- In 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 γH2A, 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 γH2A followed by next-generation sequencing
(ChIP-seq). γH2A-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 γH2A localization. In contrast, γH2A is not required for H3K9 methylation or DNA methylation. Normal
γH2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase
DIM-2. γH2A 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.