Smad6 Represses Dlx3 Transcriptional Activity through Inhibition of DNA Binding

Abstract

Dlx3 (Distal-less 3) is a homeobox-containing transcription factor required for normal placental development in mice. Here we demonstrate that Dlx3 interacts with Smad6, a member of a larger family of transcriptional regulators generally thought to regulate transforming growth factor β/bone morphogenetic protein signaling. Immunocytochemical and immunoprecipitation studies demonstrate overlapping nuclear localization and physical interaction between Dlx3 and Smad6 in human choriocarcinoma cells and in differentiated trophoblasts from human placenta. In vitro protein interaction studies mapped the Smad6 interaction domain within Dlx3 to residues 80-163, a region of Dlx3 that includes a portion of the homeodomain. Dlx3 and Dlx4 share homology within this region, and Dlx4 was also found to bind Smad6. Using the Esx1 gene promoter as a model for a Dlx3-responsive gene, studies demonstrate two near consensus Dlx3 binding sites within the proximal 2.3 kb of the transcription start site. Interestingly, binding of Dlx3 to one of these two sites was inhibited by interaction with Smad6. Consistent with this result, expression of an Esx1 promoter luciferase reporter was increased by overexpression of Dlx3; this effect was reversed with co-expression of Smad6. Further, small interference RNA-mediated knockdown of endogenous Smad6 increased Dlx3-dependent expression of the Esx1 gene promoter. Thus, Smad6 appears to functionally interact with Dlx3, altering the ability of Dlx3 to bind target gene promoters. Smad6 appears to play a modulatory role in the regulation of Dlx3-dependent gene transcription within placental trophoblasts. Dlx3 (Distal-less 3) is a homeobox-containing transcription factor required for normal placental development in mice. Here we demonstrate that Dlx3 interacts with Smad6, a member of a larger family of transcriptional regulators generally thought to regulate transforming growth factor β/bone morphogenetic protein signaling. Immunocytochemical and immunoprecipitation studies demonstrate overlapping nuclear localization and physical interaction between Dlx3 and Smad6 in human choriocarcinoma cells and in differentiated trophoblasts from human placenta. In vitro protein interaction studies mapped the Smad6 interaction domain within Dlx3 to residues 80-163, a region of Dlx3 that includes a portion of the homeodomain. Dlx3 and Dlx4 share homology within this region, and Dlx4 was also found to bind Smad6. Using the Esx1 gene promoter as a model for a Dlx3-responsive gene, studies demonstrate two near consensus Dlx3 binding sites within the proximal 2.3 kb of the transcription start site. Interestingly, binding of Dlx3 to one of these two sites was inhibited by interaction with Smad6. Consistent with this result, expression of an Esx1 promoter luciferase reporter was increased by overexpression of Dlx3; this effect was reversed with co-expression of Smad6. Further, small interference RNA-mediated knockdown of endogenous Smad6 increased Dlx3-dependent expression of the Esx1 gene promoter. Thus, Smad6 appears to functionally interact with Dlx3, altering the ability of Dlx3 to bind target gene promoters. Smad6 appears to play a modulatory role in the regulation of Dlx3-dependent gene transcription within placental trophoblasts. The Distal-less family of transcriptional regulators includes six members in mammals, arrayed in pairs and aligned with the hox gene clusters along different chromosomes (1.Bendall A.J. Abate-Shen C. Gene (Amst.). 2000; 247: 17-31Crossref PubMed Scopus (227) Google Scholar, 2.Beanan M.J. Sargent T.D. Dev. Dyn. 2000; 218: 545-553Crossref PubMed Scopus (73) Google Scholar). Dlx3 is tandemly arrayed with Dlx4 on human chromosome 17 and is involved in developmental determination of multiple tissues, including the first and second branchial arches, teeth, bone, and multiple epithelia, including the skin, mammary gland primordia, and the placenta (3.Morasso M.I. Mahon K.A. Sargent T.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3968-3972Crossref PubMed Scopus (72) Google Scholar). The relationship between convergently transcribed pairs of Dlx family members and specific hox gene clusters has suggested that, although independent of hox gene expression patterns, these homeodomain-containing transcription factors are clearly involved in important aspects of developmental morphogenesis (reviewed in Ref. 2.Beanan M.J. Sargent T.D. Dev. Dyn. 2000; 218: 545-553Crossref PubMed Scopus (73) Google Scholar). The importance of Dlx3 during development and in the adult arises from several different observations of disease states. Mutations in Dlx3 are believed to be causally related to tricho-dento osseous syndrome, a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium (4.Price J.A. Bowden D.W. Wright J.T. Pettenati M.J. Hart T.C. Hum. Mol. Genet. 1998; 7: 563-569Crossref PubMed Scopus (205) Google Scholar, 5.Price J.A. Wright J.T. Kula K. Bowden D.W. Hart T.C. J. Med. Genet. 1998; 35: 825-828Crossref PubMed Scopus (73) Google Scholar, 6.Haldeman R.J. Cooper L.F. Hart T.C. Phillips C. Boyd C. Lester G.E. Wright J.T. Bone. 2004; 35: 988-997Crossref PubMed Scopus (36) Google Scholar). The defect in Dlx3 leading to tricho-dento osseous syndrome appears to be associated with a four-nucleotide deletion just downstream of the homeodomain, resulting in a premature truncation of the protein. Amelogenesis imperfecta with taurodontism has similar characteristics as tricho-dento osseous syndrome and has also been associated with mutations with Dlx3 in some families investigated, albeit distinct from the fournucleotide deletion/mutation described above (7.Dong J. Amor D. Aldred M.J. Gu T. Escamilla M. MacDougall M. Am. J. Med. Genet. A. 2005; 133: 138-141Crossref Scopus (103) Google Scholar). Amelogenesis imperfecta is an autosomal dominant trait leading to dental enamel defects and enlarged pulp chambers and has been associated with a two-nucleotide deletion within the homeodomain of Dlx3. This deletion again results in a frameshift and premature truncation of Dlx3 in the carboxyl terminus, primarily downstream of the homeobox. In addition to tricho-dento osseous syndrome and amelogenesis imperfecta, Dlx3/Dlx4 have been identified in the gene interval thought to be involved in some forms of craniofacial abnormalities, including cleft palate (8.Juriloff D.M. Harris M.J. Brown C.J. Mamm. Genome. 2001; 12: 426-435Crossref PubMed Scopus (52) Google Scholar). The putative involvement of Dlx3 in the occurrence of cleft palate is also supported by the murine model deficient in endothelin-A receptor (the endothelin-A receptor), which results in cleft palate and hypoplasia of the mandible (9.Clouthier D.E. Williams S.C. Yanagisawa H. Wieduwilt M. Richardson J.A. Yanagisawa M. Dev. Biol. 2000; 217: 10-24Crossref PubMed Scopus (176) Google Scholar, 10.Clouthier D.E. Williams S.C. Hammer R.E. Richardson J.A. Yanagisawa M. Dev. Biol. 2003; 261: 506-519Crossref PubMed Scopus (49) Google Scholar, 11.Clouthier D.E. Schilling T.F. Birth Defects Res. C Embryol. Today. 2004; 72: 190-199Crossref PubMed Scopus (48) Google Scholar). In this model, Dlx3 expression is thought to be dependent upon endothelin-1 through a Gαq/Gα11-dependent mechanism. In the Gαq/Gα11-deficient mouse, Dlx3, among other factors, is specifically down-regulated, supporting the speculation of the importance of Dlx3 in cranio-facial morphogenesis (12.Ivey K. Tyson B. Ukidwe P. McFadden D.G. Levi G. Olson E.N. Srivastava D. Wilkie T.M. Dev. Biol. 2003; 255: 230-237Crossref PubMed Scopus (49) Google Scholar). Thus, the role and importance of Dlx3 in morphogenic aspects of development and in epithelial differentiated function is rather far reaching. The Dlx3 null mouse dies in utero by embryonic day 10 due to putative placental failure (13.Morasso M.I. Grinberg A. Robinson G. Sargent T.D. Mahon K.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 162-167Crossref PubMed Scopus (218) Google Scholar). This was associated with a failure in the development/morphogenesis of the placental labyrinth compartment of the murine placenta. Further, genetic loss of Dlx3 was correlated with reduced expression of an additional homeobox factor, Esx1, suggesting that Dlx3 may be an important transcriptional regulator of Esx1 promoter activity. Studies from our laboratory identified Dlx3 as a cell type-specific transcriptional activator in placental trophoblasts. Dlx3 binds to and transactivates the promoter for the glycoprotein hormone α subunit gene via a cis-acting element required for full basal activity of this gene (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google Scholar). The glycoprotein hormone α subunit is a subunit of the glycoprotein small interference domain transforming growth factor bone morphogenetic has been identified in and and appears to play a role in the of in to the and of J.T. J. H. A. B. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, M. J. 2004; Scopus Google Scholar). for the of the α subunit promoter and regulation of Esx1 in the Dlx3 null mouse, Dlx3 appears to function as a putative transcriptional has also been that Dlx3 as a regulator of gene transcription in M.J. Sargent T.D. Dev. Dyn. 2000; 218: 545-553Crossref PubMed Scopus (73) Google Scholar). This may be due to of Dlx3 in Ref. 2.Beanan M.J. Sargent T.D. Dev. Dyn. 2000; 218: 545-553Crossref PubMed Scopus (73) Google dependent upon cell and This was the for to binding of Dlx3 in the of the human placenta. The studies Smad6 as a binding for Dlx3 a of a human placental Dlx3 and Smad6 are in the of cells of including and trophoblasts from differentiated human placenta. between Dlx3 and Smad6 the binding of Dlx3 that Smad6 as a regulator of Dlx3-dependent gene transcription of the Esx1 promoter. and in these studies by two through for human Dlx3 was by Morasso of of deletion of the Dlx3 by the by and sites to the and The in these as and The in these as and the by the for in Smad6 expression was a from H. expression was a from The human Dlx4 was by from from cells the and The resulting Dlx4 was and by The promoter was by mouse and the and and sites the of the and The and the promoter was a luciferase reporter The of the was by was to the Dlx3 binding within the Esx1 luciferase This a for the near consensus Dlx3 binding site. The was The human α subunit gene promoter luciferase reporter has been (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google Scholar). Dlx3 as the protein with a human placental as the The human Dlx3 was the and in the human placental was in The and as was the to of was in the The and with an of The required and the required was in of the with the human placental on of on to that the was to activity to and the of with and the and a of the was by The interaction between Dlx3 and target was a the and on of cells for the of nuclear with by in with a of to as and by and in a of and and to for on by and by the cell a by for The nuclear was in a 10 and was to a of and nuclear with for by for and the nuclear was in of the nuclear by from and cell nuclear in of and the nuclear protein was to and to for with for to protein the normal Dlx3 Smad6 of protein was and to for an additional with in an of and for and for on by and to by with in the Dlx3 was in The from the Smad6 was the of Smad6 on due to similar In other the Smad6 was and the was by cells on for one with and for with in to in cells with Dlx3 again with by with a and in for in through a of and with and with of human placenta from from the and of the and the on the of in with for and to and was as described (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google that as described and Smad6, Dlx3, and of Dlx3 a transcription and the 10 portion of protein was for a to a along with Smad6 and of protein was and to for an additional with by and the was in and for with The was in and and by as described (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google Scholar, K.A. M.S. 2004; PubMed Scopus Google the for by the addition of Dlx3 binding (the element from the glycoprotein subunit promoter (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google and two putative Dlx3 binding sites identified within the Esx1 The binding an additional and on Smad6 Dlx3 in binding Dlx3 to with for to Smad6 protein was and the for an additional with binding Dlx3 and Smad6 and for the full the and by binding studies with similar The for as one binding proximal binding and cells in with cells to and cells with an Esx1 promoter luciferase reporter Dlx3 expression a and of Smad6 expression with a of by with the activity was of from the and luciferase activity was by cell protein as by a studies in on with similar are as 3) of a of expression was for of specific and expression of in cells a within the human Smad6 The as The to as Smad6 and in was a to gene and as an The and of the and also and sites the of the and The and the of the with and a for the by and of cells in and with the the the for and through a and the was for of cells in the of cells to the and cells with and to for cell for cells and in of the cell as described to of and between of was Dlx3 and Smad6 in a human Dlx3 was as a protein in a of a human placental The of independent identified from the and in a with the the The resulting in the interaction by with Dlx3 was a the region of Smad6. of the Smad6 growth on in the of of Dlx3 supported growth the of Dlx3 and Smad6 in of growth Dlx3 and Smad6 in the in and in studies on localization of Dlx3 and Smad6 in cells of Dlx3 and Smad6 and primarily in the nuclear compartment in cells a choriocarcinoma cell of Consistent with this Dlx3 and Smad6 nuclear to and within of differentiated human placenta. of Dlx3 in placenta additional the expression of Dlx3 during in have Dlx3 in placental trophoblasts in human placenta during of human (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google Scholar). The studies the that Dlx3 expression may be within cell in the human placenta. Dlx3 and Smad6 in the choriocarcinoma cell model, we to this model for have cells for of gene related to glycoprotein hormone and Dlx3 gene promoter expression (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google Scholar, K.A. M.S. 2004; PubMed Scopus Google Scholar, M.S. M. T. J.M. Mol. Biol. 2000; PubMed Scopus Google Scholar). studies nuclear from cells and Smad6 that Dlx3 and Smad6 interact in cells The Dlx3 to Smad6 was due to the of Smad6 and interference with the in the this was with of Dlx3 and Smad6 as with of Dlx3 and Smad6 with the Dlx3 specific with Smad6 Thus, the interaction in the was supported by studies in choriocarcinoma cells that these two and in vitro Dlx3 and Smad6 of the the of the we a of deletion of Dlx3 The for these mutations was on the of important within Dlx3 by J.T. Morasso M.I. J. Sci. 2000; PubMed Google Scholar, Morasso M.I. 2001; PubMed Scopus Google upon the homeodomain the binding Dlx3 deletion in vitro along with Dlx3 and Smad6. Smad6 was to the to interaction with Dlx3. we identified the importance of which Smad6 similar to Smad6 as suggesting that Smad6 interaction domain required a portion of the homeodomain upon these we that also bind Smad6. This was in our putative of these deletion of similar as the Dlx3 deletion a region of that was to bind Smad6 in vitro studies again supported the that the portion of the homeodomain was for interaction with Smad6 in of Dlx3 with Dlx4 member of the Distal-less family in placenta J. B. Gene (Amst.). PubMed Scopus Google within this region with of homology within the homeodomain, supporting the that Dlx4 may also bind Smad6. upon in vitro Dlx4 Smad6 to to bind Smad6 was in these studies as a binds Smad6. of Dlx3 and Dlx4 between and of within the Smad6 interaction domain The consensus is in the Smad6, and Dlx4 as as described in the to and to studies the Smad6 and Smad6 and of Dlx3 Dlx4 as The and by and by with Dlx3 to the of the Esx1 to the of the interaction on gene we 2.3 of the of the Esx1 promoter. The of the Dlx3 null mouse that the loss of Dlx3 in was correlated with a loss of Esx1 in mouse placenta (13.Morasso M.I. Grinberg A. Robinson G. Sargent T.D. Mahon K.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 162-167Crossref PubMed Scopus (218) Google Scholar). of this we identified two near consensus Dlx3 binding sites within the promoter The was and the proximal to the transcription start of the Esx1 as P. Dev. Biol. PubMed Scopus Google Scholar). of these two the of the binding was the two on the of this from the consensus Dlx3 binding J.A. Morasso M.I. Sargent T.D. Res. 1999; PubMed Scopus Google we in the human glycoprotein hormone α subunit promoter (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google Scholar). Using we the binding the and proximal putative Dlx3 binding sites from the Esx1 promoter and nuclear from cells a the from the α subunit promoter was of with of these binding The was similar to the binding with the proximal site. Dlx3 in sites to bind Dlx3; in the binding of Dlx3 for these binding sites The Dlx3 was larger in due to the of the the protein. The of binding for Dlx3 was the the the proximal our on the Dlx3 binding within the Esx1 gene promoter. studies nuclear and Dlx3 that endogenous Dlx3 bind to this The addition of the Dlx3 to binding in that normal The addition of the Smad6 the protein to these binding in suggesting that Smad6 may in binding with Dlx3 and that Dlx3 the importance of this to Esx1 gene the was to a and a luciferase reporter gene in studies The Esx1 promoter was by Dlx3. The in the putative Dlx3 binding reduced Esx1 expression by suggesting that this was important for promoter activity by binding of the Esx1 promoter binds Dlx3 and is for full gene the binding for Dlx3 and cell nuclear studies a Dlx3 binding of endogenous including Dlx3, to this site. on the and by The addition of a Dlx3 to binding in the of a as was as a for the Dlx3 In some binding the was and to the Dlx3 as a is by an the of the studies to the importance of the Dlx3 binding on expression of the Esx1 promoter luciferase reporter cells with Esx1 luciferase reporter an Esx1 luciferase reporter a within the Dlx3 binding site. with a Dlx3 expression are as luciferase activity for protein for a The and to the of Smad6 on Dlx3 Dlx3 and Smad6 that the interaction of Dlx3 with the Dlx3 binding within the Esx1 promoter In this Smad6 a with the Dlx3 binding site. Smad6 was the binding Dlx3, the Dlx3 binding was in a Using the as a similar of Smad6 protein reduced Dlx3 albeit to a with the of the Esx1 promoter. Studies on Dlx3 be by Smad6 in binding the addition of Dlx3 with Smad6 and Dlx3 binding was to and Smad6 was studies that for the Smad6 for Dlx3 binding of the of protein of the suggesting that Smad6 for Dlx3 on gene that have Dlx3 binding within the Esx1 gene promoter. the of the interaction on the Esx1 we again the Esx1 reporter upon the binding studies described our was that Smad6 overexpression expression of the Esx1 promoter by Dlx3. Using in overexpression of Dlx3 and Smad6 in increased of Dlx3 and Smad6 Further, Dlx3 overexpression increased Esx1 luciferase activity of Smad6 this in reduced basal activity of the Esx1 reporter gene Consistent with our with of Smad6 expression along with Dlx3 in a of Esx1 promoter activity. of the α subunit gene promoter was also reduced by overexpression of Smad6 In an to the of Smad6 on gene transcription in we the of Smad6 on the domain protein and promoter expression J.A. J.A. Biol. 2004; PubMed Scopus Google Scholar, J.A. A. S. J.A. J.A. Mol. Dev. 2003; PubMed Scopus Google is dependent upon Dlx3 expression The promoter is in In to the of Smad6 on expression of the Esx1 and α subunit Smad6 effect on the promoter and increased expression of the promoter in a studies the that Smad6 transcriptional and the of Smad6 on Esx1 and α subunit promoter activity are of Smad6 in of the Esx1 Gene overexpression studies be of studies be the of Smad6 on Dlx3-dependent gene we cell specific Smad6 of the cell found to have specific in Smad6 protein expression and with expression of Dlx3 studies in the and the Smad6 cell the Esx1 luciferase reporter that basal activity of this promoter was in the and cell in the and cell In to Dlx3, Esx1 luciferase promoter activity was in Smad6 and cell in cell and in a correlated with the loss of Smad6 in these cell Thus, with the overexpression loss of endogenous Smad6 was correlated with Dlx3-dependent transcription of the Esx1 gene promoter. The studies for the between Dlx3, a Distal-less homeobox factor required for normal placental development in the mouse, and an Smad6. The role of Dlx3 as a transcriptional activator is upon studies in as the glycoprotein hormone α subunit gene promoter (14.Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. T. J. Biol. 2001; PubMed Scopus Google the expression of the homeobox factor Esx1 in the Dlx3 null mouse (13.Morasso M.I. Grinberg A. Robinson G. Sargent T.D. Mahon K.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 162-167Crossref PubMed Scopus (218) Google and the role of Dlx3 in the regulation of the gene A. Morasso M.I. J. M. A.J. Mol. Biol. 2004; PubMed Scopus Google Scholar). Dlx3 has been to transcriptional as as transcriptional in (reviewed in Ref. 2.Beanan M.J. Sargent T.D. Dev. Dyn. 2000; 218: 545-553Crossref PubMed Scopus (73) Google Scholar). The ability of Dlx3 to in several different as a transcriptional may be by the of and cell type-specific binding The important that Dlx3 and Smad6 a in a in cells in and in vitro Dlx3 and Smad6 also overlapping expression in the of differentiated human placental suggesting that a interaction between these two may be important in The that one Dlx3 is an may also important between the transforming growth factor morphogenetic protein and important developmental that Dlx3. are transcriptional regulators that be (reviewed in K. Sci. 2000; PubMed Scopus Google Scholar, Sci. 2004; PubMed Scopus Google Scholar, D. M. 2004; PubMed Scopus Google Scholar). and are of the activity of of the with a and nuclear of the and to target have an interaction between and in the of regulation of cells S. K. K. M. S. K. S. H. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). of this family is the and In the of the activity of the and several have been to for binding to the an for activity J. 2003; PubMed Scopus Google Scholar). In Smad6 has been to with the ability of to a with independent of A. G. J. A. Dev. 1998; 12: PubMed Scopus Google Scholar). also with that to be involved in nuclear and target the and for by the again to an K. Sci. 2000; PubMed Scopus Google Scholar, Sci. 2004; PubMed Scopus Google Scholar, D. M. 2004; PubMed Scopus Google Scholar). In the nuclear Smad6 appears to the transcriptional to target related to B. M. Mol. Biol. 2003; PubMed Scopus Google Scholar). upon the Dlx3 and Smad6 to the of placental trophoblasts independent of signaling. Smad6 has been to as a transcriptional interaction with homeodomain transcription factor, S. J. Biol. 2000; PubMed Scopus Google Scholar, S. J. Biol. PubMed Scopus Google Scholar). In these Smad6 with to be to to transcription via of studies that the interaction between Dlx3 and Smad6 may an different for transcriptional in human placental Smad6 interaction clearly in a of Dlx3 binding via results demonstrate that the Smad6 interaction includes the first two α of the homeodomain. Smad6 interaction with Dlx3 these two α are to binding as This a for of transcriptional a may of Dlx3 with specific target gene promoter Smad6 protein overexpression inhibited Dlx3-dependent gene in the expression of endogenous Smad6 knockdown important that Dlx3-dependent gene expression may be to in Smad6 protein in placental the importance and of Dlx3-dependent to Smad6 This may be of Dlx3 target with binding sites that have binding The Dlx3 homeodomain is and as a binding one of the characteristics of homeobox transcription factors Gene (Amst.). PubMed Scopus Google Scholar, G. Sci. PubMed Scopus Google Scholar). The domain of Dlx3 has been in studies a nuclear localization in a region to and including a portion of the transcriptional have been to residues and and within the homeodomain is an for protein C J.T. Morasso M.I. J. Sci. 2000; PubMed Google Scholar, Morasso M.I. 2001; PubMed Scopus Google Scholar, J.A. Morasso M.I. Sargent T.D. Res. 1999; PubMed Scopus Google Scholar). The deletion in the studies residues within Dlx3 as the Smad6 interaction domain in This region of Dlx3 the first full α and of within the homeodomain. In this interaction a for protein C activity. for may the is a interference model, the interaction includes a portion of the homeodomain. In the of in this interaction domain to The of this is in six Dlx family members (1.Bendall A.J. Abate-Shen C. Gene (Amst.). 2000; 247: 17-31Crossref PubMed Scopus (227) Google with the that, Dlx3, Dlx4 is also a protein. Using Dlx3, Morasso and Morasso M.I. 2001; PubMed Scopus Google that by protein C by protein in of Dlx3 with the of Smad6 on This speculation that of Dlx3 may Smad6 leading to reduced binding Dlx3 target Studies are to this and to be in cell are to regulation by a of growth factors, as growth factor, and In the of these growth factors are through and transcriptional This has to a an are by of (reviewed in Ref. K. PubMed Scopus Google Scholar). Interestingly, Dlx3 expression during development in upon J.A. M.J. M. Sargent T.D. Dev. Biol. 1999; PubMed Scopus Google Scholar, T. M. Sargent T.D. J. Dev. Biol. 2001; Google Scholar). of these the receptor in a of Dlx3, and Dlx3 expression in mouse was also to be to regulation by Morasso M.I. Res. PubMed Scopus Google in a with This speculation that Smad6 expression as a the of increased Smad6 expression may to important of Dlx3-dependent gene have been to regulate during the that these of are and important during placental morphogenesis J. S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). and for during the of these and the the for important in and Morasso and members of the laboratory for in the of the