The Cardiac Determination Factor, Nkx2-5, Is Activated by Mutual Cofactors GATA-4 and Smad1/4 via a Novel Upstream Enhancer

Abstract

The mammalian homologue of Drosophila tinman, Nkx2-5, plays an early role in regulating cardiac genes and morphogenesis. Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-β family of signaling molecules, are involved in numerous developmental processes. BMP signaling is crucial in the regulation of Nkx2-5 expression and specification of the cardiac lineage. Constitutively active BMP type I receptor or the downstream pathway components and DNA-binding transcription factors, Smad1/4 directly activated Nkx2-5 gene transcription. We identified and characterized a novel upstream Nkx2-5 enhancer, composed of clustered repeats of Smad and GATA DNA binding sites. This composite Nkx2-5 enhancer was a direct target of BMP signaling via cooperative interactions between the downstream transducers Smad1/4 and GATA-4. In mammalian two hybrid assays, Smad factors recruited the hybrid gene GATA4-VP16 to strongly drive transcription of a reporter gene containing multimerized Smad binding sites These cofactors interacted through the second zinc finger and adjacent basic domain of GATA-4 and the N-terminal domain of Smads. Smad4 and GATA4 were also found to bind in vivo with the Nkx2-5 composite enhancer, as revealed by chromatin immunoprecipitation analysis of differentiated P19 cells. Finally, transgenic mice containing the Smad/GATA composite enhancer recapitulated early murine Nkx2-5 cardiac expression and deletion of this enhancer within a 10-kb transgene pBS-Nkx2-5 LacZ significantly reduced expression in the cardiac crescent. Thus, integration of GATA transcription factors with BMP signaling, through co-association with Smads factors, may initiate early Nkx2-5 expression; suggesting a vital role for the combination of these factors in the specification of cardiac progenitors. The mammalian homologue of Drosophila tinman, Nkx2-5, plays an early role in regulating cardiac genes and morphogenesis. Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-β family of signaling molecules, are involved in numerous developmental processes. BMP signaling is crucial in the regulation of Nkx2-5 expression and specification of the cardiac lineage. Constitutively active BMP type I receptor or the downstream pathway components and DNA-binding transcription factors, Smad1/4 directly activated Nkx2-5 gene transcription. We identified and characterized a novel upstream Nkx2-5 enhancer, composed of clustered repeats of Smad and GATA DNA binding sites. This composite Nkx2-5 enhancer was a direct target of BMP signaling via cooperative interactions between the downstream transducers Smad1/4 and GATA-4. In mammalian two hybrid assays, Smad factors recruited the hybrid gene GATA4-VP16 to strongly drive transcription of a reporter gene containing multimerized Smad binding sites These cofactors interacted through the second zinc finger and adjacent basic domain of GATA-4 and the N-terminal domain of Smads. Smad4 and GATA4 were also found to bind in vivo with the Nkx2-5 composite enhancer, as revealed by chromatin immunoprecipitation analysis of differentiated P19 cells. Finally, transgenic mice containing the Smad/GATA composite enhancer recapitulated early murine Nkx2-5 cardiac expression and deletion of this enhancer within a 10-kb transgene pBS-Nkx2-5 LacZ significantly reduced expression in the cardiac crescent. Thus, integration of GATA transcription factors with BMP signaling, through co-association with Smads factors, may initiate early Nkx2-5 expression; suggesting a vital role for the combination of these factors in the specification of cardiac progenitors. The homeobox gene, Nkx2-5 (also called Csx), a vertebrate homologue of Drosophila tinman, is one of the earliest known markers of mesoderm fated to give rise to myocardium (reviewed by Harvey, Ref.1Harvey R.P. Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (486) Google Scholar). Nkx2-5, tinman, and other related factors recognize novel NKE sequence elements (2Damante G. Fabbro D. Pellizzari L. Civitareale D. Guazzi S. Polycarpou-Schwartz M. Cauci S. Quadrifoglio F. Formisano S. Di Lauro R. Nucleic Acids Res. 1994; 22: 3075-3083Crossref PubMed Scopus (101) Google Scholar, 3Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and may require combinatorial interactions with other cardiac-restricted factors, such as serum response factor, GATA-4 and Tbx5 for regulation of myogenic-specified genes (4Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 5Sepulveda J.L. 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Development. 1999; 126: 75-84Crossref PubMed Google Scholar). A more proximal enhancer was able to drive expression in the stomach and outflow tract and the early heart tube (10Searcy R.D. Vincent E.B. Liberatore C.M. Yutzey K.E. Development. 1998; 125: 4461-4470Crossref PubMed Google Scholar). These Nkx2-5 enhancers appear to be under the control of GATA dual zinc finger transcription factors and their cognate DNA binding sites. GATA-4/5/6 have overlapping expression with Nkx2-5, and GATA-4-null mice died of severe defects in heart and foregut formation, with a failure of fusion of the bilateral heart fields (11Molkentin J.D. Lin Q. Duncan S.A. Olson E.N. Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (936) Google Scholar). However, since GATA factors are expressed in a greater diversity of embryonic tissues than those with Nkx2-5, GATA signaling alone could not provide the appropriate range of Nkx2-5 expression (12Molkentin J.D. J. Biol. 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PubMed Scopus Google Scholar). Smad sites in the proximal enhancer of Nkx2-5 C.L. J. Richardson J.A. Olson E.N. Dev. Biol. PubMed Scopus Google Scholar, C.M. R.D. Vincent E.B. Yutzey K.E. Dev. Biol. PubMed Scopus Google Scholar) were found to be required for These sites were also GATA sites (10Searcy R.D. Vincent E.B. Liberatore C.M. Yutzey K.E. Development. 1998; 125: 4461-4470Crossref PubMed Google Scholar). Thus, and vertebrate and the Smad factors are involved in embryonic heart development, and it is this signaling pathway with GATA factors to Nkx2-5 and other cardiac We found that BMP signaling activated Nkx2-5 directly through Smad1/4 transcription factors, and that was with GATA We identified a novel upstream Nkx2-5 gene enhancer composed of highly Smad and GATA binding sites. This Smad/GATA composite enhancer LacZ reporter transgene in the cardiac and in the it recapitulated embryonic Nkx2-5 gene Smad factors were also of GATA-4 to a Smad reporter and in a mammalian In GATA-4 and Smad co-association was to be in their role as of the BMP signaling expression was by was a of and with a GATA-4 was by with the in and sites. this and were a was of the second zinc finger to and of GATA-4 were by and by the upstream region of the Nkx2-5 gene to to and to were by and the J. R. D. M. V. Genes Dev. Scopus Google Scholar). a pBS-Nkx2-5 LacZ (8Reecy J.M. Li X. Yamada M. DeMayo F.J. Newman C.S. Harvey R.P. Schwartz R.J. Development. 1999; 126: 839-849Crossref PubMed Google Scholar) containing the enhancer was and sites of to pBS-Nkx2-5 which a containing the enhancer was and the to The was sites of pBS-Nkx2-5 LacZ to a DNA was for by of the by by and the was the were or were to transgenic were and for as (8Reecy J.M. Li X. Yamada M. DeMayo F.J. Newman C.S. Harvey R.P. Schwartz R.J. Development. 1999; 126: 839-849Crossref PubMed Google Scholar). were by the and LacZ and were and were in with serum and and were or to the P19 the of were in containing serum and for and for more in were in were with and by was a to activated and to and was found by to of control the and and proteins were by in cells. were as J.L. Belaguli N. Nigam V. Chen C-Y. Nemer M. Schwartz R.J Mol. Cell. Biol. 1998; 18: 3405-3415Crossref PubMed Scopus (270) Google Scholar). GATA-4 was by of by in were by GATA-4 proteins with proteins in binding and for by in the were by in were which were and to were with and and or were as Lin X. R. J. 2000; PubMed Scopus Google Scholar, M. Lin X. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The were in a containing and GATA-4 proteins were the an were a and analysis was or were as J.L. Belaguli N. Nigam V. Chen C-Y. Nemer M. Schwartz R.J Mol. Cell. Biol. 1998; 18: 3405-3415Crossref PubMed Scopus (270) Google Scholar) in of GATA binding Smad were as M. Lin X. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) in of Smad binding were and the to in with The for the were as and and were and and and in GATA-4 were for binding was as by and PubMed Scopus Google Scholar). or differentiated P19 were with in growth for with was to a of to were by and were and to chromatin for and of of chromatin was the control in the and as The were by with and immunoprecipitation was with of Smad4 a of GATA4 were with A by of were the with two with A second immunoprecipitation was to with the of the were by the in the of were with and with in the of of as a DNA were by in of and for The were as composite enhancer, and enhancer, and enhancer, and The were with and under Nkx2-5 by BMP through Smad and GATA to cis-acting of a of in the of the Nkx2-5 gene R.J. Olson E.N. Development. 1999; 126: 4187-4192Crossref PubMed Google Scholar). of the upstream of Nkx2-5 to a reporter were with active type I BMP receptor P19 which are of cardiac Nkx2-5 and BMP signaling K. I. Y. Kudoh S. D. Yazaki Y. Komuro I. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, K. Y. Kudoh S. D. M. K. S. Yazaki Y. Nagai R. Komuro I. J. Biol. 2001; PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar). The reporter with the of region was activated by the region was upstream and downstream and the upstream the response to activated BMP receptor was through the which was activated more strongly by than with the and of the BMP signaling the Smad factors, and were found to the alone and in combination the known of GATA factors for expression in GATA-4 alone was found to the reporter with and Smad4 alone and in combination with the two Smad factors and an the activation by Smad1/4 with or GATA-4 Smad1/4 activation by and by GATA-4 was Thus, the as a and Smad4 of Nkx2-5 gene GATA-4 and overlapping expression with and We which of GATA-4 were for Smad and could for GATA-4 as a Smad In a of Smad factors, a activation of the Nkx2-5 reporter by Smad1/4 or was observed GATA-4 and were of the Smads in the of However, of GATA-4 the or activation in a with to activation by the Smad of GATA-4 also an of for the in the of is not involved in GATA or more likely the Smad4 in the is for DNA binding and with the GATA that this type of the of a Smad by between the and We have the activation of the Nkx2-5 and by BMP signaling, and cooperative activation of the reporter by Smad and GATA transcription deletion analysis of the Nkx2-5 gene was in of the the upstream In the was a of between and and a of Smad between and in the and and their was a in between and and a in Smad and cooperative deletion to This in to deletion to the which a Smad Thus, a proximal Smad was and a distal and an region between and were for combinatorial for could GATA or Smad or The distal is likely the GATA identified by C.L. Wu C. Mercer B. Webb R. Richardson J.A. Olson E.N. Development. 1999; 126: 75-84Crossref PubMed Google Scholar). and of a in the Nkx2-5 identified an region within the that a of GATA and Smad sites A and were GATA sites with sites by Smad in in a as in GATA sites were found the upstream identified sites. Smad were also found in and directly adjacent to GATA sites. it is known that Smads bind to or Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google this Smad/GATA composite region was for activation and binding by GATA and Smad The Smad/GATA and GATA and Smad were upstream of an to a LacZ reporter In these and were P19 with GATA and Smad factors and for In GATA-4 and Smad1/4 GATA and Smad sites GATA-4 and Smad1/4 expression alone activated this the combination strongly transcription The Smad reporter was only activated by Smad transcription factors and activated by GATA-4 alone or the to the of Smad in P19 cells. GATA-4 could not through the GATA sites and the of Smad1/4 not have These may that GATA-4 factors were to Smad sites via Smads factors, Smad1/4 were not recruited through GATA sites. the of Smad and adjacent GATA sites for the of A containing the proximal cardiac and stomach enhancer, and to have GATA and Smad was also for This enhancer was not significantly activated by it was and it was also strongly by the combination of Smad and GATA factors of GATA and Smad to for binding of the composite enhancer by GATA and Smad transcription factors, were of the Smad/GATA composite enhancer as in was of containing repeats of GATA or Smad as of the highly sites found in the combinatorial the binding of expressed and and Smad4 to the Smad sites. by Smad4 was than that of and the combination of the two the binding and of was by with the Smad sites. the binding of to GATA sites. by GATA was than that observed for a GATA the or the and of binding of GATA-4 to direct sites. the binding Thus, the Smad/GATA composite Nkx2-5 enhancer that were strongly by GATA and Smad Smad GATA-4 in GATA factors were to Smads to GATA sites Biol. Full Text Full Text PDF PubMed Scopus Google and the were We reporter gene which contains repeats in and a Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). This reporter gene characterized in P19 cells. In and Smad4 of GATA-4 to the multiple and Smad4 this reporter and and their was was a for in with Smad4 and GATA-4. alone with the Smad reporter was the by the of the to for the Smad1/4 In GATA-4 and Smad factors were with the Smad GATA-4 with Smad4 the of Smad4 of not the activation by Smad4 with or GATA-4. and were by with GATA-4 an in the DNA-binding binding to Smad and activation of this In Smad4 was with GATA-4 or a that the of the second zinc and to bind target sites. The GATA-4 zinc finger was able to with to a as strongly as the GATA-4 is recruited to the reporter by of Smad transcription Smad with GATA the between Smad and GATA factors to reporter and the of GATA-4 to Smad sites by Smad We expressed and in GATA In A and and of proteins were for with GATA-4. is an the GATA-4 with of Smad proteins were able to with GATA-4. GATA-4 was not by with a the with the N-terminal and the more in the of by BMP Smad4 also and interacted the as for with a deletion in the domain DNA interacted more these that the DNA-binding N-terminal domain of Smad proteins was able to bind in a region overlapping the DNA binding to determine which GATA factors and were involved in Smad We that since Smad4 interacted strongly with GATA-4 and it is the for Smad that it be the Smad to for GATA is a of of the in GATA proteins and were by not by This that the domain likely in a region of the GATA one of the zinc finger analysis revealed that binding to Smad4 required the second zinc finger and adjacent basic domain of and that not with the N-terminal or the zinc finger A GATA-4 with of the of the second zinc finger was by as as the the binding is not We were that the not be of in vivo cells. in the of GATA-4 proteins or were an the proteins by of The the of Smad and GATA proteins in In with the GATA-4 was able to strongly with more with or the deletion of the N-terminal domain and the zinc finger with of the which an by those and of GATA-4 through second zinc finger and other In the between GATA and Smad proteins between the Smad and the GATA and basic In between Smad4 and GATA4 with the Nkx2-5 direct of in vivo of Smad4 and GATA4 with the Nkx2-5 composite enhancer identified in this immunoprecipitation analysis and differentiated as in and of the chromatin with and the DNA was to with the of for the Smad/GATA enhancer as as for the Nkx2-5 enhancer and enhancer that a Smad within is required for the enhancer early and of heart in vivo (10Searcy R.D. Vincent E.B. Liberatore C.M. Yutzey K.E. Development. 1998; 125: 4461-4470Crossref PubMed Google Scholar); the region as a control for Smad4 The distal enhancer was to be by GATA4 C.L. Wu C. Mercer B. Webb R. Richardson J.A. Olson E.N. Development. 1999; 126: 75-84Crossref PubMed Google Scholar). In Smad4 immunoprecipitation with differentiated P19 a and a composite enhancer were of enhancer was In Smad4 immunoprecipitation with P19 of the enhancer could be the Smad4 binding to and the Smad/GATA enhancer were cardiac a the chromatin was In these of the enhancer were the of the GATA4 was observed to bind to the Smad/GATA composite enhancer and not to the enhancer by the in vivo Smad/GATA for Nkx2-5 GATA and Smad was for in transgenic mice with the reporter In A and two transgenic for analysis of of The in only in the cardiac as for the Nkx2-5 gene this that in which also through the These transgenic were of this expression are in The of the enhancer were to expression of LacZ and also in a for between Smad and GATA sites. In a transgenic containing the composite enhancer was and were was observed the heart tube and in the heart and LacZ was also observed in the and in the myocardium and in the and the of the was found in and and in the during LacZ in these and was strongly in the of the and also in other and tissues heart is with of the proximal Nkx2-5 enhancer, enhancer in the heart C.M. R.D. Vincent E.B. Yutzey K.E. Dev. Biol. PubMed Scopus Google Scholar). determine the enhancer was for early Nkx2-5 the LacZ expression of the in which a containing the sites was in the of the Nkx2-5 upstream 10-kb the upstream 10-kb region LacZ expression during early (8Reecy J.M. Li X. Yamada M. DeMayo F.J. Newman C.S. Harvey R.P. Schwartz R.J. Development. 1999; 126: 839-849Crossref PubMed Google Scholar) in observed LacZ in the cardiac in of mouse In of transgenic not LacZ expression a transgenic LacZ expression in the cardiac than that the type in the of other Nkx2-5 enhancers within the 10-kb DNA the composite enhancer revealed a role for early Nkx2-5 expression in the cardiac crescent. of Nkx2-5 by GATA-4 and The Nkx2-5 an for the regulation of and cardiac gene Nkx2-5 is the earliest for the heart and expression the myocardium in the enhancers and repressors have identified in the Nkx2-5 upstream regulatory region orchestrating expression in These the of gene regulation heart of have that BMP signaling the expression of Nkx2-5, it not to We provide in this a of Nkx2-5 activation by This pathway in a with cardiac GATA factors through and it is that other pathways and transcription factors also with these or to the cardiac and the expression of by BMP signaling was in P19 as to other more that be differentiated by of transcription factor Belaguli D. Wu Schwartz R.J. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google P19 are more cardiogenic in 2001; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google with a of BMP signaling and the Smads as as Nkx2-5 and GATA factors K. I. Y. Kudoh S. D. Yazaki Y. Komuro I. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, K. Y. Kudoh S. D. M. K. S. Yazaki Y. Nagai R. Komuro I. J. Biol. 2001; PubMed Scopus Google Scholar). that is vital for activation of Nkx2-5, the of multiple in GATA sites in Nkx2-5 have to be required for expression in the in GATA-4 was a of Nkx2-5 reporter GATA-4 and Smad1/4 were was activation in or Smad4 alone were for in the a BMP through the receptor and between the and Smad proteins be may be BMP signaling and of the Smad in P19 it that the Smad factors to the and and or the the Finally, deletion analysis of the Nkx2-5 region revealed multiple sites for BMP activation and in proximal and distal to the enhancers (7Schwartz R.J. Olson E.N. Development. 1999; 126: 4187-4192Crossref PubMed Google Scholar, C.L. J. Richardson J.A. Olson E.N. Dev. Biol. PubMed Scopus Google Scholar, C.M. R.D. Vincent E.B. Yutzey K.E. Dev. Biol. PubMed Scopus Google Scholar). a region containing GATA and Smad binding sites was able to direct such and as in was We that be expression family signaling or GATA factors the were more the of the enhancer, as in the of this in the of other regulatory for early Nkx2-5 expression in the cardiac crescent. is that the of the GATA sites to the and In a GATA to the one identified to as a or in the R. C. B. K. D. J. 2000; PubMed Scopus Google Scholar). This is with the of multiple GATA sites to be activated in transgenic mice or by GATA-4 in The combinatorial enhancer in or an region (8Reecy J.M. Li X. Yamada M. DeMayo F.J. Newman C.S. Harvey R.P. Schwartz R.J. Development. 1999; 126: 839-849Crossref PubMed Google and it may be that the GATA sites are in the of activated Smads. the sites were in gene expression during early heart that Smad4 in vivo to the Smad/GATA composite enhancer region in differentiated P19 not in P19 cells. with (2Damante G. Fabbro D. Pellizzari L. Civitareale D. Guazzi S. Polycarpou-Schwartz M. Cauci S. Quadrifoglio F. Formisano S. Di Lauro R. Nucleic Acids Res. 1994; 22: 3075-3083Crossref PubMed Scopus (101) Google Scholar, 3Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (268) Google that Smad4 strongly to enhancer not enhancer region the of Smad binding sites in enhancer, suggesting that Smad4 binding to the enhancer of Nkx2-5 is and The that GATA4 to Smad/GATA enhancer the for adjacent GATA and Smad sites their to transcription. GATA and Smad and between and was to be during Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). these factors to an reporter in and the of to a GATA was to be through the of and We in the GATA4 with Smad factors through the second zinc finger and adjacent basic region of and may be for We have between of Smad and GATA factors in GATA-4 was recruited to Smad sites of the reporter and may the binding of the Smad proteins to their in to an activation Smad4 was to the GATA-4/5/6 and the region identified is between these and GATA factors, that the through the highly zinc finger region is a more also that Smad proteins with other zinc finger and J. G. J. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Lin X. R. J. 2000; PubMed Scopus Google Scholar, M. K. K. S. Yazaki Y. K. 1998; PubMed Scopus Google and GATA proteins are to with other factors through their zinc We the between GATA and Smad factors as a which to of the GATA family members and to or Smads. could for GATA-4 in Smad The of the between the may a of signaling and tissue-specific factors involved in developmental processes. it is that other family members through the combinatorial enhancer in Nkx2-5, or and have in of mesoderm Dev. Biol. 1998; PubMed Scopus Google Scholar, Development. 1997; PubMed Google Scholar). Smad factors with other proteins containing zinc We that the GATA homologue in with and to of the BMP pathway in a as of GATA-4/5/6 in a role of BMP signaling in the of GATA factors in and heart J.L. Dev. 2001; PubMed Scopus Google Scholar). We have of GATA transcription factors in BMP signaling through with with direct Nkx2-5, suggesting a vital role in mesoderm and specification of cardiac progenitors. We Belaguli for and and to for the reporter