Mechanisms of RNA polymerase II transcription regulation

RNA聚合酶II转录调控机制

• 批准号: 10321903
• 负责人: Dylan J Taatjes
• 金额: $ 63.3万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10321903
• 关键词: Address; Area; Biochemical; Biological Assay; Biology; Cells; Chemicals; Code; Communication; Complex; DNA Binding; DNA Polymerase II; DNA-Directed RNA Polymerase; Development; Disease; Enhancers; Enzymes; Gene Expression; Genes; Genetic Transcription; Goals; Growth and Development function; Heart Diseases; Human; Human Genome; In Vitro; Liquid substance; Location; Malignant Neoplasms; Mediator of activation protein; Methods; Modeling; Molecular; Nucleic Acids; Oncogenic; Phase; Phosphotransferases; Population; Positive Transcriptional Elongation Factor B; Proteins; RNA; RNA Processing; Regulation; Research; Signal Transduction; System; Testing; Transcription Elongation; Transcription Factor TFIIA; Transcription Initiation; Transcriptional Regulation; Untranslated RNA; Work; base; experimental study; follow-up; genome editing; genome-wide; in vitro Assay; insight; nervous system disorder; novel strategies; programs; promoter; public health relevance; reconstitution; success; therapeutic target; transcription factor; transcription factor TFIIH; transcriptomics

SUMMARY/ABSTRACT Our research program combines detailed biochemical reconstitution experiments with powerful cell-based assays, with a goal of gaining fundamental mechanistic insights about RNA polymerase II (pol II) function and its regulation. The 12-subunit human pol II enzyme transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is regulated by the 4.0 MDa Pre-Initiation Complex (PIC), which contains TFIIA, IIB, IID, IIE, IIF, IIH, pol II, and Mediator. Together with sequence-specific, DNA-binding transcription factors (TFs), the PIC helps direct the timing, location, and direction of pol II transcription, genome- wide. How TFs and the PIC work together during different stages of pol II transcription (e.g. initiation, pausing, elongation) remain incompletely understood; moreover, new insights over the past 5+ years have transformed our understanding of transcription. For instance, enhancer RNA (eRNA) transcription and enhancer-promoter communication appear to drive lineage- or signal-specific (or oncogenic) gene expression programs, and liquid phase separated molecular condensates correlate with pol II activity in cells. Although new mechanistic models have emerged, such models cannot be reliably tested using only cell-based methods, in part because of the enormous complexity of cellular systems. For instance, the identity and concentration of the proteins, nucleic acids, and biochemicals that are present at any given gene in a population of cells cannot possibly be defined. In the next 5 years, we propose to leverage our unique expertise in biochemical reconstitution with cutting- edge cellular methods to address the following high-impact areas: 1) Liquid phase-separated molecular condensates and pol II function. We seek to define how (or whether) molecular condensates regulate transcription, including whether distinct compositions help control different stages of pol II transcription (e.g. initiation vs. elongation). 2) Regulation of pol II initiation, pausing, and elongation by the transcriptional kinases CDK7 (TFIIH subunit), CDK8 (Mediator-associated kinase), and CDK9 (P-TEFb kinase). We will assess what each kinase, alone and in combination with the others, contributes to the regulation of pol II activity. This will include potential “downstream” impacts on elongation rates or RNA processing. 3) Enhancer RNA (eRNA) transcription and super-enhancer function. We will dissect the mechanistic requirements for bidirectional eRNA transcription, to determine whether they are distinct from typical protein-coding genes. Furthermore, we seek to reconstitute super-enhancer function in vitro, which would serve as a framework for understanding the “rules” by which super-enhancers drive high-level transcription in human cells. (Although this aspect is ambitious, we note our recent success with reconstitution of pol II promoter-proximal pausing, which the field long considered difficult if not impossible.) Finally, we emphasize that an equally important aspect of our research plan is to rigorously test the models that emerge from our detailed and systematic in vitro assays through targeted, follow-up cell- based assays, which will implement genome-editing, chemical biology, transcriptomics, and other approaches.

摘要/摘要 我们的研究项目结合了详细的生化重建实验和强大的基于细胞的 分析，目的是获得关于RNA聚合酶II(PolII)功能和 它的规定。人类POLII酶由12个亚基组成，可转录所有蛋白质编码和许多非编码RNA 在人类基因组中。POL II转录起始受4.0丙二醛预起始复合体(PIC)调控， 包括TFIIA、IIB、IID、IIE、IIF、IIH、POL II和调解人。以及特定于序列的DNA结合 转录因子(TF)，PIC帮助指导PolII转录的时间、位置和方向，基因组- 很宽。转录因子和PIC如何在POLII转录的不同阶段(例如，启动、暂停、 延伸率)仍然不完全被理解；此外，过去5年多来的新见解已经转变 我们对转录的理解。例如，增强子RNA(Erna)转录和增强子-启动子 交流似乎驱动特定于谱系或信号的(或致癌的)基因表达程序，以及液体 相分离的分子凝聚体与细胞中的PolII活性相关。尽管新的机械论模型 ，这样的模型不能仅使用基于细胞的方法进行可靠的测试，部分原因是 蜂窝系统的巨大复杂性。例如，蛋白质、核酸的特性和浓度 酸，以及存在于细胞群体中任何给定基因的生物化学物质都不可能被定义。 在未来5年，我们计划利用我们在生化重建方面的独特专业知识进行切割- 边缘细胞方法解决以下高影响领域：1)液体相分离的分子 凝析油和POL II功能。我们试图定义分子凝聚体如何(或是否)调节 转录，包括不同的组成是否有助于控制POLII转录的不同阶段(例如 起始期与延伸率)。2)转录酶对PolII的启动、暂停和延长的调节 CDK7(TFIIH亚单位)、CDK8(介体相关激酶)和CDK9(P-TEFb激酶)。我们将评估 每一种酶单独或与其他酶结合，对POLII的活性起到调节作用。这将是 包括对延伸率或RNA加工的潜在“下游”影响。3)增强子RNA(Erna) 转录和超级增强子功能。我们将剖析双向ERNA的机械要求 转录，以确定它们是否与典型的蛋白质编码基因不同。此外，我们寻求 在体外重建超级增强子功能，这将作为一个框架，通过 哪些超级增强剂可以驱动人类细胞的高水平转录。(尽管这方面雄心勃勃，但我们注意到 我们最近成功地重建了PolII启动子--近端停顿，这一点长期以来一直被业界认为是困难的 如果不是不可能的话。)最后，我们强调，我们的研究计划的一个同样重要的方面是严格地 通过有针对性的后续细胞，测试我们详细和系统的体外测试产生的模型- 基于分析，它将实施基因组编辑、化学生物学、转录组学和其他方法。