Control and Function of Histone H3 Lysine 27 Methylation in Neurospora crassa
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Date
2020-02-27
Authors
McNaught, Kevin
Journal Title
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Publisher
University of Oregon
Abstract
In higher eukaryotes, proper development and maintenance of cellular identity requires the activity of Polycomb repressive complexes (PRCs). PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), which serves as a repressive chromatin mark. PRC2 is conserved in the filamentous fungus, Neurospora crassa, and H3K27 methylation covers around 7% of the genome, repressing scores of genes. My research focused on two basic questions: 1. What controls the distribution of H3K27 methylation? and 2. How does H3K27 methylation function as a repressive mark?
As one approach to identify factors that influence the deposition of H3K27 methylation, I screened gene knock-outs of nst (neurospora sir two) homologues for defects in H3K27 methylation. I discovered that loss of NST-3, a putative H3K56 deacetylase, results in accumulation of H3K27 methylation at centromeres, and that this is suppressed by strains lacking rtt109 or bearing histones with the H3K56R substitution.
In collaboration with Kirsty Jamieson, we investigated what role chromosome position had on the deposition of H3K27 methylation by examining chromosomal rearrangement strains. We identified both position-dependent and position-independent H3K27 methylation. We further found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive H3K27 methylation, and that this is effect is mediated by telomere repeats, (TTAGGG)n.
As a part of a forward-genetics selection for factors necessary for Polycomb silencing, spearheaded by Elizabeth Wiles, I identified two alleles of a gene, NCU04278, that is required for the telomere-dependent H3K27 methylation uncovered in our previous work. Immunoprecipitation followed by mass spectrometry analyses of NCU04278 and known PRC2 members established that NCU04278 is a PRC2 accessory subunit and was therefore designated PAS.
The aforementioned forward genetic selection identified another gene, epr-1 (effector of polycomb repression 1; NCU07505). EPR-1 associates with H3K27 methylation in vivo and in vitro, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway.
This dissertation includes both previously published and unpublished co-authored material.