MyoD belongs to a family of transcription factors - the myogenic bHLH- which is required for the formation of skeletal muscle in the animal. The transcriptional activity of MyoD is regulated at the DNA-binding level, by post-translational modifications, and by interaction with several regulatory proteins. Among the latter, acetyltransferases and deacetylases promote and inhibit the activity of MyoD, respectively, through acetylation/deacetylation of histones and of MyoD itself. Recently, we have shown that acetylation of MyoD not only increases its affinity for DNA but also induces a conformational change in its structure. We speculate that such conformational change may affect the ability of MyoD to interact with protein partners. To isolate proteins interacting with MyoD we have developed a bicistronic retroviral system to deliver MyoD to cells growing is suspension. We have generated cells stably expressing a double-tagged recombinant MyoD (FLAG and HA sequence tags). Large quantities of cells were grown and protein purification performed by affinity chromatography using FLAG-immobilized agarose beads. The MyoD-associated factors (MAFs) were resolved on SDS-PAGE and the corresponding polypeptides were identified by mass spectrometry.We are currently testing the functional relevance of some of the MAFs. These experiments should help elucidating the molecules and mechanisms employed to achieve cell-type specific gene expression in skeletal muscle.
