曲古抑菌素A对核移植供体细胞组蛋白H3乙酰化水平的影响

目的探讨组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)对组蛋白H3乙酰化水平的影响,进而提高他们重编程的能力。方法用不同浓度的TSA处理胎儿成纤维细胞,应用免疫荧光和WesternBlot方法对细胞乙酰化水平进行量化分析。结果0.5nM,5nM,50nM和100nM浓度的TSA均引起供体成纤维细胞形态学的变化,对供体成纤维细胞组蛋白H3乙酰化有明显的剂量效应,随着TSA浓度增加,组蛋白H3乙酰化水平逐渐增加,在TSA浓度5nM时比0.5nM增加尤为明显。结论TSA能够提高胎儿成纤维细胞组蛋白H3乙酰化水平,提高核移植重编程的能力。     

Heterochromatin in interphase nuclei of Arabidopsis thaliana

The eukaryotic nucleus represents a complex arrangement of heterochromatic and euchromatic domains, each with their specific nuclear functions. Somatic cells of a multicellular organism are genetically identical, yet they may differ completely in nuclear organization and gene expression patterns. Stable changes in gene expression without modifying the sequence are the result of epigenetic changes and include covalent modifications in cytosine residues of DNA and in histone tails giving rise to altered chromatin protein complexes, remodeling of chromatin and changes in chromatin compaction. Large-scale differences in chromatin structure are visible at the microscopic level as euchromatin and heterochromatin. Arabidopsis thaliana chromosomes display a relatively simple distribution of euchromatic and heterochromatic segments overlapping with gene-rich and repeat-rich regions, respectively. Recently, we have shown that Arabidopsis provides a well-defined system to study individual chromosomes and chromatin domains in interphase nuclei as well as the relationship between chromatin condensation and epigenetic mechanisms of gene silencing. This overview focuses on the organization and composition of heterochromatin in Arabidopsis nuclei.     

Methylation of histone H4 by arginine methyltransferase PRMT1 is essential in vivo for many subsequent histone modifications

PRMT1 is a histone methyltransferase that methylates Arg3 on histone H4. When we used siRNA to knock down PRMT1 in an erythroid cell line, it resulted in nearly complete loss of H4 Arg3 methylation across the chicken beta-globin domain, which we use as a model system for studying the relationship of gene activity to histone modification. We observed furthermore a domain-wide loss of histone acetylation on both histones H3 and H4, as well as an increase in H3 Lys9 and Lys27 methylation, both marks associated with inactive chromatin. To determine whether the effect on acetylation was directly related to the loss of H4 Arg3 methylation, we performed an in vitro acetylation reaction on chromatin isolated from PRMT1-depleted cells. We found that nucleosomes purified from these cells, and depleted in methylation at Arg3, are readily acetylated by nuclear extracts from the same cells, if and only if the nucleosomes are incubated with PRMT1 beforehand. Thus, methylation of histones by PRMT1 was sufficient to permit subsequent acetylation. Consistent with earlier reports of experiments in vitro, H4 Arg3 methylation by PRMT1 appears to be essential in vivo for the establishment or maintenance of a wide range of "active" chromatin modifications.     

Fission yeast CENP-B homologs nucleate centromeric heterochromatin by promoting heterochromatin-specific histone tail modifications

Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1. However, the primary determinants of the positioning of heterochromatin are still unclear. The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric alpha-satellite DNA and associate with centromeric heterochromatin. We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin. In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region. CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6. Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3. These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails.     

The effects of acetaldehyde exposure on histone modifications and chromatin structure in human lung bronchial epithelial cells

As the primary metabolite of alcohol and the most abundant carcinogen in tobacco smoke, acetaldehyde is linked to a number of human diseases associated with chronic alcohol consumption and smoking including cancers. In addition to direct DNA damage as a result of the formation of acetaldehyde‐DNA adducts, acetaldehyde may also indirectly impact proper genome function through the formation of protein adducts. Histone proteins are the major component of the chromatin. Post‐translational histone modifications (PTMs) are critically important for the maintenance of genetic and epigenetic stability. However, little is known about how acetaldehyde‐histone adducts affect histone modifications and chromatin structure. The results of protein carbonyl assays suggest that acetaldehyde forms adducts with histone proteins in human bronchial epithelial BEAS‐2B cells. The level of acetylation for N‐terminal tails of cytosolic histones H3 and H4, an important modification for histone nuclear import and chromatin assembly, is significantly downregulated following acetaldehyde exposure in BEAS‐2B cells, possibly due to the formation of histone adducts and/or the decrease in the expression of histone acetyltransferases. Notably, the level of nucleosomal histones in the chromatin fraction and at most of the genomic loci we tested are low in acetaldehyde‐treated cells as compared with the control cells, which is suggestive of inhibition of chromatin assembly. Moreover, acetaldehyde exposure perturbs chromatin structure as evidenced by the increase in general chromatin accessibility and the decrease in nucleosome occupancy at genomic loci following acetaldehyde treatment. Our results indicate that regulation of histone modifications and chromatin accessibility may play important roles in acetaldehyde‐induced pathogenesis. Environ. Mol. Mutagen. 59:375–385, 2018. © 2018 Wiley Periodicals, Inc.     

Nano-scale analyses of the chromatin decompaction induced by histone acetylation

The acetylation of histone tails is a key factor in the maintenance of chromatin dynamics and cellular homeostasis. The hallmark of active chromatin is the hyper-acetylation of histones, which appears to result in a more open chromatin structure. Although short nucleosomal arrays have been studied, the structural dynamics of relatively long acetylated chromatin remain unclear. We have analyzed in detail the structure of long hyper-acetylated chromatin fibers using atomic force microscopy (AFM). Hyper-acetylated chromatin fibers isolated from nuclei that had been treated with Trichostatin A (TSA), an inhibitor of histone deacetylase, were found to be thinner than those from untreated nuclei. The acetylated chromatin fibers were more easily spread out of nuclei by high-salt treatment, implying that hyper-acetylation facilitates the release of chromatin fibers from compact heterochromatin regions. Chromatin fibers reconstituted in vitro from core histones and linker histone H1 became thinner upon acetylation. AFM imaging indicated that the gyration radius of the nucleosomal fiber increased after acetylation and that the hyper-acetylated nucleosomes did not aggregate at high salt concentrations, in contrast to the behavior of non-acetylated nucleosomal arrays, suggesting that acetylation increases long-range repulsions between nucleosomes. Based on these data, we considered a simple coarse grained model, which underlines the effect of remaining electric charges inside the chromatin fiber.     

The roles of histone modifications and small RNA in centromere function

Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi). Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function. The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion. The chromatin structure of both domains is regulated epigenetically. In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role. In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein. Swi6 then directly mediates cohesin binding to the centromeric heterochromatin. Recently, a surprising link was made between heterochromatin formation and RNAi.     

Epigenome and chromatin structure in human embryonic stem cells undergoing differentiation

Epigenetic histone (H3) modification patterns and the nuclear radial arrangement of select genetic elements were compared in human embryonic stem cells (hESCs) before and after differentiation. H3K9 acetylation, H3K9 trimethylation, and H3K79 monomethylation were reduced at the nuclear periphery of differentiated hESCs. Differentiation coincided with centromere redistribution, as evidenced by perinucleolar accumulation of the centromeric markers CENP‐A and H3K9me3, central repositioning of centromeres 1, 5, 19, and rearrangement of other centromeres at the nuclear periphery. The radial positions of PML, RARα genes, and human chromosomes 10, 12, 15, 17, and 19 remained relatively stable as hESCs differentiated. However, the female inactive H3K27‐trimethylated X chromosome occupied a more peripheral nuclear position in differentiated cells. Thus, pluripotent and differentiated hESCs have distinct nuclear patterns of heterochromatic structures (centromeres and inactive X chromosome) and epigenetic marks (H3K9me3, and H3K27me3), while relatively conserved gene density‐related radial chromatin distributions are already largely established in undifferentiated hES cells. Developmental Dynamics 237:3690–3702, 2008. © 2008 Wiley‐Liss, Inc.     

Release of yeast telomeres from the nuclear periphery is triggered by replication and maintained by suppression of Ku-mediated anchoring

The perinuclear localization of Saccharomyces cerevisiae telomeres provides a useful model for studying mechanisms that control chromosome positioning. Telomeres tend to be localized at the nuclear periphery during early interphase, but following S phase they delocalize and remain randomly positioned within the nucleus. We investigated whether DNA replication causes telomere delocalization from the nuclear periphery. Using live-cell fluorescence microscopy, we show that delaying DNA replication causes a corresponding delay in the dislodgment of telomeres from the nuclear envelope, demonstrating that replication of individual telomeres causes their delocalization. Telomere delocalization is not simply the result of recruitment to a replication factory in the nuclear interior, since we found that telomeric DNA replication can occur either at the nuclear periphery or in the nuclear interior. The telomere-binding complex Ku is one of the factors that localizes telomeres to the nuclear envelope. Using a gene locus tethering assay, we show that Ku-mediated peripheral positioning is switched off after DNA replication. Based on these findings, we propose that DNA replication causes telomere delocalization by triggering stable repression of the Ku-mediated anchoring pathway. In addition to maintaining genetic information, DNA replication may therefore regulate subnuclear organization of chromatin.