VIMENTINS REGULATORY FACTORS INVOLVED IN DEVELOPMENT

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Myogenesis requires the coordinate expression of genes unique to muscle and the inactivation of others which are not required for the muscle phenotype. Although considerable information exists about what controls skeletal gene expression, less is known about what governs cardiac expression, and virtually nothing about how non-muscle-specific genes are "turned-off" during myogenesis, despite the fact that gene inactivations equally important to maintaining the muscle phenotype. The cytoskeletal proteins, vimentin and desmin, provide an excellent model system for determining how genes are differentially expressed during development. Vimentin synthesis is first detected at the delineation of the mesoderm. A number of cell types differentiate from this lineage and continue to synthesize vimentin. Others, like muscle, inactivate the vimentin gene and "turn-on" desmin. The goal of this proposal is to delineate how the vimentin gene is specifically inactivated during myogenesis and how aberrant expression could affect the developmental program. Obviously, any deviation in this decision could result in compromised development. In analyzing vimentin gene expression, we have found unique positive and negative regulatory factors which control the downregulation of the vimentin gene. In chick skeletal myoblasts, the DNA binding activity of the negative (silencer) factor is low, the positive (antisilencer) protein is high, and vimentin mRNA is abundant. As myogenesis proceeds the activity of the silencer factor increases dramatically, whereas antisilencer activity virtually disappears and the level of vimentin mRNA decreases dramatically. Therefore, the interplay of these two factors is crucial for determining the physiologically correct expression of the vimentin gene. In heart (day 14) we find a 3-fold higher level of the silencer factor than in skeletal (breast) muscle. Therefore, we conclude that a similar program must be occurring during cardiac myogenesis. In this proposal we aim to further define the mechanism. We will continue with the characterization and cloning of both the unique silencer and antisilencer factor. We refer to this protein as an antisilencer, because it only functions in concert with the silencer element and does not contribute to gene expression on its own unlike the typical enhancer protein. With the acquisition of nucleotide and protein sequence information, we plan to alter the cellular content of these factors in both normal and myogenic cells. We will determine the effect of these genetic alterations on vimentin gene expression and more importantly the myogenic program in both skeletal and cardiac cells. A screen of the genomic data base reveals several genes (from chick to man) with homologous silencer and antisilencer elements. As antibodies or cRNA probes become available, we will examine the expression of these key regulatory factors during embryogenesis. Finally, we will determine the mechanism by which these regulatory factors control expression of vimentin as well as other genes. It is hoped that by understanding how these genetic controls contribute to development, we can determine how abnormalities may develop in the heart.