FUNCTIONAL ANALYSIS OF A NOVEL CHROMATIN REGULATOR, SET4, IN SACCHAROMYCES CEREVISIAE

Abstract

Gene expression regulation is tightly controlled by chromatin structure regulators via diverse mechanisms that ensure proper functioning of stress-response pathways during environmental changes. Many chromatin modifiers play key roles in controlling transcriptional programs; however, there are several poorly-characterized chromatin regulators which are predicted to play important roles in cellular responses to different environmental conditions. Functional analysis of uncharacterized chromatin factors may lead to a better understanding of mechanisms, hierarchy, and changes in regulatory networks of cells in response to stress. In budding yeast, Set4 and Set3 contain two domains known as SET and PHD domains, which are common signatures of chromatin regulators in mammals. Set4 and Set3 are members of a SET domain subfamily, which also includes the fly protein UpSET and mammalian proteins MLL5 and SETD5. These protes share conserved but divergent SET domain sequences. Set4 is not well-characterized in budding yeast, although genome-wide studies suggested it might be important for cellular responses to stress. The goal of my dissertations was to investigate the biological and biochemical roles for Set4 in yeast cells. We determined that, while in normal growth conditions, SET4 is expressed at low levels, increased expression of SET4 is toxic to cells. However, under oxidative stress, higher survival was observed for cells with high levels of Set4. We found that Set4 is associated with chromatin, and many stress response genes are regulated by Set4 in response to oxidative stress, suggesting that Set4 is a stress-regulated chromatin protein. Although Set4 possesses a SET domain, there are divergent residues from canonical SET domain in critical sites for substrate binding and catalysis of methyl transfer. Our biochemical investigation of potential catalytic activity of the SET domain of Set4 indicated that the domain does not have methyltransferase activity in vitro. To further investigate the role of Set4 in gene expression control genome-wide, RNAseq experiments were employed to identify genes regulated by Set4 under normal and stress conditions. A large number of genes were found up- or down-regulated in either wildtype or set4? strains, particularly in the presence of oxidative stress. Many of these genes were found to be located at the telomere, or to be near or overlapping with non-coding transcripts. Follow-up qPCR experiments suggests that Set4 regulates genes near telomeres, most likely in a stress-independent manner. Altogether, we propose two models for Set4 function: Set4 get activated under oxidative stress and become associated with specific chromatin loci possibly in cooperation with other transcription factors to control stress response genes, and Set4 may have house keeping roles to maintain appropriate gene expression level of genes at the telomere.