Regulation of transcriptional initiation by the 19S proteasome subcomplex.

19S 蛋白酶体亚复合体对转录起始的调节。

• 批准号: 8497039
• 负责人: Sukesh R. Bhaumik
• 金额: $ 33.63万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2017-09-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8497039
• 关键词: 26S proteasome; ATP phosphohydrolase; Acetyltransferase; Address; Applications Grants; Biochemical; Cell Cycle; Cell physiology; Cells; Complex; Cyclin-Dependent Kinase Inhibitor; Cyclins; DNA Microarray Chip; Development; Disease; Eukaryota; Eukaryotic Cell; Funding; Galactose; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Histone H4; Human; Investigation; Life; Link; Malignant Neoplasms; Mediator of activation protein; Methodology; Modeling; Nucleosome Core Particle; Nucleosomes; Oncogene Proteins; Ornithine Decarboxylase; Outcomes Research; Play; Proteins; RNA Polymerase II; Recruitment Activity; Regulation; Research; Research Proposals; Resources; Role; Series; Structure; Study Section; Therapeutic Agents; Transcription Initiation; Transcriptional Regulation; United States National Institutes of Health; Yeasts; base; chromatin immunoprecipitation; genome-wide; in vivo; multicatalytic endopeptidase complex; particle; promoter; protein degradation; public health relevance; research study; transcription factor

DESCRIPTION (provided by applicant): The 26S proteasome complex is essential for targeted protein degradation. It is composed of a 20S core particle (CP) and a 19S regulatory particle (RP). The 20S CP confers the proteolytic activity to the proteasome. The 19S RP has six different ATPase components, and regulates the function of the 20S CP in an ATP-dependent manner. Intriguingly, several studies have implicated the non-proteolytic role of the 19S RP in transcriptional initiation or activation independently of the 20S CP. However, the detailed regulatory mechanisms of transcriptional initiation by the 19S RP remained largely unknown in living cells. Based on our current NIH R15 funding, we have demonstrated in yeast that the 19S RP, but not 20S CP, is recruited to the GAL1 UAS (upstream activating sequence), and establishes a specific interaction network of Gal4-SAGA- Mediator (SAGA, Spt-Ada-Gcn5-acetyltransferase) at the UAS by enhancing the interaction of activator Gal4 with coactivator SAGA, which promotes the formation of preinitiation complex (PIC) at the core promoter to initiate transcription. We subsequently found similar results at several other genes. Overall, we find that 19S RP promotes the interaction of activator with coactivator for facilitating the formation of PIC, and hence transcriptional initiation. However, it is not clearly understood how the 19S RP promotes the interaction of activator with coactivator. Further, the roles of different ATPases of the 19S RP in such regulation are yet unknown. Moreover, it is not known whether the 19S RP functions as a global regulator of transcriptional initiation. Answers to these important questions will significantly contribute to the regulatory roles and mechanisms of the 19S RP in transcriptional initiation. Therefore, in this NIH R15 renewal application, we propose to address these questions. We will specifically determine (i) the role of the 19S RP in global transcriptional initiation, and (ii) how the 19S RP or its ATPase activity promotes the activator-coativator interaction, and hence transcriptional initiation. We will address these specific research aims primarily using the ChIP (chromatin immunoprecipitation), mutational, transcriptional, biochemical, biophysical, and ChIP- seq methodologies. The outcomes of this research proposal will provide important information on the 19S RP regulation of eukaryotic transcriptional initiation, and hence will significantly contribute to our current understanding of eukaryotic gene regulation. Since the transcription machinery as well as the 19S RP is conserved throughout eukaryotes from yeast to humans, these results in yeast will provide important resources for research in humans. Thus, the results will assist in the development of transcription-based therapeutic agents, as a growing number of diseases including cancer are linked to aberrant transcriptional initiation and/or are characterized by altered patterns of gene expression.

描述（由申请人提供）：26S 蛋白酶体复合物对于靶向蛋白质降解至关重要。它由20S核心颗粒（CP）和19S调节颗粒（RP）组成。 20S CP 赋予蛋白酶体蛋白水解活性。 19S RP 具有六种不同的 ATP 酶成分，并以 ATP 依赖性方式调节 20S CP 的功能。有趣的是，一些研究表明 19S RP 在转录起始或激活中的非蛋白水解作用独立于 20S CP。然而，活细胞中 19S RP 转录起始的详细调控机制仍然很大程度上未知。基于我们目前的 NIH R15 资助，我们在酵母中证明了 19S RP（而不是 20S CP）被招募到 GAL1 UAS（上游激活序列），并建立了 Gal4-SAGA-Mediator（SAGA， Spt-Ada-Gcn5-乙酰转移酶）通过增强激活剂 Gal4 与共激活剂 SAGA 的相互作用，促进预起始的形成复合物（PIC）位于核心启动子处以启动转录。我们随后在其他几个基因中发现了类似的结果。总的来说，我们发现 19S RP 促进激活子与共激活子的相互作用，从而促进 PIC 的形成，从而促进转录起始。然而，目前尚不清楚19S RP如何促进激活剂与共激活剂的相互作用。此外，19S RP 的不同 ATP 酶在这种调节中的作用尚不清楚。此外，尚不清楚 19S RP 是否作为转录起始的全局调节因子发挥作用。这些重要问题的答案将对 19S RP 在转录起始中的调控作用和机制做出重大贡献。因此，在这份 NIH R15 续展申请中，我们建议解决这些问题。我们将具体确定 (i) 19S RP 在全局转录起始中的作用，以及 (ii) 19S RP 或其 ATP 酶活性如何促进激活剂-辅激活剂相互作用，从而促进转录起始。我们将主要使用 ChIP（染色质免疫沉淀）、突变、转录、生化、生物物理和 ChIP-seq 方法来解决这些具体研究目标。这项研究计划的结果将提供关于真核转录起始的 19S RP 调控的重要信息，因此将极大地有助于我们目前对 真核基因调控。由于转录机制以及 19S RP 在从酵母到人类的真核生物中都是保守的，因此酵母中的这些结果将为人类研究提供重要资源。因此，这些结果将有助于开发基于转录的治疗剂，因为包括癌症在内的越来越多的疾病与异常转录起始有关和/或以基因表达模式改变为特征。