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dc.contributor.advisorChatterjee, Champak
dc.contributor.authorShelton, Patrick Michael McPhee
dc.date.accessioned2020-02-04T19:25:05Z
dc.date.available2020-02-04T19:25:05Z
dc.date.submitted2019
dc.identifier.otherShelton_washington_0250E_20887.pdf
dc.identifier.urihttp://hdl.handle.net/1773/45137
dc.descriptionThesis (Ph.D.)--University of Washington, 2019
dc.description.abstractHistone post-translational modifications (PTM) within chromatin control many DNA-templated cellular processes, from DNA damage repair pathways to gene transcription. In order to understand the influence of PTM on these important processes and their associated regulatory enzymes, my graduate work in the Chatterjee research group at the University of Washington has focused on developing chemical techniques for the synthesis of full-length and site-specifically modified proteins. The biophysical and biochemical impacts of histone modification by the small ubiquitin-like modifier (SUMO) family of proteins in particular are poorly understood. Early studies associated histone H4 sumoylation with gene repression, but offered little insight into the precise mechanisms underlying this repression. Therefore, this thesis focuses on investigating mechanisms by which SUMO may control gene function. Toward this goal, I have synthesized numerous methylated and acetylated N-terminal histone peptides and full-length histone proteins to assess how sumoylation modulates the activity of lysine specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1) complexes associated with gene silencing. The Chatterjee lab has previously developed a new approach for the semisynthesis of site-specifically sumoylated histone H4 (suH4) by using a 2-(aminooxy)ethanethiol-mediated expressed protein ligation strategy. During the development of the 2-(aminooxy)ethanethiol ligation method, it was serendipitously discovered that ligation products still bearing the ligation handle undergo reverse native chemical ligation (NCL) in thiol-containing buffers, resulting in isolable C-terminal α-thioesters. I have harnessed this observation in the development of a novel, robust and multifaceted technique using the C-terminal mercaptoethoxyglycinamide (MEGA) handle to synthesize valuable C-terminal peptide α-thioesters and cyclic peptides (Chapter 2). Optimized conditions allow for peptide thioesterification using most amino acids at the C-terminal position under mild conditions, and I have demonstrated compatibility with a wide range of peptide lengths. The MEGA approach is also amenable to one-pot NCL reactions by introducing a N-terminal cysteine containing peptide, and is ideally suited for the synthesis of cyclic peptides via intramolecular NCL. Semisynthetic strategies developed by our lab have allowed assembly of simultaneously methylated, acetylated and sumoylated mononucleosome substrates for evaluating the effect of suH4 on chromatin-modifying enzymatic activity. Specifically, I have harnessed these new methodologies to demonstrate that suH4 stimulates the activity of the transcriptionally repressive enzymes LSD1 and HDAC1 when complexed with the scaffolding protein, co-repressor of RE1 silencing transcriptional factor 1 (CoREST1). I have demonstrated that suH4 stimulates the enzymatic activity of the HDAC1-CoREST1 sub-complex towards both mono- and polyacetylated nucleosomes relative to nucleosome containing wild-type H4 (Chapter 3). This stimulation was dependent on the interaction between SUMO and the intact non-consensus-SUMO interacting motif (ncSIM) within CoREST1. Given the importance of the CoREST1-ncSIM interaction, we have further employed 2D-NMR strategies to map the important interacting residues in SUMO and quantify the binding interaction for the first time. These interactions provide new therapeutic targets in diseases arising from the misregulation of gene function. I have also sought to more completely characterize the substrate specificity of LSD1 and to explore the effect of suH4 on LSD1-CoREST1 sub-complex activity (Chapter 4). My efforts have demonstrated that the acetylation state in H3 tail peptides finely tunes LSD1 activity towards its primary substrate, methylated H3K4 (H3K4me1/2). To corroborate these findings in the context of mononucleosomes and to assess LSD1 kinetic changes imparted by suH4 I have developed a high-throughput 384-well microplate-based demethylation assay platform. This set-up allows conservation of valuable semisynthetic materials and facilitates data collection. Using the microplate platform, I have demonstrated steady-state kinetics of LSD1-CoREST1 toward H3K4me2 and suH4 containing mononucleosomes for the first time. We hope to further delineate subtle contextual effects on the removal of H3K4 methylation by the LSD1-CoREST1 sub-complex.
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.rightsnone
dc.subjectChromatin
dc.subjectEnzymology
dc.subjectPost-translational modifications
dc.subjectSemisynthesis
dc.subjectThioesterification
dc.subjectChemistry
dc.subject.otherChemistry
dc.titleChemical Approaches to Investigate the Biochemical Crosstalk Between Histone Sumoylation, Methylation and Acetylation
dc.typeThesis
dc.embargo.termsOpen Access


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