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Authors: Daley, Sara
Advisors: Muir, Tom W.
Contributors: Chemistry Department
Subjects: Biochemistry
Issue Date: 2021
Publisher: Princeton, NJ : Princeton University
Abstract: G9a (also known as EHMT2) is a lysine histone methyltransferase that deposits mono- and dimethylation at lysine 9 of histone H3 (H3K9me1/2). G9a activity has long been linked to transcriptional regulation, as H3K9me1/2 is a histone post-translational modification (PTM) that is considered a hallmark of facultative heterochromatin, i.e. transcriptionally-silenced genomic regions. The C-terminal SET domain of G9a is responsible for catalysis, while a series of embedded ankyrin repeats (ARs) close to the SET domain preferentially bind H3K9me1/2. Given the ability for G9a to recognize and deposit H3K9me1/2, we sought to construct chromatinized substrates to assess how G9a engages with nucleosome(s). In this study, we employed a DNA-barcoded PTM library to profile the substrate scope of G9a and detailed how pre-installed PTMs, such as H3K4me3 and H4 poly-acetylation, can regulate the activity of this enzyme. We also prepared chemically- and spatially-defined nucleosome arrays to define how H3K9me1/2 anchors and positions the enzyme. We further detailed the precise geometric and domain requirements for G9a-deposited H3K9me1/2 spreading using heterotypic tetra and mononucleosomes. As part of this work, we exploited several streamlined methodologies to construct site-specifically modified MegaDalton-sized chromatin complexes with high levels of chemical control. We further attempted to extend the DNA-barcoding platform to develop a high-throughput system to study such G9a spreading. Collectively, our results shed light into how G9a senses the larger chromatin PTM landscape, ultimately leading to the establishment of silenced chromatin states.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog:
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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