We hypothesize that the conformational change induced by acetylation may affect the ability of MyoD to interact with protein partners. The objective of this study is the isolation and characterization of proteins preferentially interacting with MyoD in its acetylated versus non-acetylated forms. Since MyoD hyperacetylation accompanies muscle differentiation and temporally coincides with its transcriptional activation, it is likely that proteins that preferentially associate with hyperacetylated MyoD will positively regulate myogenesis. We are also interested in isolating proteins that interact with hypoacetylated (inactive) MyoD to understand the repressive mechanisms that silence MyoD in undifferentiated muscle cells. To isolate proteins interacting with MyoD we have developed a bicistronic retroviral system to deliver MyoD to cells growing both in suspension and on plastic supports. We have also generated cells expressing a version of MyoD that cannot be acetylated and are interested in determining and compare the pattern of the MyoD-associated factors (MAFs) isolated using both MyoD """"""""wild-type"""""""" and non acetylatable MyoD.
The specific aims of this study are:1. To isolate MAFs and perform protein purification by affinity chromatography using FLAG-immobilized agarose beads. 2. To characterize the isolated protein complexes by SDS-PAGE followed by mass spectrometry of the individual isolated bands.3. To compare the pattern of MAFs derived from cells expressing """"""""wild-type"""""""" MyoD versus non-acetylatable MyoD.In the past year, we have constructed and characterized bicistronic retroviral vectors expressing either MyoD""""""""wild-type"""""""" or non acetylatable MyoD and have generated three cells lines (HeLa, NIH 3T3 and C2C12) transduced with the above retroviruses. Furthermore, we have initiated a pilot purification of the MAFs.
