Abstract
We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5′ and 3′ ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells.
Highlights
We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR)
I-SceI induces a double-strand break (DSB) in one GFP copy (I cassette) that can be repaired from the second copy (II cassette) by homologous recombination (HR), yielding GFP+ clones. 75–90% of the cells are repaired by NHEJ with or without small deletions at the I-SceI site[6,13]
We suggest that de novo DNA methylation at the site of DSB repair stabilizes the H3K9m3 mark, which alters the chromatin structure of the entire GFP gene
Summary
We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). The altered methylation pattern is largely restricted to a segment immediately 3′to the DSB along the direction of transcription Hypermethylation of this tract significantly modifies the local chromatin structure and reduces transcription[8,11]. These data nicely account for the high polymorphism of methylation profiles in cells populations derived from individual somatic tissues[12]. Variations drive or are induced by local DNA methylation These events could generate cells with the same genotype but with various levels of gene expression. Does, the extent and pattern of methylation following repair impart variation of gene expression in cell populations with an identical genotype? Does, the extent and pattern of methylation following repair impart variation of gene expression in cell populations with an identical genotype? We chose to approach these questions in a system in which DNA damage and repair can be controlled temporally and spatially, focusing our attention on local transient as well as permanent changes induced by damage and repair
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