To achieve the larger goal of understanding the regulatory network that controls myogenesis, it is important to understand the mechanisms governing DNA binding by transcription factor MyoD, which plays a defining role in muscle development. MyoD binding at numerous muscle-specific genes is facilitated by phosphorylation of the histone acetyl transferase p300 by the protein kinases AKTs 1 and 2, the consequent association of p300 with MyoD, and the removal of HDAC1 from MyoD by Rb1. Our recent determination of precise locations of in vivo MyoD genome-wide binding in differentiated muscle cells in culture has identified binding sites in apparent association with genes that are not expressed or that are down-regulated. These results raise the possibility that histone acetylation, commonly associated with active genes, is not a contributor to MyoD binding at these sites. Thus, rather than being dependent upon Rb1 and/or AKT 1 and 2, MyoD binding at these sites may be facilitated by different coregulatory mechanisms. We propose to begin a search for alternate binding mechanisms using a combination of molecular biological and bioinformatic approaches. This proposal has 3 specific aims. 1) To informatically analyze our current genome-wide MyoD binding data to identify candidate sites for those at which binding of MyoD may be independent of Rb1 and/or AKT 1 and 2. 2) Using leads from the literature, from our preliminary results, and the proposed informatic analysis, to a) identify a small set of sites for which MyoD binding is differentially affected in vivo Rb1 at only some of the sites, and b) identify a small set of sites for which MyoD binding is differentially affected in vivo AKT1/2 at only some of the sites. MyoD binding will be assayed by ChIP followed by quantitative PCR and gene expression will be assayed by quantitative reverse transcriptase PCR. Once a set of binding sites has been identified, they will be used to vet preparations of ChIP fragments for genome-wide ChIP-Seq. 3) To use ChIP-Seq to determine on a genome- wide scale the sites at which MyoD is bound a) Rb1 and b) AKT1 and 2 activities. The results will be examined informatically for selective enrichment of one or more sequence motifs in those regions containing sites that remain bound in the absence of AKT 1and 2 activity or of Rb1. Such enrichment would suggest differential use of coregulators to facilitate DNA binding. The proposed work incorporates tests for 3 hypotheses. 1) Mechanisms of MyoD binding at sites throughout the genome differ with respect to their dependence upon the presence of Rb1 or AKT 1 and 2 activities;the difference will be apparent in sequence features near MyoD binding sites. 2) At genes for which expression has been shown to be reduced in the absence of Rb1, MyoD binding will also be reduced. If true, binding will be restored in the presence of histone deacetylase 1 inhibitors. 3) Given their mutual influence on chromatin and MyoD acetylation, the increased expression of the Rb1 and Akt2 genes during myogenesis may be regulated by crosstalk. The results of our work can be expected to generate testable hypotheses regarding alternate mechanisms of MyoD binding.
The proposed work will contribute to our understanding of muscle development at the molecular level. What we learn will also provide insights into the nature of molecular mechanisms that operate in other process of tissue formation, including muscle regeneration. The more we understand about normal processes, the more quickly we will be able to understand and address the molecular failures that occur in abnormal development and in cancers, which frequently exhibit loss of the developed nature of the tissues in which they arise.
