Heterochromatin Controls γH2A Localization in Neurospora crassa

Abstract

bIn response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called ␥H2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of ␥H2A followed by next-generation sequencing (ChIP-seq). ␥H2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal ␥H2A localization. In contrast, ␥H2A is not required for H3K9 methylation or DNA methylation. Normal ␥H2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. ␥H2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.

Genome-wide redistribution of H3K27me3 is linked to genotoxic stress and defective growth

“…Heterochromatin domains an H3 K9R or an H3 K9Q substitution allele to mimic H3 with unmodified lysine-9 or acetylated lysine-9, respectively (46). Normal H3K27 methylation patterns were abolished in these mutants, similar to the case for Δdim-5 strains (Fig.…”

“…Previous work suggests that abnormal localization of γH2A is not the cause of the MMS-sensitivity phenotype. Although site-specific enrichment of γH2A is lost in the Δdim-5 mutant, this is the result of increased γH2A in euchromatin rather than a decrease in γH2A at heterochromatin (46). Moreover, double mutants of Δdim-5 and hH2A S131A display an additive increase in MMS sensitivity (46), suggesting that loss of DIM-5 and γH2A lead to MMS sensitivity through independent mechanisms.…”

“…Although site-specific enrichment of γH2A is lost in the Δdim-5 mutant, this is the result of increased γH2A in euchromatin rather than a decrease in γH2A at heterochromatin (46). Moreover, double mutants of Δdim-5 and hH2A S131A display an additive increase in MMS sensitivity (46), suggesting that loss of DIM-5 and γH2A lead to MMS sensitivity through independent mechanisms. Hence, it is likely that elevated levels of γH2A are a response to genotoxic stress in Δdim-5 rather than the cause of genotoxic stress.…”

“…We previously showed that γH2A is enriched in heterochromatin domains in wild-type cells and that γH2A is deposited throughout the genome in the Δdim-5 mutant, leading to loss of region-specific enrichment (46). We performed ChIP-seq for γH2A in wild type, Δdim-5, Δset-7, and Δdim-5; Δset-7 double mutants (Fig.…”

“…DCDCdeficient mutants exhibit chromosome segregation defects and hypersensitivity to the DNA-damaging agent methylmethanesulfonate (MMS) (5), and mutants lacking the catalytic subunit of DCDC, defective in methylation-5/kmt1 (dim-5), exhibit increased rates of mitotic recombination (43). In addition, during normal replicative growth, dim-5 strains display induction and redistribution of γH2A, a phosphorylated form of H2A that is induced by DNA damage or DNA replication stress (44)(45)(46). These data suggest that heterochromatin is critically important in Neurospora, but the cause of genotoxic stress and impaired growth in heterochromatin-defective mutants is not understood.…”