Affinity-seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage

Abstract

BackgroundGenetic recombination plays an important role in evolution, facilitating the creation of new, favorable combinations of alleles and the removal of deleterious mutations by unlinking them from surrounding sequences. In most mammals, the placement of genetic crossovers is determined by the binding of PRDM9, a highly polymorphic protein with a long zinc finger array, to its cognate binding sites. It is one of over 800 genes encoding proteins with zinc finger domains in the human genome.ResultsWe report a novel technique, Affinity-seq, that for the first time identifies both the genome-wide binding sites of DNA-binding proteins and quantitates their relative affinities. We have applied this in vitro technique to PRDM9, the zinc-finger protein that activates genetic recombination, obtaining new information on the regulation of hotspots, whose locations and activities determine the recombination landscape. We identified 31,770 binding sites in the mouse genome for the PRDM9Dom2 variant. Comparing these results with hotspot usage in vivo, we find that less than half of potential PRDM9 binding sites are utilized in vivo. We show that hotspot usage is increased in actively transcribed genes and decreased in genomic regions containing H3K9me2/3 histone marks or bound to the nuclear lamina.ConclusionsThese results show that a major factor determining whether a binding site will become an active hotspot and what its activity will be are constraints imposed by prior chromatin modifications on the ability of PRDM9 to bind to DNA in vivo. These constraints lead to the presence of long genomic regions depleted of recombination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0024-6) contains supplementary material, which is available to authorized users.

HELLS and PRDM9 form a Pioneer Complex to Open Chromatin at Meiotic Recombination Hotspots

“…Both of these features are required for meiotic recombination (Diagouraga et al, 2018;Parvanov et al, 2017). While the number of recombination hotspots are generally depleted from repressive regions of chromatin marked by H3K9me2/me3 (Patel et al, 2019;Walker et al, 2015), hotspots that are found in these domains show nearly similar level of PRDM9-dependent histone modification as those found outside of H3K9me2/3 domains, supporting the idea that even within heterochromatin, PRDM9 and HELLS have the capacity to create open chromatin. Further evidence for a pioneer-factor role for PRDM9 comes from our ex vivo analyses.…”

Genomic and chromatin features shaping meiotic double-strand break formation and repair in mice

“…1F). This difference is partially due to higher background signal from transcription-dependent H3K36me3: when we omitted hotpots in genes previously shown to be transcribed in juvenile testes 33 (where meiotic cells are enriched because of semi-synchronous spermatogenesis), the correlation increased to ∼33% (data not shown). By multiple linear regression, combining data for both histone marks for all hotspots gave a significant but quantitatively small improvement over a model with H3K4me3 alone for predicting SPO11-oligo counts (R 2 = 0.44; p < 2.2 × 10 −16 , ANOVA; Table S1).…”

“…Pre-processing of these data was described previously 26 . H3K36me3 data (GSE76416) 32 and H3K9me2/3 and RNA-seq data (GSE61613) 33 were converted to mouse genome assembly GRCm38/mm10 sequence coordinates using the UCSC Genome Browser LiftOver tool (http://genome.ucsc.edu/cgi-bin/hgLiftOver). …”

Diverse roles for CDK‐associated activity during spermatogenesis

“…SPO11‐mediated DSB formation occurs preferentially within so‐called ‘meiotic hotspots’ or ‘recombination hot regions’ of the genome. The biased nature of this process is enforced by epigenetic modifications placed proximal to the incipient break site . These alterations are thought to create a preferential environment for the recombination machinery and are typically directed toward euchromatic regions depleted of nucleosomes .…”

“…The biased nature of this process is enforced by epigenetic modifications placed proximal to the incipient break site . These alterations are thought to create a preferential environment for the recombination machinery and are typically directed toward euchromatic regions depleted of nucleosomes . Meiotic DSB formation results in the induction of approximately 150 and 200–300 DSBs during human and murine meiosis, respectively .…”