Supplemental request for MAX-TL Ultracentrifuge and rotor

MAX-TL 超速离心机和转子的补充请求

• 批准号: 10386257
• 负责人: Dylan J Taatjes
• 金额: $ 4.92万
• 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/10386257
• 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 (for R35 GM139550) 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.

总结/摘要（适用于R35 GM 139550） 我们的研究计划结合了详细的生化重建实验和强大的基于细胞的 分析，目的是获得有关RNA聚合酶II（pol II）功能的基本机制见解， 其规定。12亚基人pol II酶转录所有蛋白质编码RNA和许多非编码RNA 在人类基因组中。Pol II转录起始受4.0 MDa前起始复合物（PIC）调节， 其中包含TFIIA、IIB、IID、IIE、IIF、IIH、pol II和Mediator。与序列特异性DNA结合 在转录因子（TF）中，PIC有助于指导pol II转录的时间、位置和方向，基因组- 宽转录因子和PIC在pol II转录的不同阶段（例如起始，暂停， 延伸）仍然不完全理解;此外，过去5年多来的新见解已经改变了 我们对转录的理解。例如，增强子RNA（eRNA）转录和增强子启动子 通讯似乎驱动谱系或信号特异性（或致癌）基因表达程序， 相分离的分子缩合物与细胞中的pol II活性相关。虽然新的机械模型 已经出现，这样的模型不能可靠地测试，只使用基于细胞的方法，部分原因是 蜂窝系统的巨大复杂性例如，蛋白质、核酸和蛋白质的特性和浓度可以被确定。 酸和生物化学物质存在于细胞群体中的任何给定基因上，不可能被定义。 在接下来的5年里，我们计划利用我们在生物化学重组方面的独特专业知识， 边缘细胞的方法，以解决以下高影响领域：1）液相分离分子 冷凝物和pol II功能。我们试图定义分子凝聚体如何（或是否）调节 转录，包括不同的组合物是否有助于控制pol II转录的不同阶段（例如， 起始对延伸）。2)转录激酶对pol II起始、暂停和延伸的调节 CDK 7（TFIIH亚基）、CDK 8（介体相关激酶）和CDK 9（P-TEFb激酶）。我们将评估 每种激酶单独或与其它激酶组合有助于调节pol II活性。这将 包括对延伸率或RNA加工潜在的“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的机制要求， 转录，以确定它们是否不同于典型的蛋白质编码基因。此外，我们力求 在体外重建超级增强子功能，这将作为理解“规则”的框架， 哪些超级增强子驱动人类细胞中的高水平转录。（虽然这方面的目标是雄心勃勃的，但我们注意到， 我们最近成功地重建了pol II启动子近端停顿，该领域长期以来一直认为这是困难的 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 通过靶向的、后续的细胞- 基于分析，这将实现基因组编辑，化学生物学，转录组学和其他方法。