UNIVERSITY OF MASSACHUSETTS
To understand normal development and differentiation, it is necessary to determine the mechanisms by which cells initiate new programs of gene expression and promote formation of specific cell lineages. Typically, this involves activation of genes that are transcriptionally silent and that are likely incorporated into repressive chromatin structure. Previously, we have modified molecular methods to allow us to examine changes in chromatin structure as well as the binding of transcription factors, chromatin remodeling enzymes, histone modifying enzymes, and modified histones at endogenous, skeletal muscle specific loci in developing embryonic tissue from stages E10.5 onward. However, formation of the somites, from which skeletal muscle tissue of the trunk and limbs originates, begins after E7.5. At E10.5 the specification the skeletal muscle differentiation program has already begun and myogenic genes that are induced at early times of skeletal muscle differentiation have already been activated. While we previously proposed to examine molecular events associated with the initiation of early gene expression using cell culture models for differentiation, we have been unable to address the initiation of skeletal muscle differentiation directly using embryonic tissue. Here, in collaboration with Dr. Jaime Rivera-P?rez, we propose to utilize molecular methods to examine the regulation of myogenic gene expression and differentiation in vivo from the onset of somite formation (Aim 1). This represents an unprecedented opportunity to understand the molecular mechanisms controlling the regulation of the initiation of the skeletal muscle differentiation pathway in vivo. The function(s) of factors implicated by these studies will be further characterized via manipulation in cell culture models for differentiation (Aim 2). Finally, we have documented that inter-chromosomal interactions occur between myogenic late genes occurs during myogenesis in culture and in vivo at E10.5 and later; we will probe earlier stages of embryogenesis to better characterize the formation of these higher order gene associations (Aim 3).