx3 is expressed in the granular layer of the epidermis and in the hair matrix cells of the hair follicle (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar,16Robinson G.W. Mahon K.A. Mech. Dev. 1994; 48: 199-215Crossref PubMed Scopus (188) Google Scholar), and there is evidence strongly supporting the critical role of the Dlx3 homeoprotein in the regulation of expression of late epidermal differentiation genes (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar). In vitro studies have shown that Dlx3 binds to an AT-rich region and acts as a positive transcriptional regulator (17Feledy J. Morasso M.I. Jang S.-I. Sargent T.D. Nucleic Acids Res. 1999; 27: 764-770Crossref PubMed Scopus (78) Google Scholar), which is activated during the Ca2+ shift in keratinocytes induced to differentiate in culture (18Morasso M. Jamrich M. Sargent T.D. Dev. Biol. 1994; 162: 267-276Crossref PubMed Scopus (27) Google Scholar). In transgenic mice, ectopic expression of Dlx3 in basal cells is accompanied by the cessation of cell proliferation and the up-regulation of expression of late epidermal differentiation structural genes including profilaggrin (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar). A potential binding site for Dlx3 has been identified in the profilaggrin gene, suggesting that the observed up-regulation of this gene in the transgenic mice may result from a direct effect of Dlx3 (15Morasso M.I. Markova N.G. Sargent T.D. J. Cell Biol. 1996; 135: 1879-1887Crossref PubMed Scopus (106) Google Scholar). Altogether, these data strongly support a role for Dlx3 as a determinant factor in the activation of expression of granular markers during the terminal differentiation of keratinocytes. During the process of terminal epiderm
During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.
The development of autoimmune disease in the MRL/MpJ-lpr inbred mouse strain depends upon the maturation of a subset of T lymphocytes that may cause sustained activation of immunological effector cells such as B cells and macrophages. We tested the hypothesis that abnormal effector cell activation reflects constitutive overexpression of a T cell cytokine. We found that a newly defined T cell cytokine, Eta-1, is expressed at very high levels in T cells from MRL/l mice but not normal mouse strains and in a CD4-8- 45R+ T cell clone. The Eta-1 gene encodes a secreted protein that binds specifically to macrophages, possibly via a cell adhesion receptor, resulting in alterations in the mobility and activation state of this cell type (Patarca, R., G. J. Freeman, R. P. Singh, et al. 1989. J. Exp. Med. 170:145; Singh, R. P., R. Patarca, J. Schwartz, P. Singh, and H. Cantor. 1990. J. Exp. Med. 171:1931). In addition, recent studies have indicated that Eta-1 can enhance secretion of IgM and IgG by mixtures of macrophages and B cells (Patarca, R., M. A. Lampe, M. V. Iregai, and H. Cantor, manuscript in preparation). Dysregulation of Eta-1 expression begins at the onset of autoimmune disease and continues throughout the course of this disorder. Maximal levels of Eta-1 expression and the development of severe autoimmune disease reflect the combined contribution of the lpr gene and MRL background genes.
Both analyses of x-ray diffraction patterns of well oriented specimens of trichocyte keratin intermediate filaments (IF) and in vitro cross-linking experiments on several types of IF have documented that there are three modes of alignment of pairs of antiparallel molecules in all IF: A11, A22 and A12, based on which parts of the major rod domain segments are overlapped. Here we have examined which residues may be important for stabilizing the A11 mode. Using the K5/K14 system, we have made point mutations of charged residues along the chains and examined the propensities of equimolar mixtures of wild type and mutant chains to reassemble using as criteria: the formation (or not) of IF in vitro or in vivo; and stabilities of one- and two-molecule assemblies. We identified that the conserved residue Arg10 of the 1A rod domain, and the conserved residues Glu4 and Glu6 of the linker L2, were essential for stability. Additionally, conserved residues Lys31 of 1A and Asp1 of 2A and non-conserved residues Asp/Asn9of 1A, Asp/Asn3 of 2A, and Asp7 of L2 are important for stability. Notably, these groups of residues lie close to each other when two antiparallel molecules are aligned in the A11 mode, and are located toward the ends of the overlap region. Although other sets of residues might theoretically also contribute, we conclude that these residues in particular engage in favorable intermolecular ionic and/or H-bonding interactions and thereby may play a role in stabilizing the A11 mode of alignment in keratin IF.
Abstract Dlx4 is a member of a family of homeobox genes with homology to Drosophila distal‐less ( dll ) gene. We show that Dlx4 expression pattern partially overlaps with its cis‐linked gene Dlx3 during mouse development as well as in neonatal and adult skin. In mice, Dlx4 is expressed in the branchial arches, embryonic limbs, digits, nose, hair follicle and in the basal and suprabasal layers of mouse interfollicular epidermis. We show that inactivation of Dlx4 in mice did not result in any overtly gross pathology. Skin development, homeostasis and response to TPA treatment were similar in mice with loss of Dlx4 compared to wild‐type counterparts.
Comparative co-localization analysis of transcription factors (TFs) and epigenetic marks (EMs) in specific biological contexts is one of the most critical areas of ChIP-Seq data analysis beyond peak calling. Yet there is a significant lack of user-friendly and powerful tools geared towards co-localization analysis based exploratory research. Most tools currently used for co-localization analysis are command line only and require extensive installation procedures and Linux expertise. Online tools partially address the usability issues of command line tools, but slow response times and few customization features make them unsuitable for rapid data-driven interactive exploratory research. We have developed PAPST: Peak Assignment and Profile Search Tool, a user-friendly yet powerful platform with a unique design, which integrates both gene-centric and peak-centric co-localization analysis into a single package. Most of PAPST's functions can be completed in less than five seconds, allowing quick cycles of data-driven hypothesis generation and testing. With PAPST, a researcher with or without computational expertise can perform sophisticated co-localization pattern analysis of multiple TFs and EMs, either against all known genes or a set of genomic regions obtained from public repositories or prior analysis. PAPST is a versatile, efficient, and customizable tool for genome-wide data-driven exploratory research. Creatively used, PAPST can be quickly applied to any genomic data analysis that involves a comparison of two or more sets of genomic coordinate intervals, making it a powerful tool for a wide range of exploratory genomic research. We first present PAPST's general purpose features then apply it to several public ChIP-Seq data sets to demonstrate its rapid execution and potential for cutting-edge research with a case study in enhancer analysis. To our knowledge, PAPST is the first software of its kind to provide efficient and sophisticated post peak-calling ChIP-Seq data analysis as an easy-to-use interactive application. PAPST is available at https://github.com/paulbible/papst and is a public domain work.
