The 26S proteasome consists of 20S proteolytic core particle (CP) and a 19S regulatory particle (RP). It is engaged in degradation of a variety of proteins, and thus, regulates many important cellular processes. Importantly, our results reveal that the 19S RP is recruited to the upstream activating sequence to facilitate transcription complex assembly at the core promoter to stimulate transcription initiation by enhancing the targeting of co-activators, SAGA (Spt-Ada-Gcn5-acetyltransferase) or TFIID (Transcription factor IID) to the activator at the SAGA or TFIID-regulated genes, respectively, in a proteolysis-independent manner. Intriguingly, transcription initiation has also been shown to be promoted by the proteolytic activity of the proteasome. However, it is not clearly understood how the 26S proteasome promotes transcription initiation. We hypothesize that proteasome controls transcription initiation by regulating co-activator via ubiquitylation and proteasomal degradation. Indeed, our preliminary results revealed that the Sgf73 component of the co- activator, SAGA, undergoes ubiquitylation and proteasomal degradation, thus supporting our hypothesis. However, the E3 ubiquitin ligase involved in such regulation of Sgf73 is yet unknown. Further, how this ligase interacts with and is targeted to Sgf73 for ubiquitylation and proteasomal degradation, and the physiological relevance of such regulation of Sgf73 on SAGA?s integrity (and hence its functions in regulation of chromatin modification and transcription initiation) remain largely elusive. Moreover, other factors such as ubiquitin protease and conjugase among others involved in Sgf73 ubiquitylation and proteasomal degradation are not known. Answer to these important questions would fundamentally develop novel ubiquitin-proteasome system regulation of SAGA in orchestrating chromatin modification and transcription, thus greatly advancing the field of gene regulation. In addition, these results would have significant impact on disease pathogenesis and future therapeutic development, since SAGA as well as Sgf73 are evolutionarily conserved from yeast to humans, and associated with various diseases. Therefore, we propose to address above questions in this application. Specifically, we will (i) identify E3 ubiquitin ligase involved in Sgf73 ubiquitylation and proteasomal degradation, (ii) determine how Sgf73 recognizes E3 ligase, (iii) determine ubiquitylation site(s) on Sgf73, (iv) determine the physiological relevance/role of Sgf73 ubiquitylation and proteasomal degradation in regulation of SAGA?s integrity, chromatin modification and transcription, and (v) identify and characterize ubiquitin conjugase and ubiquitin protease involved in regulation of Sgf73 ubiquitylation and proteasomal degradation, and hence SAGA and its functions. Collective results would identify ubiquitin ligase, conjugase and protease in regulation of ubiquitylation and proteasomal degradation of SAGA component, Sgf73, with roles in SAGA?s integrity and functions in gene expression, thus advancing our understanding of gene regulation by ubiquitin-proteasome system with implications in human health.
Proposal narrative: The 26S proteasome complex is essential for targeted protein degradation via ubiquitylation, and promotes transcription initiation. This proposal aims to delineate proteasomal regulation of transcription initiation, which is not clearly understood. The results will significantly advance our understanding of the complex regulation of transcription initiation by the ubiquitin-proteasome system, and such knowledge will provide important information for disease pathogenesis and future therapeutic development to maintain normal cellular functions, since a growing number of diseases including cancers are linked to aberrant transcription initiation as well as ubiquitin-proteasome system.
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