Transcriptional Homeostasis and Chromatin Dynamics

Abstract

Multiple regulatory mechanisms work to ensure that eukaryotic transcription maintains mRNA pools and subsequent protein synthesis. When errors in transcription occur, deleterious effects on cellular fitness can develop. RNA degradation as well as histone modifications, specifically at promoter proximal nucleosomes, play a critical role in maintaining transcription, but, exact mechanisms are not fully understood. In this dissertation, I investigate the role of RNA degradation and chromatin dynamics in transcription regulation as well as further understand, through biochemical analysis, a critical histone deacetylase. Using various genome-wide methodologies in Saccharomyces cerevisiae, we find a functional interaction between the nuclear RNA exosome and histone variant H2A.Z that maintains mRNA levels. There is a reduction in RNA polymerase II nascent transcription following RNA exosome subunit Rrp6 depletion that is further globally accentuated with H2A.Z deposition loss. To understand the mechanism leading to this global reduction, we identify the mRNA of Sirtuin histone deacetylase Hst3 as a target of the RNA exosome, revealing a means to link degradation to the transcription machinery. These findings show that even slight changes in deacetylase or acetylase activity can have significant effects on transcription. Additionally, we reveal a global impact of H2A.Z on transcription. We further investigate the functional and structural significance of human surtuin histone deacetylase SIRT6 (yeast homolog Hst3). Using histone deacetylase assays, we confirm the significance of specific residues of SIRT6 in nucleosome binding and deacetylase activity. Additionally, we show SIRT6 has reduced deacetylase activity in vitro on acetylated lysine 56 as compared to acetylated lysine 9 on histone H3. Finally, we confirm structural findings that the histone tail of H2A impacts SIRT6 H3K9Ac deacetylation activity. Together, these findings indicate a critical importance of histone deacetylase activity in maintaining transcription, a novel role of H2A.Z in global transcription regulation that furthers our understanding of SIRT6 structure and function.