Models of gene regulation suggest large-scale chromatin organization regulates transcription by restricting accessibility of large protein complexes to target sequences, controlling interactions between distant regulatory sequences, and/or modulating intranuclear gene positioning. However, the actual large-scale chromatin organization of transcriptionally active gene loci is unknown, with textbook models based largely on indirect molecular assays. Our long-term objectives are to determine the large-scale chromatin folding and intranuclear positioning of specific gene loci, to identify their cis and trans determinants, and to understand the functional significance of this level of chromatin organization with regard to transcriptional regulation. Several recent experimental developments indicate a high probability for productive investigations. We have developed methods allowing direct visualization of large-scale chromatin decondensation and intranuclear movements accompanying gene activation of BAG transgenes- first in live cells, and then at the ultrastructural level using a novel immunogold labeling procedure. These methods should be applicable to endogenous gene loci.
The specific aims for this project period will be to: (1) Directly determine changes in 3-D large-scale chromatin ultrastructure accompanying gene activation;(2) Directly visualize changes in intranuclear positioning of gene loci associated with gene activation / repression and test the dependence of these movements on actin / myosin;(3) Identify the cis and trans determinants of changes in large-scale chromatin structure and intranuclear positioning of gene loci associated with gene activation / repression;(4) Dissect the molecular sequence determinants which determine Drosophila polytene chromosome band and interband organization. Public Health Relevance: Currently a major impediment to development of gene therapy methods is our incomplete understanding of the requirements for ensuring high and sustained levels of expression from transgenes. Insight from our studies should be useful in guiding the design of future gene constructs and artificial chromosomes used in gene therapy.
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