PRDM9 binding organizes hotspot nucleosomes and limits Holliday junction migration

Abstract

In mammals, genetic recombination during meiosis is limited to a set of 1-to 2-kb regions termed hotspots. Their locations are predominantly determined by the zinc finger protein PRDM9, which binds to DNA in hotspots and subsequently uses its SET domain to locally trimethylate histone H3 at lysine 4 (H3K4me3). This sets the stage for double-strand break (DSB) formation and reciprocal exchange of DNA between chromatids, forming Holliday junctions. Here we report genomewide analyses of PRDM9-dependent histone modifications using two inbred mouse strains differing only in their PRDM9 zinc finger domain. We show that PRDM9 binding actively reorganizes nucleosomes into a symmetrical pattern, creating an extended nucleosome-depleted region. These regions are centered by a consensus PRDM9 binding motif, whose location and identity was confirmed in vitro. We also show that DSBs are centered over the PRDM9 binding motif within the nucleosome-depleted region. Combining these results with data from genetic crosses, we find that crossing-over is restricted to the region marked by H3K4me3. We suggest that PRDM9-modified nucleosomes create a permissible environment that first directs the location of DSBs and then defines the boundaries of Holliday junction branch migration.[Supplemental material is available for this article.]

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

“…1C) and significantly overlapped with regulatory elements in the genome (∼60% of common H3K4me3 peaks overlapped with annotated transcription start sites or testes-specific enhancers). Strain-specific H3K4me3 peaks (10,835 for B6 and 24,185 for RJ2) mirrored the site-specific PRDM9 methyltransferase activity, as previously reported Baker et al 2014). The difference in the number of H3K4me3 and DMC1 peaks, compared with PRDM9, could be due to various reasons, for instance, the ChIP assay sensitivity or the shorter half-life of PRDM9 association with its binding sites compared with that of DMC1 or H3K4me3.…”

“…Cst allele in the C57BL/6 background (Baker et al 2014). Therefore, we mapped DMC1 and H3K4me3 in the B6 and RJ2 strains by ChIP-seq experiments (Supplemental Table S1).…”

“…Cst allele (Baker et al 2014). Therefore, we asked whether consensus motifs were present in each PRDM9 binding site class.…”

DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination

“…In wild-type testes, we were able to detect high H3K4me3 and DMC1 enrichment at five C57Bl6/Jassociated hot spots and poor enrichment at a C57Bl6/Jassociated cold spot ( Fig. 4D; Buard et al 2009;Baker et al 2014;B De Massy, unpubl.). In Dnmt3L −/− testes, low levels were maintained at the investigated cold spot, suggesting that ectopic H3K4me3 and DMC1 enrichment does not occur globally throughout the genome during Dnmt3L −/− meiosis and could be specific to TE sequences.…”

Identification of a QTL in Mus musculus for Alcohol Preference, Withdrawal, and Ap3m2 Expression Using Integrative Functional Genomics and Precision Genetics

“…Chromatin was sheared to mononucleosomes using micrococcal nuclease digestion. Briefly, four ChIP reactions from a single preparation of spermatocyte chromatin were pooled after final DNA elution, concentrated using Agencourt AMPure XP beads (Beckman Coulter), and quantitated using the Qubit double-stranded DNA HS assay (Life Technologies) (as described by Baker et al 2014). Approximately 7-10 ng of ChIP DNA was used for the sequencing library preparation.…”