Current models of gene regulation suggest that interphase chromosomes are divided into large, functionally distinct chromatin domains, facilitating independent regulation of neighboring genes. The structural and molecular mechanisms establishing these domains remain poorly understood. Only indirect structural assays have been available to analyze the structure of these domains, with a key limitation being the technical difficulty associated with directly visualizing domain large-scale chromatin folding. Our long term objectives are to dissect the large-scale chromatin folding of specific gene loci, to analyze the cis and trans determinants of these folding motifs, and to determine the functional significance of this level of chromatin organization with regard to transcriptional regulation. ? ? The specific aims for this project period will be to address the following questions:1. What changes in large-scale chromatin structure accompany transcriptional activation/repression?2. Does large-scale chromatin condensation constrain transcriptional activation?3. By what mechanisms do transcriptional activators modify large-scale chromatin structure?4. What cis factors control large-scale chromatin structure? Using novel visualization methods, in vivo dynamics of specific gene and chromosome loci will be observed directly during transcriptional activation or silencing. A combination of light and electron microscopy will visualize changes in large-scale chromatin organization and nuclear positioning of these chromosome regions during transcriptional activation or repression and monitor the sequential recruitment of various transcription cofactors to these sites. Several approaches will be used to begin mechanisticdissection of the underlying biochemical mechanisms underlying observed reorganization of large-scale chromatin structure, and the functional importance of large-scale chromatin organization with respect to potential for transcriptional activation will be assessed. Insight from these studies should be of help in guiding the design of future constructs and artificial chromosomes used in gene therapy.
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