Transcription elongation by RNA polymerase II in vertebrates and Drosophila is controlled by the actions of positive and negative elongation factors. This application investigates the function of three negative regulators: NELF, Pcf11, and chromatin structure. NELF was recently shown to block Pol II elongation in the promoter proximal region of the hsp70 gene in Drosophila prior to heat shock induction. Pcfl 1 was recently discovered to dismantle paused elongation complexes by a mechanism that involves the CTD of Pol II. Pcf11 could be the factor that limits Pol II processivity in cells. Chromatin structure has recently been implicated in stabilizing promoter proximal pausing on the hsp70 gene in Drosophila. The goal of this proposal is to understand the roles of negative elongation factors in controlling gene expression. The project has three specific aims: 1) Determine the functions of NELF in vivo. 2) Determine the role of Pcf11 in transcription elongation. 3) Investigate the role of chromatin modulators in controlling elongation in cells. The work uses mainly Drosophila as a model system and integrates in vivo and in vitro techniques. RNA interference and available mutations will be used to perturb the activity of proteins in vivo. Genomic footprinting with permanganate will be used view of the behavior of Pol II. Chromatin immunoprecipitation and immunofluorescence staining of polytene chromosomes will be used to monitor interactions of proteins with genes in cells. Finally, elongation complexes formed from purified Pol II will provide a biochemical basis for analyzing the actions of elongation factors. Blocks to transcription elongation have been implicated in regulating transcription of several transcription units of significant importance to diseases. These include transcription of the HIV provirus which impacts on viral replication and latency, transcription of estrogen responsive genes which could impact on breast cancer, and transcription of the proto-oncogenes, c-fos and c-myc which could impact of cell proliferation and cancer.
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