Organization and roles of nucleosomes at mouse meiotic recombination hotspots

Meiotic double strand breaks (DSBs) occur at discrete regions in the genome coined hotspots. Precisely what directs site selection of these DSBs is hotly debated and in particular it is unclear which chromatin features, and regulatory factors are necessary for a genomic region to initiate and resolve DSBs as a crossover (CO) event. In human and mouse, one layer of hotspot selection control is a recognition sequence element present at these sites that is bound by the Prdm9 zinc-finger protein. Furthermore, an overall open chromatin structure is thought to be required to allow access of the recombination machinery, and this is often dictated by the packaging of DNA around nucleosomes. We recently defined the nucleosome occupancy maps of four mouse recombination hotspots throughout meiosis. These analyses revealed no obvious dynamic changes in nucleosome occupancy, suggesting an intrinsic nature of recombinogenic sites, yet they also revealed that nucleosomes define zones of exclusion for CO resolution. Here, we discuss new evidence implicating nucleosome occupancy in recombinogenic repair and its potential roles in controlling chromatin structure at mouse meiotic hotspots.

intra-chromosomal, 3 whereas HS59.4, HS59.5 and HS61.1 are sub-telomeric. 1,2 All these five hotspots have crossover (CO) rates ranging from 0.5 to 6.0 x 10 -4 per meiosis, 1,3 which is typical to about two-thirds of all mouse meiotic hotspots. 4 The 1.5-kb core of hotspot HS9 lacks polymorphisms between the two strains, whereas the cores of HS22, HS59.4, HS59.5 and HS61.1 harbor numerous single nucleotide polymorphisms (SNP) and insertions/deletions (indel), with an overall sequence divergence of 7% (49 SNPs, 5 indels), 0.3% (3 SNPs, 1 indel), 1% (15 SNPs, no indels) and 2.7% (14 SNPs, 6 indels). 1,3,5 In our earlier report, we used an improved FACS methodology to obtain highly-enriched fractions having >95% purity for pre-leptotene, leptotene-zygotene and pachytene-diplotene cells. 2 Mono-nucleosomal fractions were then isolated from native chromatin of each population by performing micrococcal nuclease (MNase) digestion. Quantitative PCR and primer pair tiling (with 80-100 bp amplicons that overlap by 30-50 bp) then mapped nucleosome positions across ~6 Kb regions surrounding various hotspot cores. 2 Using this strategy, we generated precise nucleosome occupancy maps at hotspots HS9, HS22, HS59.4 and HS61.1 throughout meiosis, as well as in somatic (thymic) cells 2 and in this study for different strain backgrounds at hotspots HS22 and HS59.5 (Fig. 1). Note though that these nucleosome maps reflect averaged sites that are occupied by specifically stable nucleosomes in the cell population, this doesn't active hotspots, i.e., HS22 and HS59.5 (with recombination rate of 2.17 × 10 -4 and 2.22 × 10 -4 , respectively) in comparison to those having large open domains; e.g., the more active HS9 and HS61.1 hotspots with recombination rates of 4.77 × 10 -4 and 4.99 × 10 -4 . 1,3 A similar trend has been observed for S. cerevisiae hotspots, where in general more active and wide hotspots, such as YAT1, NAR1 and WHI5, have overall low nucleosome occupancy and disordered chromatin structure. 6 In accord with these findings, there is a close correlation between the locale of DNase I hypersensitive (DHSS) regions and the distribution of recombination hotspots in S. cerevisiae. [7][8][9] Finally, overlapping the yeast DSB map (obtained by sequencing genomic Spo11bound DNA fragments) with whole-genome nucleosome occupancy profiles, revealed that Spo11-associated hotspots lacked nucleosomes. 6 Collectively, these findings would suggest that DSB occur exclusively on nonnucleosomal DNA and that nucleosomes would also prevent access of Spo11. Thus, loose chromatin structure and the openness of a particular genomic region would be thought necessary for establishing a hotspot. However, genomic regions with lownucleosome occupancy are not restricted to hotspots. For instance, areas of open chromatin have been observed in regions flanking mouse meiotic hotspots, both in the immediate vicinity of hotspot cores and even in recombinogenically cold flanking regions at -25 kb to +25 kb from the hotspot core. 2 Similarly, not all nuclease hypersensitive sites are DSB hotspots in yeast and there is no correlation between the degree of nuclease hypersensitivity and the frequency of DSB formation. 8,10 Therefore, there must be additional factors HS22 and HS59.4 hotspots contained from one to three nucleosomes at the center of their cores that were flanked by 300-700 bp regions of open chromatin 2 (Fig. 1). In contrast, there is only one telomeric nucleosome at the HS61.1 hotspot core 2 and no nucleosomes are present at the HS9 recombinogenic core 2 with 700-1,000 bp open regions. Interestingly, the degree of open chromatin at the hotspot cores correlates well with recombinogenic activity of these hotspots, where a more closed chromatin structure was observed with the less recombinogenically necessarily reflect the nucleosome profile of a particular cell. The obtained data revealed generally open chromatin regions within meiotic hotspot cores, with nucleosome-free domains of 300-1,000 bp. These are considerably larger than nucleosome-depleted domains identified in yeast meiotic hotspots (of ~130 bp), most of which are located in the intergenic regions containing promoters. [6][7][8] Based on these analyses, these hotspots can be subdivided into three groups according to nucleosome patterns observed at their cores, with several, one or no nucleosomes. The HS59.5, type mouse strains. Specifically, similar nucleosome profiles were observed for HS22 locus in C57Bl/6J and B6/DBA strains, where this hotspot is active, and in DBA/2J and CAST/DBA hybrids where this hotspot is inactive 3 (Fig. 1). Similarly, nucleosome profiles at the HS59.5 hotspot were identical across strains and hybrids irrespective of their active or inactive recombination status (Fig. 1). Thus, the absence of recombination activity in a particular strain background cannot be simply attributed to addition of stable nucleosomes or to alterations in the patterns of stable nucleosomes.
profiles. 6 This agrees with an earlier yeast study, showing that DNase I hypersensitive regions that overlap with DSB sites, are identical in mitotic and meiotic cells and that this pattern does not change before, during, or following DSB formation in meiotic cells. 9 Overall, these findings indicate that the sites of stably bound nucleosomes, one of the criteria for sites of opportunity for Spo11 cleavage at the leptotene stage, are pre-established in mice and appears to be conserved with what is observed in yeast. In addition, nucleosome profiles appear to be identical for a hotspot locus that is either active or inactive in different that establish a particular genomic region as a recombination hotspot.
Nucleosome profiles at mouse meiotic hotspots appear to be pre-established early on at the spermatogonia stage, well before the initiation of meiosis, and remain nearly identical throughout meiosis. 2 Remarkably, nearly identical nucleosome landscapes are also observed in somatic thymic cells, supporting the notion that no significant chromatin remodeling occurs at the hotspots during meiosis. 2 Similar findings have been described in yeast, where whole-genome nucleosome occupancy maps also showed no differences in pre-meiotic and early meiotic  12,14,21,22,24,42,45,54,55 Specific primary sequence (shown in red line) is a key component for a hotspot to be formed, where various types of pre-recombinational complexes may bind followed by Spo11 protein. these may be PrDM9 zinc-finger protein (a); H3.3/H2a.Z labile histone variants most likely forming complex with some other protein factor (B); or chromatin remodeling factors in complex with histone acetylases bound to a DNa-binding protein (C). 17,18 inactive hotspot regions may be formed due to presence of inhibiting SNPs in a primary DNa sequence (a); by epigenetic means due to high level of DNa methylation (B) or presence of repressive histone modification marks, such as H3K27Me3 and H3K9Me2/3 (a), 24,26,27 and underacetylated histones (C), 12,23,25 that lead to a closed chromatin conformation and inaccessibility of the region by Spo11.
superposition of nucleosome occupancy maps of these four loci with the CO profiles established that in all four hotspots nucleosomes are localized exactly at the sites that are resistant to CO resolution. Detection of nucleosomes at the sites of CO repulsion zones may shed new insights in the mechanism of homologous recombination.
In an earlier study, 1 we proposed a CO repulsion zone model, in which stable nucleosomes may generate constraints to D-loop extension and second end capture. This model favors the notion that the distribution of nucleosomes directs the selection of the preferential binding sites by the Spo11 complex to open chromatin regions. There are several predictions of this model. First, the local nucleosome pattern in the vicinity of a hotspot will control strand invasion and direct D-loop formation by regulating the accessibility and positioning of the nucleosome-free region on the donor homologous chromosome. Second, nucleosome positioning and possibly certain histone modifications would control hotspot activity by affecting the distribution of CO and NCO. In support of this model, recent analysis of CO profiles and the distribution of histone modification marks at Psmb9 and Hlx1 super-hotspots show an enrichment of selectively modified histones, such as H3K4Me3 and hyperacetylated H4 at their cores, in the regions corresponding to the highest CO rates. 24

Instructive Role of DNA Motifs in Hotspot Formation
Multiple DNA regulatory sites of meiotic recombination have been identified in yeast. These sites, coined α-hotspots, mostly have preferential DNA sequences for binding various transcription factors that also may promote recombination. 17,[32][33][34][35][36] Examples of these sequences are CRE-like M26 DNA site (5'-ATG ACT G-3'), 5'-CCA AT-3' and 5'-CCC CGC A-3' motifs, which are binding sites for Atf1-Pcr1, CCAAT-binding transcription factors, and Scr1, Rsv1, Hsr1 and Rst2 zinc-finger transcription factors respectively. 17,36 However, other meiotic yeast hotspot regions, the so-called β-hotspots, are not associated with transcription hotspots preferentially occur in loci that express the prl class of long polyadenylated noncoding RNAs (ncRNAs), 29 where activation of the hotspot site by ncRNAs is thought to provoke chromatin remodeling. Alternatively, ncRNA-DNA hybrids (R-loops) have been proposed to trigger events that open chromatin for the Spo11 complex. 29 Taking all of these potential mechanisms into account, it is likely that DSB initiation and hotspot activity are controlled by several different extrinsic and intrinsic cues, and that at least some of these are also dependent upon a particular genomic background. However, one commonality is the need to form an open chromatin conformation that is conducive to DSB initiation.

Crossover Repulsion Zones
The detailed crossover (CO) profiles obtained for the highly polymorphic HS22, HS59.4, HS61.1 hotspots 1,2 and in this study for the HS59.5 locus (Fig. 3), have revealed domains that are resistant to CO resolution. These regions, termed as CO repulsion zones, have been observed in many mammalian hotspots. [3][4][5]30,31 They appeared to be rather randomly distributed across hotspot regions, where they can either occur at the center of the hotspot core (e.g., in HS22, HS59.4 and HS59.5), 2 (Fig. 3) or at the 3' boundary of the core (e.g., HS61.1). 2 Strikingly, the We predict that higher order chromatin structure, of which nucleosome patterning is a critical component, should still play significant role in establishing the suitable environment for the Spo11 complex to bind a given genomic region. Nucleosome occupancy pattern may affect the distribution of chromatin loops on the axis during synaptonemal complex formation and thereby influence the positioning of favorable sites for DSB initiation (Fig. 2). In support of this notion, in yeast, DSBs are known to be depleted in cohesin-enriched regions found at the bases of chromatin loops. 6,11 Alternatively, the initiation of DSBs may be regulated by epigenetic means, for example by histone modifications and/or DNA methylation, that may cause changes in chromatin conformation via interaction with chromatin remodeling machinery, rendering DNA loops more or less accessible for the Spo11 complex (Fig. 2). 7,[12][13][14][15][16][17][18] Multiple reports studying different organisms suggest that active histone modifications, such as acetylation, methylation and ubiquitination may position meiotic recombination (Fig. 2). [17][18][19][20][21][22][23][24][25] In contrast, repressive histone modification marks are depleted in mouse meiotic hotspots (Fig.  2). 12,[23][24][25][26][27] Other modes for control of hotspots have also been suggested, for example remote cis-acting DNA motifs affecting the initiation and activity of hotspots. 15,28 Further, in S. pombe meiotic recombination for C57Bl/6J and up to 9 out of 14 matches for CAST/EiJ background, Table  1). Thus, other levels of control besides the presence of corresponding Prdm9 protein binding site must be operational in these different backgrounds to turn the HS22 region to a hotspot. These findings are consistent with data obtained for MSTM1a and MSTM1b human hotspots, which showed that major variation in their hotspot activity and the presence/absence of a hotspot can occur without any change in local DNA sequence. 15 Finally, computational approaches to predict Prdm9 binding sequences are not always being confirmed by actual biochemical binding studies with hotspot sequences. 45 In this study, 26 sequences for Prdm9 b and Prdm9 wm7 binding were generated using C 2 H 2 zinc finger prediction program and tested in vitro for three mouse hotspots, G7c, Hlx1 and Psmb9. However, only one of them could bind Prdm9 using a southwestern assay. 45 This failure to adequately determine Prdm9 binding sites indicates that the other factors besides DNA sequence might regulate its binding to a certain region, and/or that Prdm9 binding alone is not sufficient. However, primary DNA sequence by itself is not sufficient to promote meiotic recombination. 17 For example, M26 DNA sites (ATG ACG T) in fission yeast are known to regulate from 20 to 50% of meiotic recombination by different estimates. 17,43 Yet, only 26% of M26 sites are found in active recombinogenic regions. 17 In S. cerevisiae, the hotspot activity associated with the HIS4 gene was previously shown to require the Bas1p transcription factor. 35 Nonetheless, a genome-wide analysis of Bas1p transcription factor binding sites and their recombinogenic activity 44 has demonstrated that Bas1p stimulated recombination only in 16% of these sites. Finally, if instructive, the 13-mer sequence motif CCN CCN TNN CCN C, which is present in 40% of human meiotic hotspots, can only promote recombination for 10% of its locations in the genome. 40 In mice, two different 36-and 33-mer motifs have been predicted to be recognized by two distinct Prdm9 proteins for b and wm7 alleles in M. m. domesticus and M. m. molossinus strains. 42 We have screened HS61.1, HS9 and HS59.5 sequences for the presence of Prdm9 b allele motif and HS22 sequence for the presence of Prdm9 b and wm7 allele motifs ( Table 1). For HS22 and HS59.5 cores, sequences matching from 6 to 10 out of 15 highest score bases of Prdm9 b predicted recognition motif were identified (Table 1 and Fig. 3).
Notably, screening of the HS22 hotspot core DNA sequences in C57Bl/6J strain background in which this hotspot is active and in CAST/EiJ background where it is "cold" shows that sequences with highscore matches to the recognition motifs for Prdm9 b and Prdm9 wm7 proteins are present in both (up to 9 out of 15 matches factor binding. These sites rather contain intrinsically open nucleosome-excluding DNA sequences located in intergenic genomic regions. For example, the common DNA repeat (CCGNN)n motif (where n = 12, 24 or 48), which excludes nucleosomes, can either augment (i.e., the 12-mer) or repress (i.e., the 48-mer) recombination. 37,38 In humans, several short DNA motifs were identified that are enriched in hotspots. 39,40 In particular, the 7-mer motif CCT CCC T and the 9-mer motif CCC CAC CCC are each found in about 10% of human hotspots. 39 A recent breakthrough for the recombination field was the identification of a degenerate 13-mer sequence motif CCN CCN TNN CCN C that is present at the core of approximately 40% linkage disequilibrium (LD)-defined hotspots in humans. 40 These findings suggested the existence of a DNA binding factor that could direct the DSB initiation machinery to specific sites. 40 Further, genetic studies in mice identified a region that appeared to encode a major trans-acting regulator of at least some hotspots. 41 Finally, it has now been established that this motif is bound by the meiotically expressed zinc finger histone H3K4 methyltransferase Prdm9, 16,17,42 which is thought to control DSB initiation, hotspot activity and fertility in humans and mice.
Overall, these findings suggest that primary DNA sequence is a key regulatory component of DSB initiation and meiotic hotspot activity in yeast and mammals. This control could occur via binding of a transcription factor or DNA binding histone modification enzyme in such a way as to make sites accessible for recombination proteins to approach (Fig. 2).  tCN tGN tGN tNN tNN NNN NtN NNN NNN ttN tGN for  a Spo11 protein complex is anchored to the labile nucleosome sites, they are getting replaced with recombination protein machinery stimulating initiation of homologous recombination (Fig. 2). Certainly this is a hypothesis that needs to be further investigated in order to uncover the fundamental steps leading to DSB initiation.

Acknowledgments
We are indebted to Dr J. Cleveland for critical comments of the manuscript, to Dr H. Petrie and B. Torres (Scripps Florida FACS core) for crucial technical advice and assistance and to numerous colleagues for discussions and insights. This project was supported in part by funding from the State of Florida to Scripps and award number R01GM085079 from the National Institute of General Medical Sciences.
active promoters, enhancers and insulator regions. 48 In addition, double-variant nucleosomes correspond to sites of tissuespecific DNase I hypersensitivity. 48 Accessing a site occupied by a nucleosome for DNA binding, transcription, replication or repair requires nucleosome removal or sliding, and/or unraveling of the supercoiled DNA double helix. H3.3/ H2A.Z nucleosomes could be produced and destabilized as part of this dynamic process. 47 It has been proposed that they may serve as "place holders," preventing the region from being covered with stable canonical nucleosomes and nonspecific factors. 48 Alternatively, these labile nucleosomes may be displaced by transcription factors. 48 We speculate that H3.3/ H2A.Z nucleosomes may be present in the "nucleosome-free" regions of potential meiotic recombination hotspots and mark accessible sites of DSB opportunity. Once regions" are in fact not really free but are rather occupied by labile H3.3/H2A.Z nucleosomes. [46][47][48][49] Unlike most of "stable" histones, which are deposited on DNA during replication phase (S-phase), H2A.Z and H3.3 histone variants are deposited throughout the cell cycle and thus they must replace resident histones. For example, histone H3.3 slowly replaces H3 after differentiating cells have exited the cell cycle 50,51 and also during spermatogenesis before DNA becomes repackaged with protamines. 50,52,53 Nucleosomes containing both H3.3 and H2A.Z histone variants are unusually unstable at the conditions normally used for nucleosome preparations. 46,48 Intriguingly, a recent genome-wide study of H3.3/ H2A.Z distribution around transcription regulatory regions in human cells showed that H3.3/H2A.Z nucleosome core particles mark "nucleosome-free regions" of