Heterochromatic repeat clustering imposes a physical barrier on homologous recombination to prevent chromosomal translocations.

Mouse pericentromeric DNA is composed of tandem major satellite repeats, which are heterochromatinized and cluster together to form chromocenters. These clusters are refractory to DNA repair through homologous recombination (HR). The mechanisms by which pericentromeric heterochromatin imposes a barrier on HR and the implications of repeat clustering are unknown. Here, we compare the spatial recruitment of HR factors upon double-stranded DNA breaks (DSBs) induced in human and mouse pericentromeric heterochromatin, which differ in their capacity to form clusters. We show that while DSBs increase the accessibility of human pericentromeric heterochromatin by disrupting HP1α dimerization, mouse pericentromeric heterochromatin repeat clustering imposes a physical barrier that requires many layers of de-compaction to be accessed. Our results support a model in which the 3D organization of heterochromatin dictates the spatial activation of DNA repair pathways and is key to preventing the activation of HR within clustered repeats and the onset of chromosomal translocations.

CRISPR/Cas9-Induced Breaks in Heterochromatin, Visualized by Immunofluorescence.

CRISPR/Cas9 technology can be used to investigate how double-strand breaks (DSBs) occurring in constitutive heterochromatin are getting repaired. This technology can be used to induce specific breaks on mouse pericentromeric heterochromatin, by using a guide RNA specific for the major satellite repeats and co-expressing it with Cas9. Those clean DSBs can be visualized later by confocal microscopy. More specifically, immunofluorescence can be used to visualize the main factors of each DSB repair pathway and quantify their percentage and pattern of recruitment at the heterochromatic region.

How to maintain the genome in nuclear space.

Genomic instability can be life-threatening. The fine balance between error-free and mutagenic DNA repair pathways is essential for maintaining genome integrity. Recent advances in DNA double-strand break induction and detection techniques have allowed the investigation of DNA damage and repair in the context of the highly complex nuclear structure. These studies have revealed that the 3D genome folding, nuclear compartmentalization and cytoskeletal components control the spatial distribution of DNA lesions within the nuclear space and dictate their mode of repair.