The Positive Roles of Lysine Deacetylase Complexes in Regulating Transcriptional Dynamics

赖氨酸脱乙酰酶复合物在调节转录动力学中的积极作用

• 批准号: 10094469
• 负责人: Catharine Lynn Smith
• 金额: $ 33.94万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094469
• 关键词: Acetylation; Acetyltransferase; Address; Biochemical; Biological Assay; Biological Models; Cell physiology; Cells; ChIP-seq; Chromatin; Circadian Rhythms; Clinical; Collaborations; Complex; Coupled; Data; Deacetylase; Development; Disease; Doctor of Philosophy; Drug Targeting; Enhancers; Ensure; Epigenetic Process; Fluorescent in Situ Hybridization; Formulation; Frequencies; Genes; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Goals; Histone Deacetylase; Histone H3; Human Pathology; Impairment; Investigation; Kinetics; Knowledge; Life; Ligands; Lysine; Maintenance; Measures; Mediating; Metabolism; Methods; Modeling; Molecular; NuRD complex; Patients; Pharmaceutical Preparations; Physiological Processes; Proteins; Public Health; Publishing; RNA Polymerase II; Regulatory Element; Research; Resources; Role; Signal Transduction; Site; Small Interfering RNA; Steroid Receptors; Steroids; Study models; Testing; Therapeutic Uses; Toxic effect; Transactivation; Transcript; Transcription Coactivator; Transcription Elongation; Transcription Initiation; Transcription Initiation Site; Transcriptional Activation; Transcriptional Regulation; United States National Institutes of Health; Work; cancer therapy; course development; epigenome; experience; gene repression; genetic corepressor; glucocorticoid-induced orphan receptor; immune function; inhibitor/antagonist; innovation; live cell imaging; nervous system disorder; next generation sequencing; novel; prevent; promoter; recruit; single molecule; therapeutic development; transcription factor

Abstract Whereas traditional models of transcription cast lysine acetyltransferases (KATs, also known as HATs) as tran- scriptional coactivators and lysine deacetylases (KDACs, also known as HDACs) as corepressors, an abun- dance of evidence demonstrates that KDACs can facilitate transcription in a gene-dependent fashion. However, the mechanisms underlying their transcription-promoting functions are poorly understood. The long-term goal of our work is to define mechanisms by which KDACs, KATs, and acetylation regulate signaling-modulated tran- scription. The proposed study utilizes glucocorticoid signaling as a model system. Published studies from the lab demonstrate that KDACs are required not only for glucocorticoid-mediated transcriptional repression but also for transcriptional activation of target genes. Preliminary studies show that KDAC1 is required for GR-activated transcription by multiple mechanisms, depending on the target gene, acting either upstream or downstream of RNA polymerase II recruitment to the transcription start site (TSS). The next logical step is to identify the KDAC1 complexes involved and investigate their roles in glucocorticoid receptor (GR)-activated transcription. The objec- tive of the proposed study is to define the roles of KDACs in the dynamics of the transcriptional cycle at gluco- corticoid receptor (GR)-activated genes. The central hypothesis is that KDAC1-containing complexes act within regulatory elements and/or gene bodies to facilitate efficient transcriptional initiation and elongation at GR-acti- vated genes in a gene-specific fashion. This hypothesis will be tested experimentally through three specific aims. In the first specific aim, the impact of Class I KDACs on the kinetics of transcriptional bursting at GR-activated genes will be measured, working from the hypothesis that their inhibition will decrease the size and/or the fre- quency of bursts at KDAC-sensitive GR target genes. This will be tested using single molecule fluorescent in situ hybridization (smFISH) and live cell imaging of transcription. In the second specific aim, the functional impact of KDAC activity on transcriptional initiation and elongation at GR-activated enhancers and within GR target genes will be defined. Next generation sequencing approaches (ChIP- and nascent transcript-sequencing) will be used to address the hypothesis that Class I KDACs facilitate GR-induced transcriptional initiation or elonga- tion in a gene- and enhancer-specific fashion. In the third specific aim, the identity of the KDAC1-containing complexes that facilitate GR transactivation and their sites of action around GR target genes will be determined. Using biochemical, molecular, and single cell approaches, the working hypothesis that KDAC1 facilitates GR transactivation in the context of the RCOR and/or NuRD complexes active at regulatory elements or within gene bodies will be addressed. This work will generate novel mechanistic knowledge of the transcriptional functions of KDACs that is relevant to basic understanding of cellular processes as well as treatment of disease through modulation of the epigenome. The study is innovative because it will move this new paradigm of KDACs as coactivors beyond traditional static models of transcription by incorporating transcriptional dynamics.

抽象的 而传统的转录模型将赖氨酸乙酰转移酶（KAT，也称为 HAT）视为反式 脚本共激活剂和赖氨酸脱乙酰酶（KDAC，也称为 HDAC）作为辅阻遏物，是一种 abun- 大量证据表明 KDAC 可以以基因依赖性方式促进转录。然而， 人们对其转录促进功能的机制知之甚少。长期目标是 我们的工作是定义 KDAC、KAT 和乙酰化调节信号调节转录的机制 铭文。拟议的研究利用糖皮质激素信号传导作为模型系统。实验室发表的研究 证明 KDAC 不仅是糖皮质激素介导的转录抑制所必需的，而且也是糖皮质激素介导的转录抑制所必需的 靶基因的转录激活。初步研究表明KDAC1是GR激活所必需的 通过多种机制进行转录，具体取决于靶基因，作用于上游或下游 RNA 聚合酶 II 招募至转录起始位点 (TSS)。下一个逻辑步骤是识别 KDAC1 复合物参与并研究它们在糖皮质激素受体（GR）激活转录中的作用。对象- 拟议研究的目的是定义 KDAC 在葡萄糖转录周期动态中的作用 皮质激素受体（GR）激活基因。中心假设是含有 KDAC1 的复合物在 调控元件和/或基因体，以促进 GR-acti- 有效转录起始和延伸 以基因特异性方式改变基因。该假设将通过三个具体目标进行实验检验。 第一个具体目标是 I 类 KDAC 对 GR 激活转录爆发动力学的影响 基因将被测量，假设它们的抑制会减少大小和/或频率 KDAC 敏感 GR 靶基因的爆发频率。这将使用单分子荧光进行测试 原位杂交 (smFISH) 和活细胞转录成像。在第二个具体目标中，功能影响 KDAC 活性对 GR 激活增强子和 GR 靶标内转录起始和延伸的影响 基因将被定义。下一代测序方法（ChIP 测序和新生转录测序）将 用于解决 I 类 KDAC 促进 GR 诱导的转录起始或延长的假设 以基因和增强子特异性方式进行灰化。在第三个具体目标中，含有KDAC1的身份 将确定促进 GR 反式激活的复合物及其围绕 GR 靶基因的作用位点。 使用生化、分子和单细胞方法，KDAC1 促进 GR 的工作假设 在调节元件或基因内活跃的 RCOR 和/或 NuRD 复合物背景下的反式激活 机构将得到解决。这项工作将产生转录功能的新机制知识 KDAC 与细胞过程的基本理解以及疾病治疗相关 表观基因组的调节。这项研究具有创新性，因为它将推动 KDAC 的新范式 通过结合转录动力学，辅激活物超越了传统的静态转录模型。