Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

Abstract

Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11(-/-) mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.

Figure 1

Mouse strains with the Psmb9 initiation site of meiotic recombination active on both, only one or none of the two homologous chromosomes. The Psmb9 hotspot is on mouse chromosome 17. The wm7 haplotype, drawn in orange (or dark grey), is required for Psmb9 activity. Fragments from B10 or B6 origin are in black and that from the A strain is in blue (or light grey). The initiation activity at Psmb9 on each homologue is indicated (+ or −). CO frequencies (percentage of recombinant molecules per total sperm DNA) in B10.AxSGR and R209xB10 are from the study by Baudat and de Massy (2007a). In R209, B6 and RB2 × B10 frequencies are in parentheses as they are predicted based on genetic analyses that showed that the presence of the R209 or RB2 chromosome (Grey et al, 2009) was sufficient to induce Psmb9 initiation activity on both homologues (Shiroishi et al, 1991; Baudat and de Massy, 2007a). A full-colour version of this figure is available at The EMBO Journal Online.

Figure 2

Enrichment for specific histone modifications at the active Psmb9 hotspot. A 4.8 kb region covering the Psmb9 hotspot, defined by high CO rates (cM per kb) is shown together with nine sequence tagged sites (STSs; red bars). Normalized ratios of immunoprecipitated fractions of chromatin between R209 (Psmb9 active) and B6 (Psmb9 inactive) for each STS are shown for elutriated spermatocytes (dark) and for liver cells (light) for different histone post-translational modifications. For H3K9Me3, H4K20Me3 and H3, ratios are from spermatocytes only. The data plotted result from several independent experiments; the experimental values and statistical analysis are shown in Supplementary Figure S1. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 3

Enrichment for specific histone modifications on the initiating chromatid. The enrichments of histone modifications along the hotspot region in B10.AxSGR relative to B6 are shown as in Figure 2, here as black curves. On the right Y-axis, percentage of B10.A allele detected in the immunoprecipitated fractions. Values are shown for each histone modification and for each of four STSs, as vertical bars together with s.d. Student's t-test was used to test the null hypothesis according to which the percentages of B10.A allele in immunoprecipitated fractions do not differ between the 5′ flanking STS (STS 1) and the two STSs inside the hotspot region (STS 8 and 11, treated as a group). A full-colour version of this figure is available at The EMBO Journal Online.

Figure 4

Enrichment for histone modifications in Spo11^−/− mice. Bound fractions, normalized to Psmb9–STS 1 (the 5′ most flanking STS), of chromatin of whole testes cells from R209xB10 Spo11^+/− (red) and R209xB10 Spo11^−/− (green) pre-puberal littermates are shown for nine sequence tagged sites along the Psmb9 hotspot region. Mann–Whitney statistical test was used to determine the statistical significance of the difference between bound fractions of Spo11^+/− and Spo11^−/− mice for STSs included within the CO hotspot region (STSs 6, 7, 8, 11 and 13). A full-colour version of this figure is available at The EMBO Journal Online.

Figure 5

Enrichment for specific histone modifications at the active Hlx1 hotspot. A 8 kb region covering the Hlx1 hotspot, defined by high CO rates (cM per kb) is shown together with seven sequence tagged sites (STSs; red bars). Normalized ratios of immunoprecipitated fractions of H3K4Me3 chromatin between R209xCast (Hlx1 active) and B6 (Hlx1 inactive), and of H4Ac5 chromatin between R209 (Hlx1 active) and B6 are shown for each STS. Normalized ratios of immunoprecipitated fractions for each STS are shown. The data plotted result from several independent experiments; the experimental values statistical analysis are shown in Supplementary Figure S8. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 6

Absence of detectable transcription activity at the Psmb9 hotspot. Northern blot of total RNA (18 μg per lane) prepared from elutriated testes cells of RJ2 and of B10. RJ2 has a shorter wm7 fragment as compared with RB2 but confers the same CO activity at Psmb9 and Hlx1 (Grey et al, 2009). The probes used were PCR products from Psmb9 6F-13R, which includes the hotspot centre, and from Psmb9 15F-18R, which lies outside the hotspot region and includes the most 3′ exon (ex) of the Psmb9 gene. A full-colour version of this figure is available at The EMBO Journal Online.