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Identification of the Ubiquitin Ligase Bre1 and its Role in Antagonizing Gene Silencing and Promoting Mitotic Exit

  • Author(s): Hwang, William W
  • Advisor(s): Madhani, Hiten D
  • et al.
Abstract

Despite their identification over forty years ago, the importance and complexity of the post-translational modification of histones, the proteins that wrap DNA into the core component of chromatin called nucleosomes, has only been appreciated in the past decade. From the initial characterization of the enzymes responsible for these modifications in yeast to the identification of their homologs in humans, it is clear that these well-conserved modifications on histones play a crucial role in almost all cellular processes, ranging from the regulation of embryonic development to the misregulation underlying malignant transformation. In this dissertation, I detail the contributions we have made to the chromatin field in elucidating not only novel histone modifiers but also unexpected processes that are dependent on these enzymes. First, I describe our identification of Bre1, the previously unknown conserved E3 ubiquitin ligase responsible for the ubiquitination of histone H2B on lysine 123 (ubH2B). We established a requirement for Bre1 and its associated protein Lge1 in not only promoting ubH2B but also being necessary for the efficient downstream methylation of histone H3 on lysine 4 and 79 (meH3K4 and K79). Furthermore, we demonstrate that that Bre1/Lge1-mediated ubH2B is necessary for the proper control of cell size, linking chromatin regulation to this important but still mysterious process. Second, our comparison of the transcriptional profiles of mutants in the ubH2B and meH3K4/K79 pathway revealed a novel function for Bre1/Lge1 in antagonizing the spread of heterochromatic silencing that is independent of the characterized anti-silencing roles for meH3-K4 and K79. Given the striking distinction in anti-silencing phenotypes between bre1 deletion mutants and mutants unable to ubiquitinate histone H2B or methylate histone H3, our results suggest that Bre1 can target substrates other than histone H2B. Finally, we discovered an unexpected requirement for multiple histone modifications for the efficient release of the nucleolar chromatin-sequestered phosphatase Cdc14 to promote mitotic exit. In summary, the work presented in this dissertation reveals previously unappreciated complexity in the regulation of chromatin function by a histone-modifying enzyme both directly and through the modifications it promotes.

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