Combinatorial expression of GATA4, Nkx2-5, and serum response factor directs early cardiac gene activity

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 (alphaCA) 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 alphaCA promoter activity. These three factors induced endogenous alphaCA mRNA over a 100-fold in murine embryonic stem cells. In addition, the DNA-binding defective mutant N-kx2-5pm efficiently coactivated the alphaCA 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 alphaCA promoter constructions. These assays indicated a dependence upon cooperative SRF binding for facilitating the recruitment of Nkx2-5 and GATA4 to the alphaCA 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 alphaCA promoter DNA binding complexes, led to a model of SRF physical association with Nkx2-5 and GATA4.