The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo

Abstract

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we suggest that it may play the same role in meiosis.

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

“…H3K36me3 is specifically enriched at recombination sites in a Spo11-independent manner PRDM9 catalyzes H3K36me3 formation in vitro (Wu et al 2013;Eram et al 2014), and H3K36me3 enrichment is detected in vivo at the center of mouse hotspots (Davies et al 2016;Powers et al 2016). Interestingly, we only detected this PRDM9-dependent H3K36me3 enrichment in class 1 PRDM9 binding sites and not in classes 2A or 2B (Fig.…”

“…3; Buard et al 2009;Grey et al 2011;Smagulova et al 2011;Pratto et al 2014;Davies et al 2016;Powers et al 2016). These studies showed that these modifications are PRDM9 dependent and thus are predicted to be catalyzed by PRDM9.…”

“…Indeed, PRDM9 can methylate histone H3 at K4, K9, and K36 in vitro (Wu et al 2013;Eram et al 2014;Koh-Stenta et al 2014). Like H3K4me3, H3K36me3 also can be associated with nucleosomes adjacent to hotspots (Buard et al 2009;Davies et al 2016;Powers et al 2016). Hotspot-associated H3K4me3 is detected as early as 9 d post-partum (dpp) in mouse testis when spermatocytes begin to enter leptonema, the first stage of meiotic prophase (Grey et al 2011).…”

X-Chromosome Control of Genome-Scale Recombination Rates in House Mice

“…The rate of crossing over differs between species (Dumont and Payseur 2011a; Smukowski and Noor 2011), among individuals (Broman et al 1998; Thomsen et al 2001; Koehler et al 2002; Kong et al 2004, 2008; Sun et al 2004; Borodin et al 2008; Coop et al 2008; Dumont et al 2009; Wong et al 2010; Fledel-Alon et al 2011), and between the sexes (Broman et al 1998; Coop et al 2008; Dumont et al 2009; Kong et al 2010). Although recent investigations have cast light on the molecular mechanisms controlling the fine-scale distribution of recombination hotspots (Baudat et al 2010; Parvanov et al 2010; Billings et al 2013; Baker et al 2014, 2015; Powers et al 2016), the genetic and evolutionary processes that shape genome-scale crossover rates remain poorly understood.…”

Multifunctional Involvement of a C2H2 Zinc Finger Protein (PbZfp) in Malaria Transmission, Histone Modification, and Susceptibility to DNA Damage Response

“…The PRDM9 protein contains a C-terminal module comprised of multiple C 2 H 2 -type zinc fingers (ZnF) which binds the degenerate 13-bp motif that was previously found to be associated with~40% of human hotspots, formation of doublestrand breaks (DSB), and recruitment of the recombination machinery leading to exchange of chromosomal material and DNA repair. Additionally, the ZnF domain (Zfd) in PRDM9 is fused to a SET domain which catalyzes the trimethylation of histone 3 lysine 4 (H3K4) and histone 3 lysine 36 (H3K36) associated with the hotspots (18,19). The SET domain initially characterized in Drosophila is an evolutionarily well-conserved domain associated with many chromosomal proteins and with proteins necessary for histone lysine trimethylation in mammalian cells (20).…”