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.
The proposed research is relevant to public health because lysine deacetylases are targets of drugs approved for treatment of several human pathologies and are under investigation for use against additional diseases. A more thorough understanding of basic lysine deacetylase functions in regulation of transcription will promote efficient clinical use of these approved therapeutics and the development of more targeted inhibitors for regulating the epigenome in disease treatment.
