Distinct histone modifications define initiation and repair of meiotic recombination in the mouse
2009; Springer Nature; Volume: 28; Issue: 17 Linguagem: Inglês
10.1038/emboj.2009.207
ISSN1460-2075
AutoresJérôme Buard, Pauline Barthès, Corinne Grey, Bernard de Massy,
Tópico(s)RNA Research and Splicing
ResumoArticle30 July 2009free access Distinct histone modifications define initiation and repair of meiotic recombination in the mouse Jérôme Buard Corresponding Author Jérôme Buard Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Pauline Barthès Pauline Barthès Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Corinne Grey Corinne Grey Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Bernard de Massy Corresponding Author Bernard de Massy Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Jérôme Buard Corresponding Author Jérôme Buard Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Pauline Barthès Pauline Barthès Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Corinne Grey Corinne Grey Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Bernard de Massy Corresponding Author Bernard de Massy Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France Search for more papers by this author Author Information Jérôme Buard 1,‡, Pauline Barthès1,‡, Corinne Grey1 and Bernard de Massy 1 1Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France ‡These authors contributed equally to this work *Corresponding authors. Institut de Génétique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, Montpellier cedex 5, 34396, France. Tel.: +33 04 9961 9972; Fax: +33 04 9961 9901; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2009)28:2616-2624https://doi.org/10.1038/emboj.2009.207 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction During meiosis, reciprocal homologous recombination events or crossovers (COs) constitute physical connections required for the proper segregation of homologous chromosomes at the first meiotic division (Petronczki et al, 2003). In addition, COs increase genome diversity by reshuffling alleles over generations. COs result from a complex process of DNA double-strand break (DSB) formation and repair (Hunter, 2007). The induced DSBs are repaired by homologous recombination using alternative pathways. The repair using the homologous chromatid as a template and associated with a reciprocal exchange leads to CO. Alternatively, the repair can lead to a non-reciprocal exchange of genetic information, a gene conversion event, without CO (Baudat and de Massy, 2007b). The initiation step of meiotic recombination, DSB formation, is catalyzed by the evolutionarily conserved Spo11 protein (Keeney and Neale, 2006). Spo11 is homologous to the catalytic subunit of the TopoVI family of type II DNA topoisomerases (Bergerat et al, 1997). Primarily on the basis of yeast studies, DSB formation has been shown to require several other proteins, the roles of which remain to be determined. DSBs are not randomly distributed in the genome and occur preferentially in regions called hotspots (Petes, 2001). How DSB frequency and position are determined is not known. In Saccharomyces cerevisiae, most DSBs occur in intergenic regions adjacent to transcription promoters (Baudat and Nicolas, 1997; Gerton et al, 2000). DSB frequency varies greatly from one promoter region to another according to undefined rules. However, transcriptional activity is not required for DSB formation. In Schizosaccharomyces pombe, DSBs occur preferentially in large intergenic regions, and with a positive correlation with the presence of non-coding RNA loci (Cromie et al, 2007; Wahls et al, 2008). On the basis of DSB mapping, at the nucleotide resolution, in S. cerevisiae, Spo11 seems to have no or little DNA sequence specificity (Liu et al, 1995; de Massy et al, 1995; Diaz et al, 2002). Several analyses show that chromatin accessibility is one important feature of initiation sites in S. cerevisiae and S. pombe (Lichten, 2008) and DSBs occur preferentially in open chromatin (Ohta et al, 1994; Wu and Lichten, 1994). In addition, acetylations of histone H3 and H4 and trimethylation of H3 K4 have been, respectively, detected at DSB sites either by local analysis at individual sites in S. pombe (Yamada et al, 2004) or, more recently, by genome-wide analysis in S. cerevisiae (Borde et al, 2009). Several mutations in enzymes involved in histone post-translational modifications (HPTMs) have also been shown to lead to a decrease or increase of DSB activity, suggesting a role for histone modifications in initiation activity (Sollier et al, 2004; Yamashita et al, 2004; Mieczkowski et al, 2007; Hirota et al, 2008; Merker et al, 2008). In mammals, DSBs have not been directly mapped, but the molecular analysis of recombinant molecules at several meiotic CO hotspots has shown that CO hotspots are preferred initiation sites (Buard and de Massy, 2007). High-resolution mapping of CO by direct analysis of recombinant molecules at individual hotspots and by genome-wide population diversity analysis shows several striking properties of hotspot distribution in the human genome that seem to be also found in mice. COs cluster within ∼2 kb-wide regions and are spaced on average every 50–100 kb (Myers et al, 2005). In the human genome, around 25 000 CO hotspots have been mapped, with activities varying over three orders of magnitude. These hotspots are mostly located outside of genes and a substantial fraction has been found to contain a degenerated 13-mer motif (Myers et al, 2008). In addition to the potential role for a specific DNA sequence or a sequence family, chromatin features have been invoked to explain DNA sequence-independent variations in recombination rates, such as those observed between male and female meiosis for instance. The recent high-resolution mapping of male and female hotspots in mice shows indeed that several hotspots have different activities in male and female meiosis (Paigen et al, 2008). We sought to directly investigate whether HPTMs participate in defining recombination hotspots in mammals by taking advantage of the features and extensive characterization of the mouse hotspot Psmb9 (Shiroishi et al, 1991; Guillon and de Massy, 2002; Guillon et al, 2005; Baudat and de Massy, 2007a). Recombination events occur at a frequency 2000-fold higher than the genome average at this hotspot and spread over 1.5 kb with the highest density in a central 210-bp region. Depending on the presence of a region of chromosome 17 from the M. m. molossinus wm7 haplotype, hybrid mice can have active initiation at Psmb9 on both, only one or none of the homologues (Baudat and de Massy, 2007a). We first determined the level of enrichment of several HPTMs at Psmb9 and in flanking regions in strains carrying active or inactive Psmb9 hotspot in meiotic and somatic cells. We then determined whether the enriched HPTMs could be a cause or a consequence of recombination activity using two complementary approaches. First, taking advantage of single nucleotide polymorphisms (SNPs), we were able to determine whether in hybrids, in which only one homologue initiates, the HPTMs are enriched on both or only one homologue. Second, we tested the enrichment of HPTMs in Spo11−/− mice deficient for the initiation of meiotic recombination (Baudat et al, 2000; Romanienko and Camerini-Otero, 2000; Mahadevaiah et al, 2001). We thus show that H3K4Me3 is specifically enriched in the chromatid that carries an active initiation site and independently from DSB formation by Spo11. Instead, H4 hyperacetylation is a chromatin modification that appears after DSB formation, as shown by its Spo11 dependence. Comparison between Psmb9 and Hlx1, a hotspot on chromosome 1, shows distinct chromatin features and two common properties: the enrichment of H3K4Me3 and of H4 hyperacetylation. Interestingly, no transcript could be detected in the Psmb9 region and the low level of transcript detected at Hlx1 does not vary with H3K4Me3 levels. Therefore, the H3K4Me3 enrichment we detected at hotspots does not correlate with detectable transcriptional activity. Altogether, these histone modifications do highlight new features of the chromatin in intergenic regions of the mouse genome and provide a basis for understanding the distribution of meiotic recombination in eukaryotes. Results Distinct HPTMs at active or inactive Psmb9 hotspot HPTMs were measured at Psmb9 and in flanking regions in strains carrying recombinationally active (R209) and inactive (B6) Psmb9 alleles, in meiotic and somatic cells. Native chromatin from elutriated pachytene spermatocytes or from liver cells was immunoprecipitated with antibodies raised against a series of HPTMs. Immunoprecipitated DNA was quantified using real-time PCR (see Materials and methods). We observed significant enrichments for a set of HPTMs in pachytene spermatocytes from a strain carrying an active Psmb9 hotspot (R209) relative to the recombinationally inactive B6 strain (Figure 1). The enrichment was restricted to the 1.5-kb hotspot region for H3K4Me3, H3K4Me2, H3K9Ac, hyperacetylated H4 and H3K27Me1 (Figure 2). For all five modifications, the enrichment was statistically significant (Supplementary Figure S1). In contrast, there was no difference for these modifications in somatic cells (liver) between the two strains. Conversely, H3K27Me3 and H3K9Me3 were significantly enriched in B6 relative to R209 in spermatocytes (Figure 2 and Supplementary Figure S1). No difference for total histone H3 could be detected between the two strains within the hotspot region, suggesting a similar density of nucleosomes. In addition, micrococcal nuclease digests of chromatin prepared from a 1:1 mix of spermatocytes from the two strains did not show any difference in sensitivity between the B6 and R209 alleles (Supplementary Figure S2), consistent with a previous analysis of DNase I hypersensitivity at this hotspot (Mizuno et al, 1996). 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint Strains R209 and B6 not only differ in the activity of the Psmb9 hotspot but also in DNA sequence all along the most proximal 33-Mb region of mouse chromosome 17, corresponding to the wm7 haplotype, and including SNPs within the hotspot region. We have recently isolated the congenic strain RB2, which possesses a shorter fragment of the wm7 haplotype than R209, located at least 10-Mb away from the hotspot and still conferring high recombination activity on the Psmb9 hotspot (Grey et al, 2009). The RB2 × B10 hybrid has, therefore, a local DNA sequence identical to B6 at and around Psmb9 and a hotspot predicted as active as in R209 (Figure 1). We observed a similar HPTM pattern, both qualitatively and quantitatively, in RB2 × B10 hybrid mice and in R209 mice (Supplementary Figure S3), showing that the local DNA sequence is not the determinant for the HPTM pattern at the hotspot. Distinction between HPTMs that precede from those that follow DSB recombinational repair Our observations raised the question whether the different levels of histone modifications were a cause or a consequence of recombination activity. If some HPTMs contribute to determine initiation site activity, two predictions can be made: (1) in hybrids in which initiation takes place on only one homologue, these HPTMs should be specifically enriched on the chromatid that initiates; (2) these HPTMs should be present at similar levels in Spo11−/− and Spo11+/− meiotic cells. To address the first issue, we took advantage of the B10.AxSGR hybrid, in which initiation takes place only on the B10.A chromosome (Figure 1). To determine on which homologue the HPTMs occur, we added an allele-specific quantification step to native ChIP and Q-PCR. Strikingly, within the central region of the hotspot, the enrichment was significantly biased towards the actively initiating B10.A allele for H3K4Me3, H3K4Me2, H3K9Ac and H3K27Me1, and towards the inactive SGR allele for H3K27Me3 (Figure 3). In contrast, there was no bias for hyperacetylated H4. The bound chromatin for sequences outside and on either side of the hotspot showed no bias towards one allele versus the other, neither did unbound chromatin within the central region of the hotspot (Supplementary Figure S4). These data show that the combination of HPTMs found enriched at the active hotspot is preferentially associated with the allele initiating meiotic recombination, except for hyperacetylated H4. Second, the analysis of chromatin from testes of 15-day-old Spo11+/− and Spo11−/− mice showed that two modifications enriched at the centre of the hotspot, H3K4Me3 and H3K9Ac, are enriched to similar levels in the presence or absence of DSB (Figure 4). This strongly suggests that these modifications are part of the substrate for recombination initiation at Psmb9. This interpretation implies that these modifications are maintained at least from the time of initiation (leptotene) to the pachytene stage. We further directly confirmed the presence of H3K4Me3 at the active hotspot during early stages of meiotic prophase I, in pre-puberal mice testes (Supplementary Figure S5) containing spermatocytes almost exclusively at leptonema and zygonema stages (Supplementary Table S3). Whether H3K4Me2 also marks the initiation site could not be definitively answered given that the enrichment was slightly lower in Spo11−/− compared with Spo11+/−, with a difference at the limit of significance (P=0.06). Interestingly, a significantly lower level of H4 hyperacetylation was detected in Spo11−/− as compared with Spo11+/− mice, suggesting that this modification is enriched upon DSB repair, consistent with the allele-specific analysis described above showing equal levels of H4 acetylation on both chromatids. Other modifications, such as H3K27Me1 and H3K27Me3, show little variation along the hotspot region, possibly as a result of the heterogeneity of the cell population in these analyses. Global analysis of the different HPTMs in meiotic prophase nuclei by immunostaining showed highly dynamic processes that probably reflect different DNA-related events, including in particular transcription and recombination (Supplementary Figure S6). This analysis essentially indicates that H3K4Me3 and H3K9Ac are present at the stage of DSB formation (leptonema), even in the absence of SPO11, and that H4 hyperacetylation is detected at all stages during meiotic prophase, including the stage of DSB repair (zygonema and pachynema). 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint H3K4Me3 and hyperacetylated H4 enrichment at the Hlx1 hotspot To test the generality of our finding at the Psmb9 hotspot, we monitored HPTMs at the Hlx1 hotspot on chromosome 1, which is specifically active in hybrids between B6 and Cast/Eij (Paigen et al, 2008; Parvanov et al, 2009). We observed a 2.5% sperm CO rate at Hlx1 in an R209 × Cast/Eij hybrid consistent with the CO pedigree analysis in B6xCast/Eij by Paigen et al. (Supplementary Figure S7). Similar to Psmb9, specific enrichment of H3K4Me3 and hyperacetylated H4 was observed at the active Hlx1 hotspot (Figure 5 and Supplementary Figure S8). Interestingly, H3K9Ac was not enriched and H3K27Me1 was even depleted at the active Hlx1 hotspot, suggesting distinct features between hotspots. The functional role for H3K4Me3 with respect to initiation activity at Hlx1 was further supported by the enrichment of this mark in Spo11−/− mice, at a level similar to that observed in Spo11+/− littermates (Supplementary Figure S9). 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. Download figure Download PowerPoint Absence of correlation between recombination and transcription activities Several histone modifications (H3K4Me3, H3K4Me2, H3K9Ac and H4 hyperacetylation) found enriched at the active Psmb9 hotspot are known for their association with transcription and are found at promoters (Barski et al, 2007; Kouzarides, 2007; Li et al, 2007; Mikkelsen et al, 2007). This raises the question whether transcription occurs at the Psmb9 hotspot. We first verified that in spermatocytes, promoters from expressed or repressed genes carry the expected HPTMs described in somatic cells. We therefore analysed the enrichment of HPTMs at the well-characterized promoter of Sycp1 (Sage et al, 1999), a meiosis-specific gene expressed during meiotic prophase and at the promoter of Nestin, a neural marker gene (Burgold et al, 2008). Consistent with previous analysis of histone modifications, H3K4Me3, H3K4Me2, H3K9Ac and hyperacetylated H4 were present at the Sycp1 promoter and H3K27Me3 was enriched at the Nestin promoter. All modifications were detected at levels higher than those detected at the centre of the Psmb9 hotspot (Supplementary Figure S10). We then directly monitored transcription activity in spermatocytes at the Psmb9 and Hlx1 hotspots either when active or when inactive. No transcript was detected along the Psmb9 region both by northern blot (Figure 6) and by random primed RT–qPCR (data not shown). A weak transcriptional activity was detected at the Hlx1 hotspot by northern blot and at equal levels in strains in which Hlx1 was active or not, as determined by RT–qPCR (around 1% of the actin level, Supplementary Figure S11), and therefore showed no correlation with the enrichment of H3K4Me3 described in Figure 5. 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. Download figure Download PowerPoint Discussion We show here that a set of histone modifications mark the activity of mouse meiotic recombination hotspots, and we further tease apart marks that are associated with the initiation of recombination from post-repair modifications using two complementary approaches. With respect to DSB repair, acetylation of H4 seems to result from meiotic DSB formation at the Psmb9 hotspot and is also associated with Hlx1 hotspot activity, an observation consistent with H4 acetylation by the Tip60/NuA4 protein as a response to phosphorylation of H2AX and accompanying DSB repair in somatic cells (van Attikum and Gasser, 2005; Altaf et al, 2007). This modification is the first identified chromatin signature of recombinational repair at mammalian hotspots. We note that, albeit highly statistically significant, the level of enrichment of H4 acetylation is low when the hotspots are active, which could be because of the relatively low frequency of DSB repair events at the hotspots tested. With respect to initiation, our results suggest that a specific chromatin organization contributes to the definition of initiation sites for meiotic recombination in mice: From the analysis of the two hotspots, Psmb9 and Hlx1, we have identified one common feature associated with initiation, the enrichment of H3K4Me3 at active hotspots. This modification is also enriched in Spo11−/− mice, as expected for a mark that contributes to hotspot activity. This interpretation is strongly supported by the specific enrichment of H3K4Me3 on the initiating chromatid in heterozygous hybrids in which only one chromatid initiates. This modification is the first identified chromatin signature of initiation of recombination at hotspots in mammals. It is interesting to note that this modification is well known to be enriched at transcriptional promoters (Barski et al, 2007; Mikkelsen et al, 2007), and was recently found to also mark transcription start sites of long non-coding transcripts (Guttman et al, 2009). In the Psmb9 region, we did not detect any transcript by northern blot hybridization and RT–PCR. At the Hlx1 hotspot, a weak transcriptional activity is detected, which might be associated with the expression of the gene located next to the hotspot, encoding for a ribosomal protein. However, the level of transcription is not different in strains in which Hlx1 hotspot is active or not, whereas the level of H3K4Me3 differs by a factor of 5. One cannot formally exclude that small-size, low-abundance or unstable RNAs have escaped our detection. In any case, given the human CO map, which shows that most hotspots do not localize near transcription promoters (Myers et al, 2005), one expects H3K4Me3 not to be sufficient to promote hotspot activity, and that other chromatin features are most probably involved in hotspot localization. Furthermore, the chromatin configuration might slightly differ between hotspots as suggested by our comparative analysis between Psmb9 and Hlx1, in which H3K27Me1 is enriched in the former only. A large number of H3K4 methyltransferases containing a SET domain has been described in mammals (Ruthenburg et al, 2007; Shilatifard, 2008). One of them, Meisetz (Prdm9), is specifically expressed in meiotic cells and has an essential function during meiotic prophase (Hayashi et al, 2005). Interestingly, we observed strong variations of the global level of H3K4Me3 in prophase nuclei with a peak at the leptonema/zygonema stages (Supplementary Figure S6). In Meisetz−/− spermatocytes, the level of H3K4Me3 is reduced compared with wild type, but is still higher than that in somatic mutant cells (Hayashi et al, 2005). In both male and female Meisetz−/− mice, gametogenesis is disrupted at the pachytene stage associated with a defect in pairing and DSB repair. One interpretation for this is that these phenotypes are due to an altered expression of one or several genes (Hayashi and Matsui, 2006). No difference between wild type and Meistez−/− testis was detected at the level of initiation of recombination, as measured by γH2AX staining. However, given the residual H3K4Me3 and the limits of immunofluorescence, it is possible that changes in the level or localization of DSB events have been undetected by this analysis. The hypothesis that the enrichment of H3K4Me3 we detected at Psmb9 and Hlx1 results from Prdm9 activity is fully consistent with the observation that the Prdm9 gene lies within the recently described critical genomic region, defined as Dsbc1. This region has been shown to contain a locus involved in genome-wide control of the distribution of recombination and hotspot activity (Grey et al, 2009; Parvanov et al, 2009). Interestingly, in S. cerevisiae, H3K4Me3 is enriched at DSB sites before DSB formation. An analysis of the Set1 mutant, the only H3K4 methyltransferase in S. cerevisiae, shows a general decrease of DSB levels (Borde et al, 2009). These observations suggest that H3K4Me3 might be an evolutionarily conserved feature of meiotic initiation sites. The analysis of set1Δ yeast also shows a minority of sites in which DSB levels are not reduced, suggesting that DSB can occur in the absence of H3K4Me3. Either in yeast or mice, the question of what directs H3K4 methyltransferase activity to specific sites in meiotic cells remains to be answered, as well as the role of this modification in generating a substrate for the protein complex generating DSB. Whatever the mechanism, an epigenetic control of hotspot localization could provide a flexible regulation of meiotic recombination and contribute to the surprisingly marked variations of hotspot activities described, for instance, between individuals in the human population (Neumann and Jeffreys, 2006; Coop et al, 2008). Materials and methods Mouse strains Nine mouse strains were used, including C57Bl/6JCr, C57Bl/10JCr (B6 and B10; purchased from Charles River laboratories, http://www.criver.com); B10.A (purchased from The Jackson Laboratory, www.jax.org); CAST/Eij (CAST); B10.A(R209) (abbreviated R209); B10.MOL-SGR (SGR); RB2 derived in our mouse facility by back-crossing B10 × R209 F1 with B10; RJ2 (carrying a recombinant chromosome 17 from RB2 × B10.A); and Spo11+/− mice carrying the Spo11 null allele Spo11tm1M (Baudat et al, 2000). Sets of 8, 15 and 12 B6 male mice 35, 6 and 40 weeks old, respectively; 8, 11 and 10 R209 male mice 29, 40 and 33 weeks old, respectively; 10 and 9 B10A × SGR hybrid male mice 29 and 30 weeks old, respectively; 12 RB2 × B10 h
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