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Originally published In Press as doi:10.1074/jbc.M203122200 on April 30, 2002

J. Biol. Chem., Vol. 277, Issue 28, 25775-25782, July 12, 2002
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Combinatorial Expression of GATA4, Nkx2-5, and Serum Response Factor Directs Early Cardiac Gene Activity*

Jorge L. SepulvedaDagger , Spiros Vlahopoulos§, Dinakar Iyer§, Narasimhaswamy Belaguli, and Robert J. Schwartz§||**

From the Dagger  Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213 and the Departments of § Molecular and Cellular Biology, || Medicine, and  Surgery, the Center for Cardiovascular Development, Baylor College of Medicine, Houston, Texas 77030

Herein, the restricted expression of serum response factors (SRF) closely overlapped with Nkx2-5 and GATA4 transcripts in early chick embryos coinciding with the earliest appearance of cardiac alpha -actin (alpha CA) transcripts and nascent myocardial cells. The combinatorial expression of SRF, a MADS box factor Nkx2-5 (a NK4 homeodomain), and/or GATA4, a dual C4 zinc finger protein, in heterologous CV1 fibroblasts and Schneider 2 insect cells demonstrated synergistic induction of alpha CA promoter activity. These three factors induced endogenous alpha CA mRNA over a 100-fold in murine embryonic stem cells. In addition, the DNA-binding defective mutant Nkx2-5pm efficiently coactivated the alpha CA promoter in the presence of SRF and GATA4 in the presence of all four SREs and was substantially weakened when individual SREs were mutated and or serially deleted. In contrast, the introduction of SRFpm, a SRF DNA-binding mutant, blocked the activation with all of the alpha CA promoter constructions. These assays indicated a dependence upon cooperative SRF binding for facilitating the recruitment of Nkx2-5 and GATA4 to the alpha CA promoter. Furthermore, the recruitment of Nkx2-5 and GATA4 by SRF was observed to strongly enhance SRF DNA binding affinity. This mechanism allowed for the formation of higher ordered alpha CA promoter DNA binding complexes, led to a model of SRF physical association with Nkx2-5 and GATA4.

* This study was supported by National Institutes of Health Grant PO1 HL-49953 (to R. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Depts. of Molecular and Cellular Biology, Division of Cardiovascular Sciences, the Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Rm. 145E, Houston, TX 77030. Tel.: 713-798-6649; Fax: 713-798-7799; E-mail: schwartz@bcm.tmc.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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