Establishment of Histone Modifications after Chromatin Assembly

Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin ‘matures’ and adopts a variety of different conformations. This differential packaging of DNA plays an important role for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine (¹⁵N₄) that is 4 Da heavier than the naturally occurring ¹⁴N₄ isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins. This method allows a comparison of modification patterns on parental versus newly deposited histones. Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones.     

Reprogramming of Meiotic Chromatin Architecture during Spermatogenesis

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The Role of Chromatin Modifications in Progression through Mouse Meiotic Prophase

Meiosis is a key event in gametogenesis that generates new combinations of genetic information and is required to reduce the chro- mosome content of the gametes. Meiotic chromosomes undergo a number of specialised events during prophase to allow meiotic recombination, homologous chromosome synapsis and reductional chromosome segregation to occur. In mammalian cells, DNA phys- ically associates with histones to form chromatin, which can be modified by methylation, phosphorylation, ubiquitination and acetylation to help regulate higher order chromatin structure, gene expression, and chromosome organisation. Recent studies have identified some of the enzymes responsible for generating chromatin modifications in meiotic mammalian cells, and shown that these chromatin modifying enzymes are required for key meiosis-specific events that occur during meiotic prophase. This review will discuss the role of chromatin modifications in meiotic recombination, homologous chromosome synapsis and regulation of meiotic gene expression in mammals.     

Structural Variation-Associated Expression Changes Are Paralleled by Chromatin Architecture Modifications

Copy number variants (CNVs) influence the expression of genes that map not only within the rearrangement, but also to its flanks. To assess the possible mechanism(s) underlying this “neighboring effect”, we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. Using chromosome conformation capture (4C-seq), we observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together. The newly identified interacting genes include AUTS2, mutations of which are associated with autism and intellectual disabilities. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We also pinpointed concomitant changes in histone modifications between samples.We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thereby possibly modulating expression globally and modifying the phenotype.GEO Series accession number: {"type":"entrez-geo","attrs":{"text":"GSE33784","term_id":"33784"}}GSE33784, {"type":"entrez-geo","attrs":{"text":"GSE33867","term_id":"33867"}}GSE33867.     

应用原子力显微镜对玉米染色质纤丝及染色体骨架的研究(英文)

应用原子力显微镜对经化学方法预处理的玉米染色体超微结构进行了研究。原子力显微镜观察的结果揭示,经30%醋酸处理的玉米染色体表面呈现出不均一的颗粒状结构。当缩小扫描范围后,在染色体表面发现直径分别为30和100 nm的两种染色质纤丝。100 nm的染色质纤丝由30 nm染色质纤丝螺旋缠绕而成。在经25%胰酶处理的玉米染色体上发现了两种类似的螺旋状染色质纤丝结构,其直径分别为30和100～150 nm。较细的染色质纤丝螺旋缠绕成较粗的纤丝,进而构成整个染色体。经低离子浓度溶液抽提,用原子力显微镜观察到了玉米染色体的染色体骨架结构。这种染色体骨架呈不规则的纤维网状,这些网状纤维在染色体中部显得较为紧密,在染色体的边缘则显得较松散。这一结果暗示染色体是由不同级别的染色质纤丝螺旋缠绕构成,为染色体的多级螺旋结构假说提供了新的证据。同时发现染色体骨架并不是呈轴样的结构存在,而是保留了染色体的基本形态,这种骨架形状也许是由分散在染色体中的染色体骨架蛋白在低离子浓度溶液抽提的过程中凝缩形成的。     

Nonredundant Requirement for Multiple Histone Modifications for the Early Anaphase Release of the Mitotic Exit Regulator Cdc14 from Nucleolar Chromatin

In Saccharomyces cerevisiae, the conserved phosphatase Cdc14 is required for the exit from mitosis. It is anchored on nucleolar chromatin by the Cfi1/Net1 protein until early anaphase, at which time it is released into the nucleoplasm. Two poorly understood, redundant pathways promote Cdc14 release, the FEAR (Cdc fourteen early release) network and the MEN (mitotic exit network). Through the analysis of genetic interactions, we report here a novel requirement for the ubiquitination of histone H2B by the Bre1 ubiquitin ligase in the cell cycle–dependent release of Cdc14 from nucleolar chromatin when the MEN is inactivated. This function for H2B ubiquitination is mediated by its activation of histone H3 methylation on lysines 4 and 79 (meH3K4 and meH3K79) but, surprisingly, is not dependent on the histone deacetylase (HDAC) Sir2, which associates with Cdc14 on nucleolar chromatin as part of the RENT complex. We also observed a defect in Cdc14 release in cells lacking H3 lysine 36 methylation (meH3K36) and in cells lacking an HDAC recruited by this modification. These histone modifications represent previously unappreciated factors required for the accessibility to and/or action on nucleolar chromatin of FEAR network components. The nonredundant role for these modifications in this context contrasts with the notion of a highly combinatorial code by which histone marks act to control biological processes.     

Plant Chromatin Structure and Post-Translational Modifications

Although a majority of the key works on chromatin structure and function have been carried out using animal tissues, studies of plant chromatin and the characterization of the histones and nonhistone chromosomal proteins are now developing well. There are clear functional differences between plant and animal genomes, including the percentage of total DNA transcribed, levels of ploidy, and the pathways of morphogenesis and cell differentiation. It is therefore not surprising that differences are appearing between animal and plant chromatin, for example, the consensus amino acid sequence for the plant H3 globular domain; the extensions to the basic domain regions of some plant histones such as H2A, which have specific interactions with linker DNA; the larger molecular weight of the plant H1 molecule with its extended basic domains correlated with short lengths of linker DNA, and the absence of the five residue binding segment in the globular part of plant H1, which suggests differences in the organization of higher order structure in plant chromatin. There are also unifying features between plant and animal chromatin, and the nature of plant material makes its study particularly advantageous in several areas. The regular nucleosome repeat and short lengths of linker DNA in some plants should provide more regular order structures for study, in which in the near absence of linker DNA, nucleosome position is the main, if not sole, determining factor in model building. However, the improved characterization and isolation of plant chromatin and associated molecules, for example, the isolation of the SPKK kinase gene in pea, are essential if major progress is to be made in our understanding of functional activities.     

Histone Phosphorylation and Pericentromeric Histone Modifications in Oocyte Meiosis

Epigenetic regulation of pericentromeric heterochromatin is crucial for proper interactions between kinetochores and spindle microtubules governing accurate chromosome segregation. Here, we first examined the dynamic distribution of phosphorylated serine 10 and 28 on H3 during mouse oocyte maturation and early embryo development using immunofluorescent staining and confocal microscopy. Our results revealed strong signals of phosphorylated H3/ser10 and 28 in the pericentromeric heterochromatin area and continuous persistent staining of the chromosome periphery, respectively. A panel of specific antibodies against various acetylated lysine, dimethylated lysine or phosphorylated serine residues on histone H3 or H4 were used to investigate the effects of Trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), on histone modifications of pericentromeric heterochromatin. Unexpectedly, TSA treatment was unable to alter the acetylation and methylation status of pericentromeric heterochromatin, however, it resulted in significant dephosphorylation of H3/ser10 at this site during mouse oocyte meiosis, which is likely to play a role in the TSA-induced defective chromosome segregation. Furthermore, by using ZM447439, an inhibitor of Aurora kinases, we revealed that Aurora kinases may participate in the regulation of histone phosphorylation during mouse oocyte maturation.     

Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals

Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities.     

A Lamin-Associated Chromatin Model for Chromosome Organization

We propose a simple model for chromatin organization based on the interaction of the chromatin fibers with lamin proteins along the nuclear membrane. Lamin proteins are known to be a major factor that influences chromatin organization and hence gene expression in the cells. We provide a quantitative understanding of lamin-associated chromatin organization in a crowded macromolecular environment by systematically varying the heteropolymer segment distribution and the strength of the lamin-chromatin attractive interaction. Our minimal polymer model reproduces the formation of lamin-associated-domains and provides an in silico tool for quantifying domain length distributions for different distributions of heteropolymer segments. We show that a Gaussian distribution of heteropolymer segments, coupled with strong lamin-chromatin interactions, can qualitatively reproduce observed length distributions of lamin-associated-domains. Further, lamin-mediated interaction can enhance the formation of chromosome territories as well as the organization of chromatin into tightly packed heterochromatin and the loosely packed gene-rich euchromatin regions.     

Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome

The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.