Induced DNA demethylation can reshape chromatin topology at the IGF2-H19 locus

Choriocarcinomas are embryonal tumours with loss of imprinting and hypermethylation at the insulin-like growth factor 2 (IGF2)-H19 locus. The DNA methyltransferase inhibitor, 5-Aza-2′deoxycytidine (5-AzaCdR) is an approved epigenetic cancer therapy. However, it is not known to what extent 5-AzaCdR influences other epigenetic marks. In this study, we set out to determine whether 5-AzaCdR treatment can reprogram the epigenomic organization of the IGF2-H19 locus in a choriocarcinoma cancer cell line (JEG3). We found that localized DNA demethylation at the H19 imprinting control region (ICR) induced by 5-AzaCdR, reduced IGF2, increased H19 expression, increased CTCF and cohesin recruitment and changed histone modifications. Furthermore chromatin accessibility was increased locus-wide and chromatin looping topography was altered such that a CTCF site downstream of the H19 enhancers switched its association with the CTCF site upstream of the IGF2 promoters to associate with the ICR. We identified a stable chromatin looping domain, which forms independently of DNA methylation. This domain contains the IGF2 gene and is marked by a histone H3 lysine 27 trimethylation block between CTCF site upstream of the IGF2 promoters and the Centrally Conserved Domain upstream of the ICR. Together, these data provide new insights into the responsiveness of chromatin topography to DNA methylation changes.     

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.     

Binding of Drosophila maternal Mamo protein to chromatin and specific DNA sequences

Alterations in chromatin structure dynamically occur during germline development in Drosophila and are essential for the production of functional gametes. We had previously reported that the maternal factor Mamo, which contains both a BTB/POZ domain and C₂H₂ zinc-finger domains and is enriched in primordial germ cells (PGCs), is required for the regulation of meiotic chromatin structure and the production of functional gametes. However, the molecular mechanisms by which Mamo regulates germline development remained unclear. To evaluate the molecular function of Mamo protein, we have investigated the binding of Mamo to chromatin and DNA sequences. Our data show that Mamo binds to chromatin and specific DNA sequences, particularly the polytene chromosomes of salivary gland cells. Overexpression of Mamo affected the organization of polytene chromosomes. Reduction in maternal Mamo levels impaired the formation of germline-specific chromatin structures in PGCs. Furthermore, we found that the zinc-finger domains of Mamo directly bind to specific DNA sequences. Our results suggest that Mamo plays a role in regulating chromatin structure in PGCs.     

Nucleosome occupancy landscape and dynamics at mouse recombination hotspots

During meiosis, paternal and maternal homologous chromosomes recombine at specific recombination sites named hotspots. What renders 2% of the mammalian genomes permissive to meiotic recombination by allowing Spo11 endonuclease to initiate double‐strand breaks is largely unknown. Work in yeast has shown that chromatin accessibility seems to be important for this activity. Here, we define nucleosome profiles and dynamics at four mouse recombination hotspots by purifying highly enriched fractions of meiotic cells. We found that nucleosome occupancy is generally stable during meiosis progression. Interestingly, the cores of recombination hotspots have largely open chromatin structure, and the localization of the few nucleosomes present in these cores correlates precisely with the crossover‐free zones in recombinogenic domains. Collectively, these high‐resolution studies suggest that nucleosome occupancy seems to direct, at least in part, how meiotic recombination events are processed.     

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.     

Synaptonemal complex stability depends on repressive histone marks of the lateral element-associated repeat sequences

The synaptonemal complex (SC) is the central key structure for meiosis in organisms undergoing sexual reproduction. During meiotic prophase I, homologous chromosomes exchange genetic information at the time they are attached to the lateral elements by specific DNA sequences. Most of these sequences, so far identified, consist of repeat DNA, which are subject to chromatin structural changes during meiotic prophase I. In this work, we addressed the effect of altering the chromatin structure of repeat DNA sequences mediating anchorage to the lateral elements of the SC. Administration of the histone deacetylase inhibitor trichostatin A into live rats caused death of cells in the pachytene stage as well as changes in histone marks along the synaptonemal complex. The most notable effect was partial loss of histone H3 lysine 27 trimethylation. Our work describes the epigenetic landscape of lateral element-associated chromatin and reveals a critical role of histone marks in synaptonemal complex integrity.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

Structure,function and DNA composition of Saccharomyces cerevisiae chromatin loops

Recent localization of cohesin association regions along the yeast chromatin fibre suggests that compositional variability of DNA in yeast is related to the function and organization of the chromosomal loops. The bases of the loops, where the chromatin fibre is attached to the chromosomal axis, are AT-rich, bind cohesin, and are flanked by genes transcribed convergently. The hotspots of meiotic recombination are mainly found in the GC-rich parts of the loops, ‘external’ with respect to the chromosomal axis, frequently in the vicinity of the promoters of divergently transcribed genes. There are two possible reasons why the regions of the hotspots of recombination were enriched in GC content during evolution. One is a biased repair of recombination intermediates, and the second is a selective advantage due to an increased chromatin accessibility, which may have the carriers of GC-enriched alleles over the carriers of AT-rich alleles.     

Global dissection of the recombination landscape in soybean using a high-density 600K SoySNP array

Recombination is crucial for crop breeding because it can break linkage drag and generate novel allele combinations. However, the high-resolution recombination landscape and its driving forces in soybean are largely unknown. Here, we constructed eight recombinant inbred line (RIL) populations and genotyped individual lines using the high-density 600K SoySNP array, which yielded a high-resolution recombination map with 5636 recombination sites at a resolution of 1.37 kb. The recombination rate was negatively correlated with transposable element density and GC content but positively correlated with gene density. Interestingly, we found that meiotic recombination was enriched at the promoters of active genes. Further investigations revealed that chromatin accessibility and active epigenetic modifications promoted recombination. Our findings provide important insights into the control of homologous recombination and thus will increase our ability to accelerate soybean breeding by manipulating meiotic recombination rate.     

Interstitial telomeric sequence blocks in constitutive pericentromeric heterochromatin from Pyrgomorpha conica (Orthoptera) are enriched in constitutive alkali-labile sites

The long interstitial telomeric repeat sequence (ITRS) blocks located in the pericentromeric chromosomal regions of most of Chinese hamster chromosomes behave as hot spots for spontaneous and induced chromosome breakage and recombination. The DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) procedure demonstrated that these ITRS are extremely sensitive to alkaline unwinding, being enriched in constitutive alkali-labile sites (ALS). To determine whether this chromatin modification occurs in other genomes with large ITRS that are not phylogenetically related to mammalian species, the grasshopper Pyrgomorpha conica was analyzed. We chose this species because, with conventional FISH, their chromosomes yield extremely small telomeric signals when probed with the (TTAGG)n polynucleotide, but large ITRS blocks as part of their pericentromeric constitutive heterochromatin. A high density of constitutive ALS was evidenced in the ITRS when intact meiotic cells or somatic cells were subjected to the DBD-FISH technique and probed with the specific telomeric DNA. DBD-FISH with simultaneous hybridization using telomeric and whole genome DNA probes showed that the ITRS tend to colocalize with areas of stronger signal from the whole genome probe. Nevertheless, the signal from the whole genome was more widespread than that from the ITRS, thus providing evidence that a high frequency of constitutive ALS was present in more than one DNA sequence type. Furthermore, stretched DNA fibers processed with DBD-FISH, revealed a distribution of telomeric sequences alternating interspersed with other possible highly repetitive DNA sequences. The abundance of ALS varied from one meiotic stage to another. Interestingly, most of the breakage and meiotic recombination in males takes place close to the constitutive heterochromatin, particularly enriched in ALS. These results provide further evidence of a particular, and possible universal, chromatin structure enriched in constitutive ALS at constitutive heterochromatic regions.     

Correlation Between DNase I Hypersensitive Site Distribution and Gene Expression in HeLa S3 Cells

Mapping DNase I hypersensitive sites (DHSs) within nuclear chromatin is a traditional and powerful method of identifying genetic regulatory elements. DHSs have been mapped by capturing the ends of long DNase I-cut fragments (>100,000 bp), or 100-1200 bp DNase I-double cleavage fragments (also called double-hit fragments). But next generation sequencing requires a DNA library containing DNA fragments of 100-500 bp. Therefore, we used short DNA fragments released by DNase I digestion to generate DNA libraries for next generation sequencing. The short segments are 100-300 bp and can be directly cloned and used for high-throughput sequencing. We identified 83,897 DHSs in 2,343,479 tags across the human genome. Our results indicate that the DHSs identified by this DHS assay are consistent with those identified by longer fragments in previous studies. We also found: (1) the distribution of DHSs in promoter and other gene regions of similarly expressed genes differs among different chromosomes; (2) silenced genes had a more open chromatin structure than previously thought; (3) DHSs in 3'untranslated regions (3'UTRs) are negatively correlated with level of gene expression.     

Activation of V(D)J recombination at the IgH chain JH locus occurs within a 6-kilobase chromatin domain and is associated with nucleosomal remodeling

IgH genes are assembled during early B cell development by a series of regulated DNA recombination reactions in which DH and JH segments are first joined followed by V(H) to DJH rearrangement. Recent studies have highlighted the role of chromatin structure in the control of V(D)J recombination. In this study, we show that, in murine pro-B cell precursors, the JH segments are located within a 6-kb DNase I-sensitive chromatin domain containing acetylated histones H3 and H4, which is delimited 5' by the DQ52 promoter element and 3' by the intronic enhancer. Within this domain, the JH segments are covered by phased nucleosomes. High-resolution mapping of nucleosomes reveals that, in pro-B cells, unlike recombination refractory nonlymphoid cells, the recombination signal sequences flanking the four JH segments are located in regions of enhanced micrococcal nuclease and restriction enzyme accessibility, corresponding to either nucleosome-free regions or DNA rendered accessible within a nucleosome. These results support the idea that nucleosome remodeling provides an additional level of control in the regulation of Ig locus accessibility to recombination factors in B cell precursors.