Phds, Stress and Starvation: The Identification of a New Rpd3 Deacetylase Complex Involved in the Yeast Oxidative Stress and Metabolism Pathways
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
The cellular pathways that govern survival in the face of diverse stresses rely on gene expression changes as one mechanism to respond to or protect against internal and external threats. Because eukaryotic DNA is packaged into chromatin, these gene expression changes depend on the targeting of regulatory proteins to specifi c regions of the genome to alter chromatin structure, promoting or repressing transcription. One protein domain involved in targeting chromatin regulators is the plant homeodomain, or PHD fi nger, a module that preferentially interacts with either methylated or unmethylated lysines on histones, and has important functions in human health. Despite recent advances in identifying the histone ligands for some PHD fi ngers as well as the functions of the proteins that contain them, for many other PHD fi ngers, including some of the 17 PHD fi ngers of the budding yeast saccharomyces cerevisiae, these questions remain unanswered. In the research presented in this thesis, I sought to gain insight into the ligands and functions for three yeast PHD fi nger proteins, the Yng1 subunit of the NuA3 acetyltransferase complex, Jhd2, and Ecm5, the latter two both being homologous to the mammalian JARID family of histone demethylases. In Chapter 2, I demonstrate that the PHD fi ngers of these proteins interact with histone H3 enriched for different sites of methylation depending on the PHD and present results of an in vitro assay used to test whether any yeast PHD fi ngers possess ubiquitin E3 ligase activity, a function ascribed to the PHD-related RING domain. In Chapter 3, I discuss experiments performed to identify the protein interaction partners of Jhd2 and Ecm5, culminating in the discovery that Ecm5 interacts with the PHD fi nger protein Snt2 as well as the Rpd3 deacetylase, forming a complex I have named Rpd3(T). I also discuss experiments showing that the ecm5 knockout strain does not have obvious defects in many yeast pathways. In Chapter 4, I present evidence that Rpd3(T) complex members are involved in the cellular oxidative stress and metabolism pathways, and discuss chromatin immunoprecipitation experiments followed by high-throughput sequencing which were performed to map Ecm5 and Snt2 localization before and after hydrogen peroxide-mediated oxidative stress. I then discuss how the Ecm5 and Snt2 localization patterns relate to gene expression changes in wild-type cells after oxidative stress and in snt2 knockout cells. I compare Ecm5 and Snt2 localization patterns in rich media before and after oxidative stress to patterns in less rich media before and after nutrient stress induced by the TOR pathway inhibitor rapamycin. Finally, I discuss potential mechanisms through which Ecm5 and Snt2, either as a pair or as a part of the Rpd3(T) complex, may help to coordinate the cellular responses to oxidative and nutrient stresses, and the greater implications of this work.