Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A

Repair of DNA double-strand breaks (DSBs) must be properly organized within diverse nuclear chromatin regions to maintain genome stability. Euchromatin and heterochromatin display major differences in histone modifications, biophysical properties and spatiotemporal dynamics of DSBs. However, whether differential histone modifying activities are required to promote DSB repair in these two distinct domains remains unknown. Using locus-specific single DSB induction in Drosophila melanogaster animal tissue, as well as CRISPR/Cas9 and radiation-induced DSBs in cultured cells, we find that the histone demethylase dKDM4A is specifically required for timely DSB repair and pathway choice in heterochromatin, but not euchromatin. Sequencing of DSB repair products, live analysis of Homologous Recombination (HR) repair protein recruitment, as well as synthetic lethality assays reveal that loss of dKDM4A results in a heterochromatin-specific increase in HR repair. In addition, we find that dKDM4A promotes the demethylation of heterochromatin-associated histone marks upon DSB induction in heterochromatin, but not euchromatin. Our results demonstrate that heterochromatin depends on DSB induced dKDM4A-dependent chromatin changes to promote proper repair and -pathway choice.