Analysis of genome integrity and chromatin architecture in heterochromatin-defective mutants of Neurospora crassa
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Eukaryotic genomes are partitioned into functionally distinctive domains: euchromatin and heterochromatin. Euchromatin corresponds to transcriptionally active domains of chromosomes whereas heterochromatin is transcriptionally inert. Constitutive heterochromatin, typically marked by H3K9me3, is required for normal growth and development in eukaryotes. Previous studies in Drosophila showed that loss of heterochromatin mediated by H3K9 histone methyltransferases leads to genome instability characterized by global chromosomal rearrangements and increased copy number of repeats. Previous research in Neurospora indicates that loss of a component required for H3K9 methylation leads to sensitization of cells to a DNA damaging agent and growth defects. These observations suggest that heterochromatin components are required for normal genome integrity. A major goal of my dissertation has been to elucidate a role of heterochromatin components such as DIM-5 in genome maintenance and to identify the underlying mechanisms by which heterochromatin components ensure proper DNA repair and/or chromosomal replication in Neurospora crassa. In this dissertation, I describe how loss of DIM-5 or other heterochromatin components affect global chromatin architecture. I show evidence that Δdim-5 strains suffer from genotoxic stress presumably caused by stalled DNA replication forks or double-stranded DNA breaks. The endogenous stress activates the DNA damage checkpoint. I also show that Δdim-5 strains form aberrant facultative heterochromatin that leads to spontaneous induction of the DNA damage response and growth defects. I demonstrate that, in Δdim-5, H3K27me3 distribution is completely switched to centromeres and AT-rich repetitive domains where constitutive heterochromatin normally occurs. This suggests that the recruitment process of the PRC2 complex responsible for H3K27me3 has changed in Δdim-5, disrupting global genome architecture. Removal of the PRC2 catalytic subunit set-7 suppresses the MMS-hypersensitive phenotype and partially relieves the growth defects of Δdim-5. Moreover, I demonstrate that Δdim-5 exhibits genetic interactions with Δmi-2 and Δcrf-6. These findings show in greater detail that heterochromatin is required for genome integrity and defects in heterochromatin affect the global landscape of chromatin components and chromatin modification. This dissertation broadens our perspective of heterochromatin function and its importance in cell growth and development in eukaryotes.