The Positive Roles of Lysine Deacetylase Complexes in Regulating Transcriptional Dynamics

摘要 而传统的转录模型将赖氨酸乙酰转移酶（KAT，也称为HAT）作为转基因生物。 作为辅阻遏物的转录辅激活因子和赖氨酸脱乙酰酶（KDAC，也称为HDAC）， 大量证据表明，KDAC可以以基因依赖的方式促进转录。然而，在这方面， 其转录促进功能的机制知之甚少。的长期目标 我们的工作是确定KDAC，KAT和乙酰化调节信号调节的转录的机制， 笔迹。该研究利用糖皮质激素信号作为模型系统。实验室发表的研究 表明KDAC不仅是糖皮质激素介导的转录抑制所必需的，而且也是 靶基因的转录激活。初步研究表明，KDAC 1是GR激活所必需的。 通过多种机制转录，这取决于靶基因，作用于上游或下游。 RNA聚合酶II募集到转录起始位点（TSS）。下一个合乎逻辑的步骤是识别KDAC 1 复合物参与，并探讨其在糖皮质激素受体（GR）激活的转录中的作用。目标- 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。核心假设是，含有KDAC 1的复合物在 调节元件和/或基因体，以促进GR-活性的有效转录起始和延伸。 以基因特异性的方式改变基因。这一假设将通过三个具体目标进行实验检验。 在第一个具体目标中，研究了I类KDAC对GR激活的转录爆发动力学的影响。 基因将被测量，从假设它们的抑制将减少大小和/或频率， KDAC敏感性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）和转录的活细胞成像。在第二个具体目标中， 在GR激活的增强子和GR靶内，KDAC对转录起始和延伸的活性 基因将被定义。下一代测序方法（ChIP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的方式表达。在第三个具体目标中，鉴定含有KDAC 1的 将确定促进GR反式激活的复合物及其在GR靶基因周围的作用位点。 使用生物化学，分子和单细胞方法，KDAC 1促进GR的工作假设 在RCOR和/或NuRD复合物在调节元件处或基因内有活性的情况下的反式激活 机构将得到处理。这项工作将产生新的机制知识的转录功能 KDAC与细胞过程的基本理解以及疾病的治疗有关， 表观基因组的调节。这项研究是创新的，因为它将推动这种新的KDAC范式， 通过整合转录动力学，超越了传统的转录静态模型。

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.