Acetylated Histone H3K9 is associated with meiotic recombination hotspots,and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast.     

Scale matters

During meiosis in many organisms, homologous chromosomes engage in numerous recombination events initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. DSBs are distributed nonrandomly, which governs how recombination influences inheritance and genome evolution. The chromosomal features that shape DSB distribution are not well understood. In the budding yeast Saccharomyces cerevisiae, trimethylation of lysine 4 of histone H3 (H3K4me3) has been suggested to play a causal role in targeting Spo11 activity to small regions of preferred DSB formation called hotspots. The link between H3K4me3 and DSBs is supported in part by a genome-wide spatial correlation between the two. However, this correlation has only been evaluated using relatively low-resolution maps of DSBs, H3K4me3 or both. These maps illuminate chromosomal features that influence DSB distributions on a large scale (several kb and greater) but do not adequately resolve features, such as chromatin structure, that act on finer scales (kb and shorter). Using recent nucleotide-resolution maps of DSBs and meiotic chromatin structure, we find that the previously described spatial correlation between H3K4me3 and DSB hotspots is principally attributable to coincident localization of both to gene promoters. Once proximity to the nucleosome-depleted regions in promoters is accounted for, H3K4me3 status has only modest predictive power for determining DSB frequency or location. This analysis provides a cautionary tale about the importance of scale in genome-wide analyses of DSB and recombination patterns.     

Scale matters: The spatial correlation of yeast meiotic DNA breaks with histone H3 trimethylation is driven largely by independent colocalization at promoters

During meiosis in many organisms, homologous chromosomes engage in numerous recombination events initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. DSBs are distributed nonrandomly, which governs how recombination influences inheritance and genome evolution. The chromosomal features that shape DSB distribution are not well understood. In the budding yeast Saccharomyces cerevisiae, trimethylation of lysine 4 of histone H3 (H3K4me3) has been suggested to play a causal role in targeting Spo11 activity to small regions of preferred DSB formation called hotspots. The link between H3K4me3 and DSBs is supported in part by a genome-wide spatial correlation between the two. However, this correlation has only been evaluated using relatively low-resolution maps of DSBs, H3K4me3 or both. These maps illuminate chromosomal features that influence DSB distributions on a large scale (several kb and greater) but do not adequately resolve features, such as chromatin structure, that act on finer scales (kb and shorter). Using recent nucleotide-resolution maps of DSBs and meiotic chromatin structure, we find that the previously described spatial correlation between H3K4me3 and DSB hotspots is principally attributable to coincident localization of both to gene promoters. Once proximity to the nucleosome-depleted regions in promoters is accounted for, H3K4me3 status has only modest predictive power for determining DSB frequency or location. This analysis provides a cautionary tale about the importance of scale in genome-wide analyses of DSB and recombination patterns.     

Changes in chromatin structure at recombination initiation sites during yeast meiosis.

Transient double-strand breaks (DSBs) occur during Saccharomyces cerevisiae meiosis at recombination hot spots and are thought to initiate most, if not all, homologous recombination between chromosomes. To uncover the regulatory mechanisms active in DSB formation, we have monitored the change in local chromatin structure at the ARG4 and CYS3 recombination hot spots over the course of meiosis. Micrococcal nuclease (MNase) digestion of isolated meiotic chromatin followed by indirect end-labeling revealed that the DSB sites in both loci are hypersensitive to MNase and that their sensitivity increases 2- to 4-fold prior to the appearance of meiotic DSBs and recombination products. Other sensitive sites are not significantly altered. The study of hyper- and hypo-recombinogenic constructs at the ARG4 locus, also revealed that the MNase sensitivity at the DSB site correlates with both the extent of DSBs and the rate of gene conversion. These results suggest that the local chromatin structure and its modification in early meiosis play an important role in the positioning and frequency of meiotic DSBs, leading to meiotic recombination.     

Dot1-Dependent Histone H3K79 Methylation Promotes the Formation of Meiotic Double-Strand Breaks in the Absence of Histone H3K4 Methylation in Budding Yeast

Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs) in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation) in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.     

The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae.

Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over. The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases. To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I. We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented. We present a model for the initiation of meiotic recombination taking into account the implications of these results.     

Use of a Recombination Reporter Insert To Define Meiotic Recombination Domains on Chromosome III of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-strand breaks (DSBs). DSBs usually occur in intergenic regions that display nuclease hypersensitivity in digests of chromatin. DSBs are distributed nonuniformly across chromosomes; on chromosome III, DSBs are concentrated in two "hot" regions, one in each chromosome arm. DSBs occur rarely in regions within about 40 kb of each telomere and in an 80-kb region in the center of the chromosome, just to the right of the centromere. We used recombination reporter inserts containing arg4 mutant alleles to show that the "cold" properties of the central DSB-deficient region are imposed on DNA inserted in the region. Cold region inserts display DSB and recombination frequencies that are substantially less than those seen with similar inserts in flanking hot regions. This occurs without apparent change in chromatin structure, as the same pattern and level of DNase I hypersensitivity is seen in chromatin of hot and cold region inserts. These data are consistent with the suggestion that features of higher-order chromosome structure or chromosome dynamics act in a target sequence-independent manner to control where recombination events initiate during meiosis.     

Dot1-Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor

During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along zip1 meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.     

Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites

The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome‐wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double‐strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild‐type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB‐prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.     

Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination‐poor region comprises more than half of barley chromosome 3H

Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single‐copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination‐poor region is preferentially marked by a heterochromatin‐typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin‐typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.     

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

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.     

The CAF-1 and Hir Histone Chaperones Associate with Sites of Meiotic Double-Strand Breaks in Budding Yeast

In the meiotic prophase, programmed DNA double-strand breaks (DSB) are introduced along chromosomes to promote homolog pairing and recombination. Although meiotic DSBs usually occur in nucleosome-depleted, accessible regions of chromatin, their repair by homologous recombination takes place in a nucleosomal environment. Nucleosomes may represent an obstacle for the recombination machinery and their timely eviction and reincorporation into chromatin may influence the outcome of recombination, for instance by stabilizing recombination intermediates. Here we show in budding yeast that nucleosomes flanking a meiotic DSB are transiently lost during recombination, and that specific histone H3 chaperones, CAF-1 and Hir, are mobilized at meiotic DSBs. However, the absence of these chaperones has no effect on meiotic recombination, suggesting that timely histone reincorporation following their eviction has no influence on the recombination outcome, or that redundant pathways are activated. This study is the first example of the involvement of histone H3 chaperones at naturally occurring, developmentally programmed DNA double-strand breaks.     

C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination

Initiation of meiotic recombination by double-strand breaks (DSBs) must occur in a controlled fashion to avoid jeopardizing genome integrity. Here, we identify chromatin-associated protein HIM-17 as a link between chromatin state and DSB formation during C. elegans meiosis. Dependencies of several meiotic prophase events on HIM-17 parallel those seen for DSB-generating enzyme SPO-11: HIM-17 is essential for DSB formation but dispensable for homolog synapsis. Crossovers and chiasmata are eliminated in him-17 null mutants but are restored by artificially induced DSBs, indicating that all components required to convert DSBs into chiasmata are present. Unlike SPO-11, HIM-17 is also required for proper accumulation of histone H3 methylation at lysine 9 on meiotic prophase chromosomes. HIM-17 shares structural features with three proteins that interact genetically with LIN-35/Rb, a known component of chromatin-modifying complexes. Furthermore, DSB levels and incidence of chiasmata can be modulated by loss of LIN-35/Rb. These and other data suggest that chromatin state governs the timing of DSB competence.     

Meiotic Recombination Initiation in and around Retrotransposable Elements in Saccharomyces cerevisiae

Meiotic recombination is initiated by large numbers of developmentally programmed DNA double-strand breaks (DSBs), ranging from dozens to hundreds per cell depending on the organism. DSBs formed in single-copy sequences provoke recombination between allelic positions on homologous chromosomes, but DSBs can also form in and near repetitive elements such as retrotransposons. When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats. A prior study in budding yeast demonstrated that insertion of a Ty retrotransposon into a DSB hotspot can suppress meiotic break formation, but properties of Ty elements in their most common physiological contexts have not been addressed. Here we compile a comprehensive, high resolution map of all Ty elements in the rapidly and efficiently sporulating S. cerevisiae strain SK1 and examine DSB formation in and near these endogenous retrotransposable elements. SK1 has 30 Tys, all but one distinct from the 50 Tys in S288C, the source strain for the yeast reference genome. From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence. Surprisingly, deletion of two Ty elements weakened adjacent DSB hotspots, revealing that at least some Ty insertions promote rather than suppress nearby DSB formation. Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles.