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.
Gupta
M
M
Heart Institute for Children and Department of Physiology and Biophysics, University of Illinois, Chicago 60612, USA. mgupta@surgery.bsd.uchicago.edu
Kogut
P
P
Davis
F J
FJ
Belaguli
N S
NS
Schwartz
R J
RJ
Gupta
M P
MP
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003101:Collodion / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000378:metabolism; D008957:Models, Genetic; D009206:Myocardium / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D026362:Serum Response Factor; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors; D003101:Collodion; D004247:DNA; D005982:Glutathione Transferase