Investigations of Novel Mechanisms of Epigenetic Regulation on Chromatin
Date of Award
Doctor of Philosophy (PhD)
The epigenetic regulation of gene expression, mediated by chromatin, is a critical component of eukaryotic cellular function. Chromatin regulates gene expression through a variety of mechanisms including histone post-translational modifications, chromatin remodeling, and recruitment of non-coding RNAs. Together, the chromatin states enable cells to organize the genome and efficiently carry out cellular functions. Given the implications of chromatin regulation in normal, developmental, and disease states, an understanding of the mechanisms of chromatin regulation is of critical importance. In this dissertation, I describe three projects towards furthering the field’s understanding of the mechanisms of chromatin regulation.First, I propose Kacme, a novel histone post-translational modification (PTM) that incorporates lysine methylation and lysine acetylation, two of the most important histone PTMs. I synthesized peptides containing this PTM and used them to raise a specific antibody. I then show that this modification is present largely on the histone H4 tail at residues K5 and K12, and is disrupted by changes is cellular acetylation and methylation pathways. Kacme is found in all cell types and tissues tested and appears to be found specifically on histone H3 in mouse testes. On chromatin, Kacme is associated with features of active transcription and shows broad overlaps with H4Kac, with some significant differences. Under stress conditions, a subset of genes show changes in Kacme occupancy that are not mirrored by H4Kac. These genes may represent targets of unique Kacme regulation. Next, I performed chromatin occupancy experiments on mammalian epigenetic factors that have been implicated in SARS-CoV-2 infection. I found that while the chromatin remodeling complexes mSWI/SNF and PRC2 target many genomic regions, they do not appear to directly target genes known to be associated with viral entry. On the other hand, I helped to confirm that HMGB1 is a novel regulator of the critical SARS-CoV-2 entry factor ACE2 and is a promising target for further exploration of host regulation of factors involved in viral pathogenesis. Finally, I describe a novel RNA chemical probing method aimed at detecting non-coding RNAs that may carry out biochemical functions in cells, especially on chromatin. I demonstrate the first chiral chemical probing reagent and show that this method identifies small but reproducible differences between structured and unstructured RNAs. The regulation of gene expression by chromatin is an extremely active field of study. The investigations shown here provide significant advances in our understanding of histone PTMs and host epigenetic factors during infection, and work towards developing ways to detect RNA regulation on chromatin.
Lu-Culligan, William J., "Investigations of Novel Mechanisms of Epigenetic Regulation on Chromatin" (2021). Yale Graduate School of Arts and Sciences Dissertations. 151.