The histone modification reader ZCWPW1 links histone methylation to PRDM9-induced double-strand break repair

Abstract

The histone modification writer Prdm9 has been shown to deposit H3K4me3 and H3K36me3 at future double-strand break (DSB) sites during the very early stages of meiosis, but the reader of these marks remains unclear. Here, we demonstrate that Zcwpw1 is an H3K4me3 reader that is required for DSB repair and synapsis in mouse testes. We generated H3K4me3 reader-dead Zcwpw1 mutant mice and found that their spermatocytes were arrested at the pachytene-like stage, which phenocopies the Zcwpw1 knock–out mice. Based on various ChIP-seq and immunofluorescence analyses using several mutants, we found that Zcwpw1's occupancy on chromatin is strongly promoted by the histone-modification activity of PRDM9. Zcwpw1 localizes to DMC1-labelled hotspots in a largely Prdm9-dependent manner, where it facilitates completion of synapsis by mediating the DSB repair process. In sum, our study demonstrates the function of ZCWPW1 that acts as part of the selection system for epigenetics-based recombination hotspots in mammals.

ZCWPW1 is recruited to recombination hotspots by PRDM9 and is essential for meiotic double strand break repair

“…We conclude that H3K4me3 alone endows only relatively weak binding, while PRDM9 is therefore able to recruit ZCWPW1 with a much greater efficiency than sites marked by H3K4me3 alone, suggesting that both histone modifications might aid efficient binding. While the increase in ZCWPW1 enrichment at sites with pre-existing H3K4me3 is consistent with both marks contributing to recruitment, we have no evidence that these weaker binding sites serve a functional role, and indeed concurrent investigations did not find strong evidence of ZCWPW1-driven transcriptional changes ( Huang et al, 2020 ).…”

“…In parallel with this work, two other studies of ZCWPW1 Huang et al, 2020 ; Mahgoub et al, 2020 have performed complementary analyses, and together with our own results allow some clear conclusions to be drawn. We mapped positions of ZCWPW1 recruitment (of the human protein) in mitotic HEK293T cells, in the presence of human or chimpanzee PRDM9 and compared to assayed peaks for PRDM9, H3K4me3 and H3K36me3 ( Altemose et al, 2017 ).…”

“…Like DMC1, RPA2 levels were also significantly elevated in the Zcwpw1 −/− mouse from zygotene onwards ( Figure 4—figure supplement 2 ). In Zcwpw1 −/− males, like in the Prdm9 −/− mutant, DSBs form and recruit RPA2, RAD51 and DMC1 in similar numbers ( Huang et al, 2020 ; Li et al, 2019a ; Mahgoub et al, 2020 ), but fail to repair efficiently, accompanied by asynapsis and meiotic arrest at pseudo-pachytene. Indeed, we observe late unrepaired DMC1 foci mainly on HORMAD2-positive asynapsed chromosomes ( Figure 4—figure supplement 3 ).…”

PRDM9 activity depends on HELLS and promotes local 5-hydroxymethylcytosine enrichment

“…As no evidence of HELLS/TET interaction in meiotic cells is available, a PRDM9-dependent chromatin modification might be implicated in recruiting the putative TET activity. ZCWPW1, a reader of H3K4me3 and H3K36me3 that is required for efficient DSB repair ( Huang et al, 2020 ; Mahgoub et al, 2020 ; Wells et al, 2019 ), might be directly or indirectly involved in this recruitment.…”

“…In addition, these histone modifications may play a role in DSB repair. Indeed, ZCWPW1, a protein reader of H3K4me3 and H3K36me3, is required for efficient DSB repair ( Huang et al, 2020 ; Li et al, 2019 ; Mahgoub et al, 2020 ; Wells et al, 2019 ). Whatever their exact role, the presence of both histone modifications leads to a unique chromatin landscape at PRDM9 sites that is not present at promoters, where H3K36me3 is depleted ( Grey et al, 2017 ; Lam et al, 2019 ; Powers et al, 2016 ).…”