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(KATS，也称为HATS)转换为反式-乙酰基转移酶。 作为辅抑制子的书面辅活化子和赖氨酸脱乙酰酶(KDAC，也称为HDAC)，是一种- 大量证据表明，KDAC可以以基因依赖的方式促进转录。然而， 其转录促进功能的潜在机制还知之甚少。的长期目标是 我们的工作是确定KDAC、KATS和乙酰化调节信号调节的转导的机制。 脚本。这项拟议的研究利用糖皮质激素信号作为模型系统。该实验室发表的研究成果 证明KDAC不仅是糖皮质激素介导的转录抑制所必需的，也是 靶基因的转录激活。初步研究表明，GR激活需要KDAC1 根据靶基因的不同，通过多种机制转录，作用于 RNA聚合酶II募集到转录起始点(TSS)。下一个合乎逻辑的步骤是识别KDAC1 并研究它们在糖皮质激素受体(GR)激活转录中的作用。客体- 这项研究的目的是确定KDAC在GLUGO-2转录循环动力学中的作用。 皮质激素受体(GR)激活的基因。中心假设是含有KDAC1的复合体在 调节元件和/或基因体，以促进在GR-ACTI-ACTI中有效的转录起始和延伸 以特定于基因的方式激活基因。这一假设将通过三个具体目标进行实验验证。 在第一个特定目标中，I类KDAC对GR激活的转录爆发动力学的影响 基因将被测量，根据他们的抑制将减少大小和/或FRE的假设工作。 KDAC敏感的GR靶基因的突发率。这将用单分子荧光素进行测试。 原位杂交和转录的活细胞成像。在第二个具体目标中，功能影响 KDAC在GR激活的增强子和GR靶区转录起始和延伸中的活性 基因将被定义。下一代测序方法(芯片测序和新生的转录测序)将 被用来解决这一假说，即I类KDAC促进GR诱导的转录起始或伸长- 以特定于基因和增强子的方式表达。在第三个具体目标中，KDAC1的身份--包含 促进GR反式激活的复合体及其在GR靶基因周围的作用部位将被确定。 使用生化、分子和单细胞方法，KDAC1促进GR的工作假说 RCOR和/或NuRD复合体在调节元件或基因内的反式激活 身体将会被处理。这项工作将产生转录功能的新的机械知识 与细胞过程的基本了解以及疾病的治疗有关的KDAC 表观基因组的调制。这项研究具有创新性，因为它将推动KDAC的这一新范式 通过结合转录动力学，超越传统静态转录模式的辅活化子。

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.