Molecular Analysis of Transcriptional Repression

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DESCRIPTION (provided by applicant): This project's long-term aim is to identify the molecular mechanisms of short-range transcriptional repressors. Repressors play a central role in the control of gene expression in many biological processes. While repression has become a topic of intense investigation, we lack a mechanistic understanding of how repression is utilized in vivo, especially in metazoans, whose developmental programs involve transcriptional regulation of considerable complexity. The project will use the unique advantages of Drosophila to apply biochemical and genetic approaches to gain a molecular understanding of the action of short-range repressors in the embryo. First, we will characterize the CtBP-dependent and -independent repression activities of the Drosophila Knirps factor using transgenic embryo assays to study repression in the physiological context, and extend this work with cell culture assays. Second, we will characterize the repression activity of wild-type CtBP cofactor in embryo and ceil-culture assays to identify residues required for activity, and to determine the effect of promoter context on activity. Third, we will analyze in the embryo transcriptional switches containing well-defined activator and repressor sites to identify the requirements for effective repression by endogenous Knirps and Giant repressors. Fourth, we will identify Knirps- and CtBP-associated factors by purifying these proteins from embryo extracts, and test for the presence of histone deacetylases. Fifth, we will examine the nature of protein complexes at genes in embryos and cell culture before and after repression, using chromatin immunoprecipitation techniques. Transcriptional repressors are involved in host of regulatory processes linked to human diseases such as cancer and developmental abnormalities, therefore knowledge of repressor activity will facilitate design of therapeutic interventions. Bioinformatic interpretation of putative regulatory regions will also benefit from a deeper understanding of factors affecting repressor activity.