Myogenesis requires the coordinate expression of genes unique to muscle and the inactivation of others, which are not require for the muscle phenotype. Although considerable information exists about what controls skeletal and now cardiac gene expression, virtually nothing is known about how non-muscle-specific genes are """"""""turned-off' during myogenesis, despite the fact that gene inactivation is 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 of these controlling regulatory factor could affect the developmental program. Obviously, any deviation in this decision could compromise development. In analyzing vimentin gene expression, the investigators have found a unique combination of positive and negative regulatory factors, which control the down-regulation of the vimentin gene. First, both basal and regulated gene expression is absolutely dependent on a GC box, which binds the regulatory factor, Sp2. They know of no other gene which uses Sp2 instead of Spl/Sp3 proteins. Upstream the investigators have located a silencer element, which binds the zinc finger protein, ZBP-89. Further upstrearn is a positive-acting element, which the investigators call an anti-silencer, because it only functions in concert with the silencer and does not contribute to gene expression on its own unlike the typical enhancer. Previously, the investigators have shown in chick skeletal myoblasts, that the binding activity of the silencer factor is low, the antisilencer is high, and vimentin mRNA is abundant. As myogenesis proceeds the activity of the silencer factor increases dramatically, whereas antisilencer activity disappears and the level of vimentin mRNA decreases dramatically. Therefore, the interplay of these two factors and Sp2 is crucial for determining the physiologically correct expression of the vimentin gene. Because silencer factor activity is more predominant in heart, the investigators conclude a similar program must be occurring during cardiac myogenesis. In this proposal the investigators aim to determine the mechanism by which these regulatory factors interact and control transcription. With the acquisition of cDNAs, the investigators plan to alter the cellular content of these factors in both normal and muscle cells and determine the resulting effect on myogenesis. They suspect that ZBP-89 is a tumor suppressor. Therefore, an alteration in its cellular content could affect cell cycle and terminal differentiation. They will examine the expression of these key regulatory factors during embryogenesis. Finally, the investigators will continue our investigation of post-transcriptional controls of vimentin mRNA localization, stability, and translation. It is hoped that by a thorough understanding of how these genetic controls contribute to development, the investigators can ultimately determine how abnormalities may develop in the heart.
Showing the most recent 10 out of 22 publications
