Covalent modifications of chromatin regulate genomic structure and accessibility in diverse biological processes such as transcriptional regulation, cell cycle progression, and the DNA damage response. Whereas many histone modifications have been characterized, understanding the interactions between these and their combinatorial effects remains an active area of investigation, including dissecting functional interactions between enzymes mediating these modifications. In budding yeast, the histone acetyltransferase Gcn5 interacts with Rts1, the regulatory subunit of protein phosphatase 2A (PP2A), in part via the dynamic phosphorylation of conserved residues on histone H2B and the centromeric-specific histone H3 variant, Cse4. To further probe these dynamics, we sought to identify kinases which might contribute and found a role for a stress-activated protein kinase, Hog1. Deletion of HOG1 (hog1∆) rescues gcn5∆ sensitivity to the microtubule poison nocodazole and the lethality of the gcn5∆ rts1∆ double mutant. Dynamic phosphorylation of histone H2B-T91 also functions in the Hog1-Gcn5 interaction. Furthermore, Loss of HOG1 decreases aneuploidy and apoptotic populations in gcn5∆ cells. Together, these results suggest that Hog1 functionally opposes Gcn5 and Rts1 in the context of the spindle assembly checkpoint and raise the possibility of further kinase involvement in GCN5’s functions.

The organization and regulation of eukaryotic DNA is a critical genomic process that is controlled by many key enzymes and multimeric complexes. One mode of regulation involves the acetylation of histones, as coordinated by the opposing enzymatic activities of histone acetyltransferases and deacetylases. Some of the enzymatic and regulatory subunits involved in chromatin regulation are essential, and thus present additional challenges to comprehensively study their function. Subunits within the NuA4 complex, such as the catalytic subunit Esa1, and non-catalytic Epl1 subunit, are examples of essential chromatin-modifying components. In this thesis, the powerful genetic tool of bypass suppression, is used to study null ESA1 and EPL1 mutants by concurrent deletion of the Rpd3L deacetylase complex, promoting a more balanced cellular acetylation state. Upon bypass, critical functions of Epl1 were defined for the first time in vivo, and implicated Epl1 as a key co-factor required for Esa1 catalytic activity. Here, Epl1 is also found to be important for targeting Esa1 to chromatin, thereby supporting Esa1’s critical activity as an acetyltransferase. Furthermore, bypass suppression of NuA4 revealed a key interaction network between NuA4 and the Rpd3L and Hda1 histone deacetylases. Despite deleting an essential acetyltransferase and two important deacetylases, growth of the epl1∆ sds3∆ hda1∆ mutant is significantly more robust than the epl1∆ sds3∆ mutant and can withstand various stresses such as high temperature and exposure to genotoxic agents. The strong genetic interaction between NuA4, Rpd3L, and Hda1 underscores the importance of understanding how acetyltransferases and deacetylases act both in opposition and cooperatively. Finally, bypass suppression of ESA1 enabled the first transcriptomics study of esa1∆ cells, defining Esa1 as a critical regulator of ribosome biogenesis. Together, the studies presented and discussed in this thesis highlight the power of bypass suppression and define key functions and regulatory targets of critical chromatin-modifiers in Saccharomyces cerevisiae.

The ends of linear chromosomes are composed of a long array of highly conserved repeat sequences that are essential for genome stability. These chromosome ends, called telomeres, act as a molecular clock that dictates cell division potential. With every cell division, a small amount of DNA erodes from each chromosome terminus, and while this erosion process is initially insignificant, the cumulative effect after many cell divisions can be catastrophic, ultimately resulting in cell death or cancerous transformations. At a certain point, so much telomeric DNA is lost that a signal is sent to halt further cell division. This block to indefinite cell division can be alleviated by the enzyme telomerase which adds telomeric DNA to chromosome ends, thereby preventing telomere shortening. Telomerase has been the subject of intensive studies since its discovery, as the contribution of telomere length regulation to human health, with regard to both cancer biology and human aging, is substantial. A major goal of the Lundblad lab is to achieve a full understanding of how this enzyme is regulated, in order to uncover novel insights into how chromosome ends can make such wide-spread contributions to human biology. In budding yeast, a telomeric dedicated RPA complex (the t-RPA complex), formed by the three essential proteins Cdc13, Stn1 and Ten1 is central to ensuring telomere homeostasis as it ensures the elongation of the telomeres by recruiting the cellular retro-transcriptase telomerase. Furthermore, it promotes efficient replication of the telomere as a telomeric specific complex that becomes part of the replisome as the replication fork advances through the duplex telomeric DNA. This dissertation describes my efforts to uncover how a third complex, the canonical RPA complex, performs a role in telomere maintenance with t-RPA and telomerase.

Post-translational histone modifications have a critical role in regulating transcription, the cell cycle, and stress responses. These histone modifications can occur in complex patterns depending on the activity of multiple chromatin modifiers. Histone acetyltransferases (HAT) are one of the classes of chromatin modifiers important for altering chromatin structure and activating transcription. Gcn5 is a highly conserved catalytic subunit found in multiple HAT complexes that regulate gene expression. In addition to HAT activity, phosphorylation of histones and non-histone proteins by protein kinases is also important. Kinases are necessary for signaling pathways such as a rapid response to stress. Here we define previously unsuspected interactions between Gcn5 and different isoforms of the protein kinase CK2, in Saccharomyces cerevisiae. We show that although CK2 has hundreds of targets in vivo, distinctions can be made between the individual catalytic subunits encoded by CKA1 and CKA2. This distinction was found in cells lacking Gcn5. Loss of CKA1 and CKA2 suppresses gcn5∆ mutant sensitivity to elevated temperatures and DNA damage induced by hydroxyurea, respectively. Suppression is lost when either CKB1 or CKB2 are also deleted. Loss of CKA1, but not CKA2 partially recovers the decrease in H3K9,K14ac in gcn5∆ cells. A directed screen of tyrosine residues of core histones demonstrated novel roles of multiple residues, but left unanswered key questions into the interactions seen between Gcn5 and CK2. Together, this work has shown distinct functions of the CK2 isoforms in their roles in specific stress responses and interactions with Gcn5.

αB-crystallin (CryAB) is a ~20-kD small heat shock protein commonly expressed in heart tissue. However, the CryAB R120G missense mutation causes misfolding of the protein, leading to the formation of insoluble CryAB aggregates in cardiomyocytes linked to the development of desmin-related cardiomyopathy. Typically, cells utilize mechanisms such as the ubiquitin-proteasome system (UPS) and autophagic-lysosome pathway to maintain proteostasis, while growing evidence shows that secretion of extracellular vesicles (EVs) may also play a role in protein quality control. Here, we analyze how these mechanisms function in the context of CryAB R120G. After confirming presence of insoluble CryAB protein in mouse hearts from a CryAB R120G KI mutant line, we observed that UPS activity seemed to decrease in mice aged 3-4 months. We identified CryAB in secreted extracellular vesicles from R120G KI heart tissue and observed the presence of autophagic markers, indicating a relationship between EV secretion and autophagy mechanisms. We also found that inhibition of the UPS decreases EV secretion. Further, secreted EVs carrying R120G CryAB do not appear to cause a pro-inflammatory response in macrophages. Our findings suggest insoluble CryAB R120G aggregates impact quality control in cardiomyocytes, and that EVs assist in clearing misfolded CryAB from the cell.