Bivalent Regions Research Articles

A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin

BackgroundBivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes.ResultsHere, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics.ConclusionsOur optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells.

Cytological Study of Family Aeshnidae (Odonata: Anisoptera) From India: A Review

ABSTRACT: Cytological review of 59 aeshnid species and cytogenetic investigations on Anax ephippiger, Anax immaculifrons, Anax indicus, Anax nigrofasciatus nigrolineatus, Anax parthenope, Gynacantha subinterrupta of the family Aeshnidae by carbol fuchsin staining and C - banding have been under taken. All the species posses 2n = 27m with X0 - XX sex determination except Anax ephippiger with 2n = 14 + neo XY, resulted by the 13 simultaneous fusions among the autosomes and between autosome and sex chromosome. The structure and behaviour of chromosomes, variation in size of m chromosomes and X chromosome and distribution of C - heterochromatin have been studied and compared among the species. C - bands are mostly present at the terminal regions of autosomal bivalents, while Anax ephippiger and Anax parthenope also possess C - bands at the interstitial and sub-terminal regions of the bivalents. Moreover, sex chromosome and m bivalent show variation in distribution of C-heterochromatin in the species. Out of these, chromosome complement of Anax indicus Lieftinck, 1942 and C - banding on Anax ephippiger and Anax indicus have been investigated for the first time. List of cytologically studied species of family Aeshnidae has been updated to 60 species.

ZFP281-BRCA2 prevents R-loop accumulation during DNA replication

R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to genome instability. Here, we show that ZFP281 cooperates with BRCA2 in preventing R-loop accumulation to facilitate DNA replication in embryonic stem cells. ZFP281 depletion reduces PCNA levels on chromatin and impairs DNA replication. Mechanistically, we demonstrate that ZFP281 can interact with BRCA2, and that BRCA2 is enriched at G/C-rich promoters and requires both ZFP281 and PRC2 for its proper recruitment to the bivalent chromatin at the genome-wide scale. Furthermore, depletion of ZFP281 or BRCA2 leads to accumulation of R-loops over the bivalent regions, and compromises activation of the developmental genes by retinoic acid during stem cell differentiation. In summary, our results reveal that ZFP281 recruits BRCA2 to the bivalent chromatin regions to ensure proper progression of DNA replication through preventing persistent R-loops.

Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone

The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.

Transition to na\xefve human pluripotency mirrors pan-cancer DNA hypermethylation

Epigenetic reprogramming is a cancer hallmark, but how it unfolds during early neoplastic events and its role in carcinogenesis and cancer progression is not fully understood. Here we show that resetting from primed to naïve human pluripotency results in acquisition of a DNA methylation landscape mirroring the cancer DNA methylome, with gradual hypermethylation of bivalent developmental genes. We identify a dichotomy between bivalent genes that do and do not become hypermethylated, which is also mirrored in cancer. We find that loss of H3K4me3 at bivalent regions is associated with gain of methylation. Additionally, we observe that promoter CpG island hypermethylation is not restricted solely to emerging naïve cells, suggesting that it is a feature of a heterogeneous intermediate population during resetting. These results indicate that transition to naïve pluripotency and oncogenic transformation share common epigenetic trajectories, which implicates reprogramming and the pluripotency network as a central hub in cancer formation.

GATA Factor-Mediated Gene Regulation in Human Erythropoiesis.

SummaryErythroid commitment and differentiation are regulated by the coordinated action of a host of transcription factors, including GATA2 and GATA1. Here, we explored GATA-mediated transcriptional regulation through the integrative analysis of gene expression, chromatin modifications, and GATA factors' binding in human multipotent hematopoietic stem/progenitor cells, early erythroid progenitors, and late precursors. A progressive loss of H3K27 acetylation and a diminished usage of active enhancers and super-enhancers were observed during erythroid commitment and differentiation. GATA factors mediate transcriptional changes through a stage-specific interplay with regulatory elements: GATA1 binds different sets of regulatory elements in erythroid progenitors and precursors and controls the transcription of distinct genes during commitment and differentiation. Importantly, our results highlight a pivotal role of promoters in determining the transcriptional program activated upon erythroid differentiation. Finally, we demonstrated that GATA1 binding to a stage-specific super-enhancer sustains the expression of the KIT receptor in human erythroid progenitors.

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver.

Post‐translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP‐Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP‐Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.

Common genetic variants associated with Parkinson\u2019s disease display widespread signature of epigenetic plasticity

Parkinson disease (PD) is characterized by a pivotal progressive loss of substantia nigra dopaminergic neurons and aggregation of α-synuclein protein encoded by the SNCA gene. Genome-wide association studies identified almost 100 sequence variants linked to PD in SNCA. However, the consequences of this genetic variability are rather unclear. Herein, our analysis on selective single nucleotide polymorphisms (SNPs) which are highly associated with the PD susceptibility revealed that several SNP sites attribute to the nucleosomes and overlay with bivalent regions poised to adopt either active or repressed chromatin states. We also identified large number of transcription factor (TF) binding sites associated with these variants. In addition, we located two docking sites in the intron-1 methylation prone region of SNCA which are required for the putative interactions with DNMT1. Taken together, our analysis reflects an additional layer of epigenomic contribution for the regulation of the SNCA gene in PD.

An annotated list of bivalent chromatin regions in human ES cells: a new tool for cancer epigenetic research.

CpG islands (CGI) marked by bivalent chromatin in stem cells are believed to be more prone to aberrant DNA methylation in tumor cells. The robustness and genome-wide extent of this instructive program in different cancer types remain to be determined. To address this issue we developed a user-friendly approach to integrate the stem cell chromatin signature in customized DNA methylation analyses. We used publicly available ChIP-sequencing datasets of several human embryonic stem cell (hESC) lines to determine the extent of bivalent chromatin genome-wide. We then created annotated lists of high-confidence bivalent, H3K4me3-only and H3K27me3-only chromatin regions. The main features of bivalent regions included localization in CGI/promoters, depletion in retroelements and enrichment in specific histone modifications, including the poorly characterized H3K23me2 mark. Moreover, bivalent promoters could be classified in three clusters based on PRC2 and PolII complexes occupancy. Genes with bivalent promoters of the PRC2-defined cluster displayed the lowest expression upon differentiation. As proof-of-concept, we assessed the DNA methylation pattern of eight types of tumors and confirmed that aberrant cancer-associated DNA hypermethylation preferentially targets CGI characterized by bivalent chromatin in hESCs. We also found that such aberrant DNA hypermethylation affected particularly bivalent CGI/promoters associated with genes that tend to remain repressed upon differentiation. Strikingly, bivalent CGI were the most affected by aberrant DNA hypermethylation in both CpG Island Methylator Phenotype-positive (CIMP+) and CIMP-negative tumors, suggesting that, besides transcriptional silencing in the pre-tumorigenic cells, the bivalent chromatin signature in hESCs is a key determinant of the instructive program for aberrant DNA methylation.

Bivalent Regions of Cytosine Methylation and H3K27 Acetylation Suggest an Active Role for DNA Methylation at Enhancers

The role of cytosine methylation in the structure andfunction of enhancers is not well understood. In this study, we investigate the role of DNA methylation at enhancers by comparing the epigenomes of the HCT116 cell line and its highly demethylated derivative, DKO1. Unlike promoters, a portion of regular and super- or stretch enhancers show active H3K27ac marks co-existing with extensive DNA methylation, demonstrating the unexpected presence of bivalent chromatin in both cultured and uncultured cells. Furthermore, our findings also show that bivalent regions have fewer nucleosome-depleted regions and transcription factor-binding sites than monovalent regions. Reduction of DNA methylation genetically or pharmacologically leads to a decrease of the H3K27ac mark. Thus, DNA methylation plays an unexpected dual role at enhancer regions, being anti-correlated focally at transcription factor-binding sites but positively correlated globally with the active H3K27ac mark to ensure structural enhancer integrity.

Initiation and progression of homologous chromosome synapsis in Crepis capillaris: Variations on a theme

The model cytogenetic plant species Crepis capillaris (2x = 6), in which all 3 chromosomes are readily distinguished, was used to analyse the initiation and progression of meiotic synapsis in a large sample of spread and silver-stained pollen mother cells. Particular emphasis was placed on detecting general patterns or trends of synaptic order, both among different bivalents and within (along) individual bivalents, and investigating the consistency or otherwise of these synaptic patterns. The order of synaptic progression and completion was partly related to chromosome length; as in other species, shorter bivalents tended to complete synapsis ahead of longer ones. Individual bivalents also showed distinct patterns of synapsis, with a tendency for subterminal regions to initiate synapsis early, followed by multiple synaptic initiations in internal bivalent regions. However, the analysis showed that these synaptic patterns are only general trends and significant variations in synaptic order and pattern, among and within bivalents, occur in individual cells.Key words: meiosis, synapsis, synaptonemal complex, Crepis.

Differential elongation of autosomal pachytene bivalents related to their DNA content in human spermatocytes.

The establishment of the complete karyotype of human pachytene spermatocytes reveals differences in stretching of chromosomes between meiosis and mitosis. Bivalents or specific regions of bivalents which exhibit many R-bands are particularly elongated. In mitotic chromosomes, the DNA contained in such bands is known to be early replicating. The study of variations in the total length and the centromeric index of bivalent 1 suggests that differential elongation of pachytene bivalents is a premeiotic event, taking place during the last DNA replication.

Chromosome pairing, recombination nodules and chiasma formation in the basidiomycete coprinus cinereus

Meiosis in the basidiomycete Coprinus cinereus was analyzed by three dimensional reconstructions of nuclei covering the period from leptotene to telophase II. Crosses involving three different strains (JR52, PR2301 and E991) were used.The analysis of 94 completely reconstructed nuclei arranged in a temporal sequence according to the morphology of the synaptonemal complex, the centromeres and the centrosomes permitted the following observations and conclusions: (1) The haploid chromosome number of Coprinus cinereus is 13. (2) Reciprocal translocations have been identified in strains PR2301 and E991. In the former strain, the translocation is between chromosomes 3 and 5 and in the latter between chromosomes 1 and 9. (3) Interlockings and chromosome breaks are present during zygotene but at a lower frequency than in organisms with longer chromosomes. The translocation quadrivalents are more often than normal bivalents involved in interlockings and have more chromosome breaks. (4) Transformation of a translocation quadrivalent into two heteromorphic bivalents was only observed once in agreement with the contention that the turnover of the synaptonemal complex required for this transformation is prevented in bivalent regions where crossing over has occurred. (5) Correction of interlockings by the «breakage-reunion» mechanism is complete before mid-late pachytene. (6) The presence of two apparently normal bivalents replacing the translocation quadrivalent in at least one, possibly several, cases suggests that a «retranslocation» has taken place, possibly by a mechanism similar to that responsible for the resolution of interlockings. The implications of this possibility are discussed. (7) During early diplotene the synaptonemal complexes are eliminated from the bivalent arms while synaptonemal complex constituents often remain associated with the centromeres and the chiasmata until late diplotene. (8) Homologous centromere regions remain fused at least until early diakinesis. It is the suggested that this association may serve the same function as chiasmata in maintaining the bivalent configuration up to metaphase I and hence improve the chances of a regular disjunction in bivalents without chiasmata. (9). Recombination nodules are readily identified in the central region of the synaptonemal complex from early zygotene to late diplotene. The total number of nodules expected upon completion of synaptonemal complex formation at late zygotene amounts to 37 and is the same as that observed at early pachytene. The total number of nodules is reduced to 26 before midlate pachytene, a reduction similar to that reported in other organisms. (10) An increasing fraction of the nodules becomes larger and surrounded by chromatin during pachytene—diplotene and by late diplotene, all nodules are replaced by small chromatin condensations—chiasmata. (11) The distribution of nodules among and along the bivalents has been analyzed by comparing the observed distributions and those produced by computer simulation of a random positioning of nodules. The analysis reveals a nearly random distribution at late zygotene while during early pachytene and especially pronounced at mid-late pachytene, the distribution of nodules deviates from a random distribution. The comparison furthermore indicates that the placement of recombination nodules on the bivalents of a nucleus is controlled by nodule/bivalent (bivalent arm) interactions while the interaction between nodules appears to be of less importance.

A nuclear body in meiosis of Bromus

1. A structure which is tentatively called a “nuclear body” has been observed in microsporocytes in a large number of species of Bromus. It was found in diploid and polyploid species and in species with complements of large (L-)chromosomes, medium (M-)chromosomes or L- and M-chromosomes. These species were collected in North and South America and Europe. It is suggested that the nuclear body occurs regularly in species of the genus Bromus. 2. The nuclear body was first visible at pachytene. It was generally single. In some individuals it disappeared at the end of prophase; in others it was present during later stages of meiosis, in some instances as late as the quartet. The nuclear body was not observed when the quartet cells had separated. Before it disappeared the nuclear body underwent a sudden or gradual diminution. It decreased in size, or became faint-staining and faded away; or it decreased in size and faded. The nuclear body generally disappeared at the end of prophase in the diploid and tetraploid species and was present at metaphase-I and later stages in higher polyploid species. 3. When the nuclear body was present in later stages of meiosis it behaved as follows: It was extruded from the spindle at metaphase-I and frequently came to lie in the polar region of the cell; it was not included in either nucleus at telophase-I. A single nuclear body was present in one dyad cell; it lay outside the spindle at metaphase-II, and was included in one of the cells of the quartet. 4. A very great range in the size of the nuclear body was found in early stages of meiosis. Some variation in size was found within individuals and among individuals of the same species. Present data indicate some relationship between chromosome number and size of the nuclear body. The largest nuclear bodies were found in duodecaploid and octoploid and the smallest in diploid species; however an exact correspondence was not observed. 5. The nuclear body is extremely variable in shape. It has a vesicular appearance; there is great variation in the number, size and staining of the vesicles. The appearance of the nuclear body may vary within and among anthers and individuals. 6. The nuclear body is not associated with the nucleolus at pachytene, nor with the nucleolar organizing regions of specific bivalents. The nuclear body is often touching a bivalent or attached by fine strands to a bivalent during prophase. However, there appears to be no specifically differentiated region of any bivalent with which it is generally associated. The nuclear body may be associated with terminal or interstitial regions of bivalents which differ in size or chromomere pattern. It was more frequently associated with bivalents in some individuals than others. 7. The nuclear body was not found in mitotic divisions in shoot or root tips and thus appears to be related specifically to the meiotic divisions. It may arise as an exudate from some chromosomes, or it may be extruded from the nucleolar material; it seems less likely to arise from the karyolymph. It may be composed of some materials discarded in preparation for meiosis or during its earliest stages, which are perhaps related to concurrent changes in chromosome organization. 8. Observations are considered which suggest that a structure similar to the nuclear body observed in Bromus may be present in other plant genera.