REGULATION OF MUSCLE GENE EXPRESSION

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During myogenesis there is a coordinate increase in the synthesis of muscle-specific proteins essential for sarcomeric development. At least in part this increase reflects transcriptional activation of the genes coding for these proteins. A central question in biology is how cells transcribe only a certain part of their genome thereby achieving a differentiated state. The long-term objective of this proposed research is to determine how regulatory singals govern muscle gene expression during myoblast differentiation. A prerequisite for these studies is to isolate related genes which are differentially expressed during myogenesis. We shall focus on a family of genes which code for the intermediate filament proteins, desmin and vimentin. Desmin is a muscle-specific protein which is synthesized concommitant with muscle cell differentiation. However, vimentin synthesis is not restricted to muscle and its expression does not reflect muscle-specific regulation. Clones of these two genes provide at least two approaches for determining fundamental control mechanisms governing gene expression in muscle. DNA sequencing allows one to search for homologous sequence elements within the genes and differences within the surrounding regulatory regions. In addition, the sequence of the desmin gene (5 feet end) will be compared to other genes exhibiting muscle-specific regulation which may reveal common structural elements required for regulated expression. The functionality of these presumptive regulatory regions will be tested by the introduction of both normal and modified genes via eukaryotic viral vectors into precursor cells (myoblasts). Myoblasts will be stimulated to differentiate and the expression of the introduced genes and host genes will be quantitated and comapred by S1 mapping experiments. Muscle-specific genes (desmin) should be expressed only in differentiated cells. Modification of the desmin gene in putative regulatory regions by deletion mapping or site-specific mutagenesis will be employed to identify sequences required for correct regulation. The long range goal of this proposed research is to achieve an understanding of the molecular mechanisms affecting transcription of a muscle-specific gene. This may involve the identification, and isolation of regulatory factors which interact with chromatin and control gene expression.