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.

No description available.

The objective of this study was to examine the influence of age on food intake, tissue and organ mass and NF-ĸB and SIRT6 levels in various tissues. The transcription factor, Nuclear Factor Kappa-B (NF-ĸB), is associated with both catabolic and anabolic pathways of muscle metabolism and may be involved in age-related muscle loss. SIRT6 is a member of the sirtuin family of proteins that function as protein lysine deacetylases and are associated with longevity in a number of organisms. Male Sprague-Dawley rats, aged 6 months (Adult) and 21 months (Old) were fed a commercially available diet for 10-17 days. Old rats consumed less food per body weight (BW) each day than Adult rats (1.45% g diet/g BW vs. 2.4% g diet/g BW). However, when intake data were expressed as g/diet per day there was no significant difference between groups. For skeletal muscle tissue, the average mass of gastrocnemius and soleus (g muscle/g BW) was significantly lower in Old rats. Levels of NF-ĸB (p65/RelA) and SIRT6 were measured by Western blot analysis in gastrocnemius, tibialis anterior, quadriceps, soleus, lung, heart, kidney and liver. NF-ĸB levels were higher in gastrocnemius of Old rats compared to Adult rats. No significant age-specific differences in SIRT6 protein levels were noted in the tissues examined. Interestingly, when examined independent of age, levels of SIRT6 were significantly different between certain tissues. Data from this study suggest that age affects muscle loss and NF-ĸB in a tissue-specific manner. Furthermore, these findings indicate tissue-specific but not age-specific differences in SIRT6 protein levels.

Cardiovascular diseases are one of the leading causes of mortality. A common denominator across most of the cardiovascular diseases like diabetic cardiomyopathy, hypertrophic cardiomyopathy, myocardial infarction and dilated cardiomyopathy is the pathological remodelling of heart leading to fibrosis. Cardiac fibrosis is characterized by the excessive production and deposition of extracellular matrix components due to unwarranted proliferation of fibroblasts. Under normal conditions, following cardiac remodelling, my fibroblasts undergo programmed cell death. However, this does not happen under pathological conditions ultimately leading to fibrosis. Although the molecular events and signalling pathways that contribute to the development of cardiac fibrosis is well established, there are limited studies which try to understand the mechanisms by which fibroblasts persist and resist programmed cell death. Here we demonstrate that SIRT6, one of the members of sirtuin family of histone deacetylases, plays an important role in regulating my fibroblast cell death. When we analysed the mice hearts and isolated fibroblasts deficient in SIRT6, we observed increased expression of my fibroblast markers, suggesting that SIRT6 deficient hearts might have a high proportion of resident my fibroblasts. Also, when SIRT6 deficient fibroblasts were subjected to genotoxic stress, they showed reduced cell death and impaired mitochondrial to nuclear AIF translocation as compared to WT controls. An important regulator of AIF mediated cell death is the protein PARP-1. When we checked the expression levels of this protein under SIRT6 deficient conditions, it was found to be low. PARP-1 was also found to degrade faster under SIRT6 deficient conditions. Further qPCR analysis revealed that the transcript levels of PARP-1 were unaffected by SIRT6 suggesting that the regulation might not be at the transcriptional level. When we studied the acetylation of PARP-1 under SIRT6 deficient conditions we found the protein to be hypo-acetylated indicating a more complex mechanism of regulation.

No description available.