The Positive Roles of Lysine Deacetylase Complexes in Regulating Transcriptional Dynamics (Diversity Supplement)

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PROJECT SUMMARY 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 (RNA Pol 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 objective of the parent grant (R01GM139829) is to define the roles of KDACs in the dynamics of the tran- scriptional cycle at glucocorticoid receptor (GR)-activated genes. The objective of the research plan described in this diversity supplement is to investigate the impact of KDAC activity on the dynamics of RNA polymerase II association with GR-activated genes. 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 to identify GR-activated genes that are regulated by KDAC activity upstream or down- stream of transcriptional initiation, working from the hypothesis that the mechanism by which KDACs facilitate transcription is gene-specific. In the second specific aim, the impact of KDAC activity on RNA pol II distribution and the pattern of nascent transcription at enhancers, promoters, and gene bodies of GR-activated genes will be determined genome-wide. The working hypothesis is that KDAC activity regulates transcription and RNA pol II dynamics at distinct gene regions in a gene-specific fashion. In the third specific aim the dynamics of histone acetylation and RNA pol II will be investigated at a GR-activated gene at which transcription can be visualized in live cells. This will be performed using nanobodies to detect histone acetylation and RNA pol II in live cells. 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 epige- nome. Importantly, in the process of doing this research the graduate student who will be supported by the supplement will gain skills critical to a successful career in the biomedical workforce.