The goal of the proposed research is to understand how cells use sumoylation to regulate transcription by RNA polymerase II (RNAP II). Analyses will be performed in both yeast and mammalian systems, and the following Specific Aims are proposed. I. Determine the role of sumoylation in gene deactivation in yeast. We recently found that sumoylation of promoter-bound factors takes place during gene activation, but paradoxically has a negative effect on transcription. At the induced ARG1 gene, sumoylation facilitates clearance of promoter-bound factors, enabling it to be shut off. This is due at least in part to sumoylation of Gcn4, the activator that binds ARG1. This modification and its effects will be characterized in detail. This analysis will be extended to other genes to establish whether sumoylation plays a general role in deactivation of induced genes. II. Determine the effect of sumoylation of yeast RNAP II. We recently determined that the largest subunits of yeast RNAP II, Rpb1 and Rpb2, are sumoylated. Rpb1 is sumoylated at Lys 1487, which is proximal to the Rpb4 subunit. Whether sumoylation at Rpb1 K1487 functions to enhance the interaction of core RNAP II with the Rpb4/7 heterodimer, and/or in some other way, will be investigated. The unusual requirement of yeast RNAP II sumoylation for Ulp2, a SUMO protease, will be examined. The function of Rpb2 sumoylation will be explored. III. Determine how transcription is regulated by sumoylation in mammalian systems. ChIP assays and siRNA knockdown experiments will be performed to determine whether sumoylation affects transcription in human cells. Effects of sumoylation on transcription in HeLa nuclear extracts will also be determined. Experiments will initially employ naked DNA templates, but will subsequently be extended to chromatin templates. IV. Determine the effect of sumoylation of human RNAP II and general transcription factors. Human RNAP II, as well as GTFs from both yeast and human cells, will be purified to identify components regulated by sumoylation. Non-sumoylatable mutants will be generated to determine whether blocking sumoylation affects activity or recruitment to active genes. Direct sumoylation of purified RNAP II will be performed to determine its effect on activity.
The experiments described in this proposal are designed to increase our understanding of the mechanism and regulation of gene transcription by protein sumoylation. Numerous studies have revealed that changes in both transcription and sumoylation occur during diseases, especially cancer. The proposed experiments will provide a mechanistic understanding of how sumoylation impacts transcription, which in turn will provide insight into the role of these processes in disease.
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