Molecular Analysis of Transcriptional Repression

项目描述（由申请人提供）：本项目的长期目标是确定短程转录抑制因子的分子机制。在许多生物过程中，抑制因子在控制基因表达方面起着核心作用。虽然抑制已成为一个深入研究的主题，但我们缺乏对抑制如何在体内发挥作用的机制理解，特别是在后生动物中，其发育程序涉及相当复杂的转录调节。该项目将利用果蝇的独特优势，应用生化和遗传方法来获得对胚胎中短程抑制因子作用的分子理解。

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.