Factors Involved in Transcription by RNA Polymerase II

描述(由申请人提供)： 摘要RNA聚合酶II(RNAPII)的伸长调控在多细胞生物体的发育和分化过程中起着重要的调控作用。这一过程的特征是负延长因子的默认作用，包括NELF和DSIF，它们阻止启动子附近启动的聚合酶的转录。这些稳定的聚合酶或者通过TTF2或其他终止因子从模板中释放，或者通过正延伸因子P-TEFb的选择性作用而允许进入生产性延伸。P-TEFb的细胞周期蛋白依赖性激酶活性受HEXIM蛋白介导的7SK SnRNP可逆结合的调节。这项提案的主要目标是进一步定义用于控制RNAPII延伸的机制。该方法将在人类和小鼠细胞以及模式生物果蝇中使用生化和分子方法。在第一个目标中，将研究P-TEFb的功能以及提前终止在调控转录中的潜在作用。该计划的一部分包括体外生化方法；然而，计划进行全球分析，其中将使用染色质免疫沉淀和大规模平行测序(CHIP-SEQ)检查所有基因的转录。第二个目标集中在用于控制P-TEFb的细胞机制，以及这些机制如何参与赋予P-TEFb激活的基因的选择性。在这里，将再次进行P-TEFb调节因子的全基因组作图。在最终目标中，将讨论终止因子TTF2的作用。除了它在有丝分裂抑制转录延长中的作用外，研究还将探讨它在转录偶联DNA修复、基因52端的提前终止以及正常32端形成后的终止中的作用。首席研究员是爱荷华大学的一名教授，该项目将在他的实验室和爱荷华大学医学院设施提供的良好环境中进行。公共卫生相关性：项目叙述本申请中的拟议研究将对理解控制转录的基本细胞机制做出重大贡献。了解转录是如何调控的至关重要，因为这一过程中的异常是负责任的发育缺陷、癌症和其他病理。

DESCRIPTION (provided by applicant): ABSTRACT It has become apparent that RNA polymerase II (RNAPII) elongation control plays a major role in regulating transcription throughout development and differentiation of multi-cellular organisms. The process is characterized by the default action of negative elongation factors, including NELF and DSIF that halt transcription of initiated polymerases near promoters. These poised polymerases are either released from the template by TTF2 or other termination factors or are allowed to enter productive elongation through the selective action of the positive elongation factor, P-TEFb. The cyclin dependent kinase activity of P-TEFb is regulated by reversible association with the 7SK snRNP mediated by HEXIM proteins. The main goal of this proposal is to further define the mechanisms utilized to control RNAPII elongation. The approach will employ biochemical and molecular methods in human and mouse cells and in the model organism, Drosophila. In the first aim, the function of P-TEFb and the potential role of premature termination in regulating transcription will be studied. Part of the plan includes in vitro biochemical approaches; however global analyses are planned in which transcription of all genes will be examined using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). The second aim focuses on cellular mechanisms utilized to control P- TEFb and how these may involved in conferring selectivity to the genes activated by P-TEFb. Here again genome wide mapping of P-TEFb regulatory factors will be carried out. In the final aim the roles of the termination factor TTF2 will be addressed. In addition to its role in mitotic repression of transcription elongation, studies will examine its role in transcription coupled DNA repair, premature termination at the 52 end of genes, and in termination following normal 32 end formation. The principle investigator is a professor at the University of Iowa and this project will be carried out in the excellent environment offered by his lab and the facilities in the University of Iowa, College of Medicine. PUBLIC HEALTH RELEVANCE: Project Narrative The proposed research in this application will make a major contribution toward understanding the basic cellular mechanisms that control transcription. It is critical to understand how transcription is regulated because abnormalities in this process are responsible developmental defects, cancer and other pathologies.