Experimental verification of Myocyte Enchancer Factor 2 (MEF2) and Serum Response Factor (SRF) interaction predicted by computational methods

In this work, a bioinformatic approach for identification of co-occuring transcription factor binding sites is presented. Computational analysis combined with an enhanced method which uses optimized matrix thresholds revealed co-occurrence of binding sites between several transcription factors and Myocyte Enhancer Factor 2 (MEF2). A predicted interaction between MEF2A and Serum Response Factor (SRF) was chosen for further analysis. The muscle creatine kinase (MCK) enhancer contains the Serum Response Element (SRE) and two putative MEF2 binding sites. Co-expression of MEF2A and SRF proteins showed that the MCK enhancer is synergistically activated by this combination of transcription factors. Analysis of the c-jun and c-fos promoters as well as a GAL4- based one hybrid reporter gene assay indicated a requirement of both binding sites for synergistic transcriptional activation. Mutation analysis indicated that "loss of binding" mutations of the MEF2 or SRE potently eradicated the synergistic effect observed on the MCK enhancer. Co-immunoprecipitation experiments revealed a physical interaction between SRF and MEF2 proteins. These results validate the in silico approach of predicting transcription factor interactions and suggest this approach could be a versatile method for prediction of transcription factor interactions.