THE CARDIAC TISSUE-RESTRICTED HOMEOBOX PROTEIN CSX NKX2.5 PHYSICALLY ASSOCIATES WITH THE ZINC-FINGER PROTEIN GATA4 AND COOPERATIVELY ACTIVATES ATRIAL-NATRIURETIC-FACTOR GENE-EXPRESSION/

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscl e-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the pre cardiac mesoderm as well as the embryonic and adult heart. Targeted di sruption of Csx causes embryonic lethality due to abnormal heart morph ogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube format ion. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate an d cooperatively activate transcription of a target gene. Coimmunopreci pitation experiments demonstrate that Csx and GATA4 associate intracel lularly. Interestingly, in vitro protein protein interaction studies i ndicate that helix III of the homeodomain of Csx is required to intera ct with GATA4 and that the carboxy-terminal zinc finger of GATA4 is ne cessary to associate with Csx. Both regions are known to directly cont act the cognate DNA sequences. The promoter-enhancer region of the atr ial natriuretic factor (ANF) contains several putative Csx binding sit es and consensus GATA4 binding sites. Transient-transfection assays in dicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpre ssion of Csx and GATA4 synergistically activates ANF reporter gene exp ression. Mutational analyses suggest that this synergy requires both f actors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first exampl e of homeoprotein and zinc finger protein interaction in vertebrates t o cooperatively regulate target gene expression. Such synergistic inte raction among tissue-restricted transcription factors may be an import ant mechanism to reinforce tissue-specific developmental pathways.