Elucidating the function of the histone H4 basic patch in SAGA-mediated histone H2B deubiquitination and histone acetylation

The eukaryotic genome is packaged into the nucleus by the wrapping of DNA around histones to form the nucleosome—the fundamental repeating unit of chromatin. In addition to its structural role, chromatin regulates DNA accessibility and thereby DNA-templated processes such as DNA replication and transcription. Chromatin structure and function is mediated by the covalent modification of the histone proteins with a vast array of post-translational modifications (PTMs) including acetylation, methylation, phosphorylation, and monoubiquitylation. Histone PTMs can affect chromatin structure directly or serve as binding sites for effector proteins and complexes that mediate a specific function. Proper regulation of histone PTMs is critical for many cellular and developmental processes and their aberrant placement or removal is a hallmark of many diseases including cancer and neurodegenerative disorders. Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes including gene transcription, DNA repair, and replication. In addition, H2Bub1 is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report a novel regulator of H2Bub1 – the histone H4 basic patch. The H4 basic patch is a hub for chromatin modifiers, yet the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, LINX (light-inducible nuclear exporter), to control the subcellular location of the H2Bub1 E3-ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected by a H4 basic patch mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased histone acetylation by SAGA in H4 basic patch mutants. The work in this dissertation discusses the discovery a new regulatory mechanism of the H4 basic patch in SAGA-mediated functionsDoctor of Philosoph

Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14

Membrane holder

Membrane holde

An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and -independent dynamics of H3K36 methylation.

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively

Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14