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.     

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.     

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: 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.     

Analysis of biological features associated with meiotic recombination hot and cold spots in Saccharomyces cerevisiae

Meiotic recombination is not distributed uniformly throughout the genome. There are regions of high and low recombination rates called hot and cold spots, respectively. The recombination rate parallels the frequency of DNA double-strand breaks (DSBs) that initiate meiotic recombination. The aim is to identify biological features associated with DSB frequency. We constructed vectors representing various chromatin and sequence-based features for 1179 DSB hot spots and 1028 DSB cold spots. Using a feature selection approach, we have identified five features that distinguish hot from cold spots in Saccharomyces cerevisiae with high accuracy, namely the histone marks H3K4me3, H3K14ac, H3K36me3, and H3K79me3; and GC content. Previous studies have associated H3K4me3, H3K36me3, and GC content with areas of mitotic recombination. H3K14ac and H3K79me3 are novel predictions and thus represent good candidates for further experimental study. We also show nucleosome occupancy maps produced using next generation sequencing exhibit a bias at DSB hot spots and this bias is strong enough to obscure biologically relevant information. A computational approach using feature selection can productively be used to identify promising biological associations. H3K14ac and H3K79me3 are novel predictions of chromatin marks associated with meiotic DSBs. Next generation sequencing can exhibit a bias that is strong enough to lead to incorrect conclusions. Care must be taken when interpreting high throughput sequencing data where systematic biases have been documented.     

Spp1 at the crossroads of H3K4me3 regulation and meiotic recombination

In Saccharomyces cerevisiae, all H3K4 methylation is performed by a single Set1 Complex (Set1C) that is composed of the catalytic (Set1) and seven other subunits (Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1 and Shg1). It has been known for quite some time that trimethylated H3K4 (H3K4me3) is enriched in the vicinity of meiotic double-strand breaks (DSBs), but the link between H3K4me3 and the meiotic nuclease Spo11 was uncovered only recently. The PHD-containing subunit Spp1, by interacting with H3K4me3 and Mer2, was shown to promote the recruitment of potential meiotic DSB sites to the chromosomal axis allowing their subsequent cleavage by Spo11. Therefore, Spp1 emerged as a key regulator of the H3K4 trimethylation catalyzed by Set1C and of the formation of meiotic DSBs. These findings illustrate the remarkable multifunctionality of Spp1, which not only regulates the catalytic activity of the enzyme (Set1), but also interacts with the deposited mark, and mediates its biological effect (meiotic DSB formation) independently of the complex. As it was previously described for Swd2, and now for Spp1, we anticipate that other Set1C subunits, in addition to regulating H3K4 methylation, may participate in diverse biological functions inside or outside of the complex.     

Genome-wide evaluation of histone methylation changes associated with leaf senescence in Arabidopsis

Leaf senescence is the orderly dismantling of older tissue that allows recycling of nutrients to developing portions of the plant and is accompanied by major changes in gene expression. Histone modifications correlate to levels of gene expression, and this study utilizes ChIP-seq to classify activating H3K4me3 and silencing H3K27me3 marks on a genome-wide scale for soil-grown mature and naturally senescent Arabidopsis leaves. ChIPnorm was used to normalize data sets and identify genomic regions with significant differences in the two histone methylation patterns, and the differences were correlated to changes in gene expression. Genes that showed an increase in the H3K4me3 mark in older leaves were senescence up-regulated, while genes that showed a decrease in the H3K4me3 mark in the older leaves were senescence down-regulated. For the H3K27me3 modification, genes that lost the H3K27me3 mark in older tissue were senescence up-regulated. Only a small number of genes gained the H3K27me3 mark, and these were senescence down-regulated. Approximately 50% of senescence up-regulated genes lacked the H3K4me3 mark in both mature and senescent leaf tissue. Two of these genes, SAG12 and At1g73220, display strong senescence up-regulation without the activating H3K4me3 histone modification. This study provides an initial epigenetic framework for the developmental transition into senescence.     

Extensive allele‐level remodeling of histone methylation modification in reciprocal F1 hybrids of rice subspecies

Epigenetic mechanisms play a major role in heterosis, partly as a result of the remodeling of epigenetic modifications in F₁ hybrids. Based on chromatin immunoprecipitation‐sequencing (ChIP‐Seq) analyses, we show that at the allele level extensive histone methylation remodeling occurred for a subset of genomic loci in reciprocal F₁ hybrids of Oryza sativa (rice) cultivars Nipponbare and 93‐11, representing the two subspecies japonica and indica. Globally, the allele modification‐altered loci in leaf or root of the reciprocal F₁ hybrids involved ?12–43% or more of the genomic regions carrying either of two typical histone methylation markers, H3K4me3 (>21 000 genomic regions) and H3K27me3 (>11 000 genomic regions). Nevertheless, at the total modification level, the majority (from ?43 to >90%) of the modification‐altered alleles lay within the range of parental additivity in the hybrids because of concerted alteration in opposite directions, consistent with an overall attenuation of allelic differences in the modifications. Importantly, of the genomic regions that did show non‐additivity in total modification level by either marker in the two tissues of hybrids, >80% manifested transgressivity, which involved genes enriched in specific functional categories. Extensive allele‐level alteration of H3K4me3 alone was positively correlated with genome‐wide changes in allele‐level gene expression, whereas at the total level, both H3K4me3 and H3K27me3 remodeling, although affecting just a small number of genes, contributes to the overall non‐additive gene expression to variable extents, depending on tissue/marker combinations. Our results emphasize the importance of allele‐level analysis in hybrids to assess the remodeling of epigenetic modifications and their relation to changes in gene expression.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w^m4 PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w^m4 backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

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.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w(m4) PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w(m4) backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

Functional analysis of histone methyltransferase g9a in B and T lymphocytes

Lymphocyte development is controlled by dynamic repression and activation of gene expression. These developmental programs include the ordered, tissue-specific assembly of Ag receptor genes by V(D)J recombination. Changes in gene expression and the targeting of V(D)J recombination are largely controlled by patterns of epigenetic modifications imprinted on histones and DNA, which alter chromatin accessibility to nuclear factors. An important component of this epigenetic code is methylation of histone H3 at lysine 9 (H3K9me), which is catalyzed by histone methyltransferases and generally leads to gene repression. However, the function and genetic targets of H3K9 methyltransferases during lymphocyte development remain unknown. To elucidate the in vivo function of H3K9me, we generated mice lacking G9a, a major H3K9 histone methyltransferase, in lymphocytes. Surprisingly, lymphocyte development is unperturbed in G9a-deficient mice despite a significant loss of H3K9me2 in precursor B cells. G9a deficiency is manifest as modest defects in the proliferative capacity of mature B cells and their differentiation into plasma cells following stimulation with LPS and IL-4. Precursor lymphocytes from the mutant mice retain tissue- and stage-specific control over V(D)J recombination. However, G9a deficiency results in reduced usage of Iglambda L chains and a corresponding inhibition of Iglambda gene assembly in bone marrow precursors. These findings indicate that the H3K9me2 epigenetic mark affects a highly restricted set of processes during lymphocyte development and activation.