The cardiac determination factor, Nkx2-5, is activated by mutual cofactors GATA-4 and Smad1/4 via a novel upstream enhancer.
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)-beta 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.
Brown
Carl O
CO
Department of Molecular and Cellular Biology, The Center for Cardiovascular Development, Baylor College of Medicine, Houston, Texas 77030, USA.
Chi
Xuan
X
Garcia-Gras
Eduardo
E
Shirai
Manabu
M
Feng
Xin-Hua
XH
Schwartz
Robert J
RJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D050991:GATA4 Transcription Factor; D017353:Gene Deletion; D017930:Genes, Reporter; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D051785:Smad Proteins; D051898:Smad1 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers
D019485:Bone Morphogenetic Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D051785:Smad Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases; D005982:Glutathione Transferase