Physical interaction between the MADS box of serum response factor and the TEA/ATTS DNA-binding domain of transcription enhancer factor-1

Serum response factor is a MADS box transcription factor that binds to consensus sequences CC(A/T)(6)GG found in the promoter region of several serum-inducible and muscle-specific genes. In skeletal myocytes serum response factor (SRF) has been shown to heterodimerize with the myogenic basic helix-loop-helix family of factors, related to MyoD, for control of muscle gene regulation. Here we report that SRF binds to another myogenic factor, TEF-1, that has been implicated in the regulation of a variety of cardiac muscle genes. By using different biochemical assays such as affinity precipitation of protein, GST-pulldown assay, and coimmunoprecipitation of proteins, we show that SRF binds to TEF-1 both in in vitro and in vivo assay conditions. A strong interaction of SRF with TEF-1 was seen even when one protein was denatured and immobilized on nitrocellulose membrane, indicating a direct and stable interaction between SRF and TEF-1, which occurs without a cofactor. This interaction is mediated through the C-terminal subdomain of MADS box of SRF encompassing amino acids 204-244 and the putative 2nd and 3rd alpha -helix/beta -sheet configuration of the TEA/ATTS DNA-binding domain of TEF-1, In the transient transfection assay, a positive cooperative effect of SRF and TEF-1 was observed when DNA-binding sites for both factors, serum response element and M-CAT respectively, were intact; mutation of either site abolished their synergistic effect. Similarly, an SRF mutant, SRFpm-1, defective in DNA binding failed to collaborate with TEF-1 for gene regulation, indicating that the synergistic trans-activation function of SRF and TEF-1 occurs via their binding to cognate DNA-binding sites. Our results demonstrate a novel association between SRF and TEF-1 for cardiac muscle gene regulation and disclose a general mechanism by which these two super families of factors are likely to control diversified biological functions.