Comprehensive Analysis of Novel E2F regulated Genes

Massachusetts General Hospital, Boston MA

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Abstract

E2F is a key downstream target of the retinoblastoma protein. It has a dual role as an oncogene and a tumor suppressor, and can promote DNA replication and apoptosis. The regulation of E2F activity has been studied in great detail. However, knowledge about the mechanisms behind its diverse biological activities is limited, but suggests that E2F may also regulate the expression of genes other then those involved in cell cycle control. To test this hypothesis and to gain insight into the mechanism of E2F function, I will perform a comprehensive analysis of genes regulated by these transcription factors in Drosophila using several approaches. Differential gene expression in e2f mutants will be analyzed using cDNA microarrays. The promoters of genes identified by this method will be examined for the presence of E2F binding sites and analyzed for in vivo binding by E2F and RB proteins using a chromatin immunoprecipitation assay. In addition, novel E2F binding sites will be identified using two methods. Cloning and analysis of co- immunoprecipitated DNA will follow chromatin immunoprecipitation with anti-E2F antibodies. A genetic selection for E2F binding sites using the yeast one-hybrid system will also be performed.