Establishment and maintenance of a heterochromatin domain

The higher-order assembly of chromatin imposes structural organization on the genetic information of eukaryotes and is thought to be largely determined by posttranslational modification of histone tails. Here, we study a 20-kilobase silent domain at the mating-type region of fission yeast as a model for heterochromatin formation. We find that, although histone H3 methylated at lysine 9 (H3 Lys9) directly recruits heterochromatin protein Swi6/HP1, the critical determinant for H3 Lys9 methylation to spread in cis and to be inherited through mitosis and meiosis is Swi6 itself. We demonstrate that a centromere-homologous repeat (cenH) present at the silent mating-type region is sufficient for heterochromatin formation at an ectopic site, and that its repressive capacity is mediated by components of the RNA interference (RNAi) machinery. Moreover, cenH and the RNAi machinery cooperate to nucleate heterochromatin assembly at the endogenous mat locus but are dispensable for its subsequent inheritance. This work defines sequential requirements for the initiation and propagation of regional heterochromatic domains.     

Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery

Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV). Loss-of-function mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase. Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9 methylation and delocalization of HP1 and HP2, most dramatically in spindle-E mutants.     

Chromatin and histones: binding of tritiated actinomycin D to heterochromatin in mealy bugs

The degree of actinomycin D binding to DNA in chromatin is dependent upon the state of repression of chromatin. Living cells bind three times more tritiated actinomycin to euchromatin than to genetically inactive heterochromatin. Extraction of histone results in a general increase in tritiated actinomycin binding and in a ratio of the uptake in heterochromatin to that in euchromatin approaching unity.     

Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly

The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails. We provide in vivo evidence that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. Moreover, an H3-specific deacetylase Clr3 and a beta-propeller domain protein Rik1 are required for H3 Lys9 methylation by Clr4 and Swi6 localization. These data define a conserved pathway wherein sequential histone modifications establish a "histone code" essential for the epigenetic inheritance of heterochromatin assembly.     

Requirement of heterochromatin for cohesion at centromeres

Centromeres are heterochromatic in many organisms, but the mitotic function of this silent chromatin remains unknown. During cell division, newly replicated sister chromatids must cohere until anaphase when Scc1/Rad21-mediated cohesion is destroyed. In metazoans, chromosome arm cohesins dissociate during prophase, leaving centromeres as the only linkage before anaphase. It is not known what distinguishes centromere cohesion from arm cohesion. Fission yeast Swi6 (a Heterochromatin protein 1 counterpart) is a component of silent heterochromatin. Here we show that this heterochromatin is specifically required for cohesion between sister centromeres. Swi6 is required for association of Rad21-cohesin with centromeres but not along chromosome arms and, thus, acts to distinguish centromere from arm cohesion. Therefore, one function of centromeric heterochromatin is to attract cohesin, thereby ensuring sister centromere cohesion and proper chromosome segregation.     

Heterochromatin and epigenetic control of gene expression

Eukaryotic DNA is organized into structurally distinct domains that regulate gene expression and chromosome behavior. Epigenetically heritable domains of heterochromatin control the structure and expression of large chromosome domains and are required for proper chromosome segregation. Recent studies have identified many of the enzymes and structural proteins that work together to assemble heterochromatin. The assembly process appears to occur in a stepwise manner involving sequential rounds of histone modification by silencing complexes that spread along the chromatin fiber by self-oligomerization, as well as by association with specifically modified histone amino-terminal tails. Finally, an unexpected role for noncoding RNAs and RNA interference in the formation of epigenetic chromatin domains has been uncovered.     

Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1

The Arabidopsis gene DDM1 is required to maintain DNA methylation levels and is responsible for transposon and transgene silencing. However, rather than encoding a DNA methyltransferase, DDM1 has similarity to the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes, suggesting an indirect role in DNA methylation. Here we show that DDM1 is also required to maintain histone H3 methylation patterns. In wild-type heterochromatin, transposons and silent genes are associated with histone H3 methylated at lysine 9, whereas known genes are preferentially associated with methylated lysine 4. In ddm1 heterochromatin, DNA methylation is lost, and methylation of lysine 9 is largely replaced by methylation of lysine 4. Because DNA methylation has recently been shown to depend on histone H3 lysine 9 methylation, our results suggest that transposon methylation may be guided by histone H3 methylation in plant genomes. This would account for the epigenetic inheritance of hypomethylated DNA once histone H3 methylation patterns are altered.     

RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation

Facultative heterochromatin that changes during cellular differentiation coordinates regulated gene expression, but its assembly is poorly understood. Here, we describe facultative heterochromatin islands in fission yeast and show that their formation at meiotic genes requires factors that eliminate meiotic messenger RNAs (mRNAs) during vegetative growth. Blocking production of meiotic mRNA or loss of RNA elimination factors, including Mmi1 and Red1 proteins, abolishes heterochromatin islands. RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly. Heterochromatin islands disassemble in response to nutritional signals that induce sexual differentiation. This process involves the antisilencing factor Epe1, the loss of which causes dramatic increase in heterochromatic loci. Our analyses uncover unexpected regulatory roles for mRNA-processing factors that assemble dynamic heterochromatin to modulate gene expression.     

Chromosome organization and heterochromatin elimination in parascaris

A cytological analysis by modern banding techniques of gonial metaphases in two Parascaris forms that have been considered varieties but now seem to be two species [P. univalens (karyotype 2n = 2) and P. equorum (karyotype 2n = 4)] reveals a different chromosome organization in each. Parascaris univalens chromosomes contain only terminal heterochromatin, while P. equorum chromosomes also contain intercalary heterochromatin. In the somatic cells of both species during early embryogenesis, chromatin diminution occurs in and consists of the elimination of all heterochromatin independent of its localization in the chromosomes.     

Control of genic DNA methylation by a jmjC domain-containing protein in Arabidopsis thaliana

Differential cytosine methylation of repeats and genes is important for coordination of genome stability and proper gene expression. Through genetic screen of mutants showing ectopic cytosine methylation in a genic region, we identified a jmjC-domain gene, IBM1 (increase in bonsai methylation 1), in Arabidopsis thaliana. In addition to the ectopic cytosine methylation, the ibm1 mutations induced a variety of developmental phenotypes, which depend on methylation of histone H3 at lysine 9. Paradoxically, the developmental phenotypes of the ibm1 were enhanced by the mutation in the chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), which is necessary for keeping methylation and silencing of repeated heterochromatin loci. Our results demonstrate the importance of chromatin remodeling and histone modifications in the differential epigenetic control of repeats and genes.     

The ligase PIAS1 restricts natural regulatory T cell differentiation by epigenetic repression

CD4(+)Foxp3(+) regulatory T (T(reg)) cells are important for maintaining immune tolerance. Understanding the molecular mechanism that regulates T(reg) differentiation will facilitate the development of effective therapeutic strategies against autoimmune diseases. We report here that the SUMO E3 ligase PIAS1 restricts the differentiation of natural T(reg) cells by maintaining a repressive chromatin state of the Foxp3 promoter. PIAS1 acts by binding to the Foxp3 promoter to recruit DNA methyltransferases and heterochromatin protein 1 for epigenetic modifications. Pias1 deletion caused promoter demethylation, reduced histone H3 methylation at Lys(9), and enhanced promoter accessibility. Consistently, Pias1(-/-) mice displayed an increased natural T(reg) cell population and were resistant to the development of experimental autoimmune encephalomyelitis. Our studies have identified an epigenetic mechanism that negatively regulates the differentiation of natural T(reg) cells.     

马立克氏病毒DNA转录区和非转录区甲基化及脱甲基化的探讨

实验以鸡马立克氏病脾脏淋巴瘤继代细胞系MDCC MSB 1和感染马立克氏病毒的鸡胚成纤维细胞(CEF)为主要试验材料,作为参考系也使用了鸡马立克氏病卵巢淋巴瘤继代细胞系MDCC JP1、MDCC JP2、MDCC HP1及MDCC HP2细胞。用Suothern斑点分子杂交放射自显影等方法,验证了MDCC MSB 1等细胞株DNA的转录区和非转录区都存在着甲基化现象。转录区甲基化程度高于非转录区。用5 氮胞苷可以阻断DNA甲基化,阻断效果在转录区最好。     

植物LHP1同源基因研究进展

拟南芥LIKE HETEROCHROMATIN PROTEIN 1 (LHP1)/TERMINAL FLOWER 2(TFL2)基因被认为是后生动物HP1的同系物.在动物中,HP1通常参与异染色质形成及基因沉默.与HP1不同,LHP1定位于常染色质并抑制位于常染色质中众多基因的表达.拟南芥LHP1/TFL2突变体表现出多重表型,如早花、降低对光周期敏感性、顶端花序和植株矮小.目前对LHP1的研究特别是在单子叶植物水稻中的研究,仅限于蛋白表达谱差异,而未在基因序列及基因表达调控水平上作深入的研究.鉴于拟南芥LHP1的重要功能,今后需对LHP1同源基因在不同物种特别是单子叶植物水稻中的功能进行深入研究;此外,还应加强不同物种LHP1进化的关系分析、LHP1在不同物种整个生育期的表达谱、单子叶植物水稻中lhp1突变体的功能表达及LHP1在开花调控网络中的定位等方面的研究.     

A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells

E2F-6 contributes to gene silencing in a manner independent of retinoblastoma protein family members. To better elucidate the molecular mechanism of repression by E2F-6, we have purified the factor from cultured cells. E2F-6 is found in a multimeric protein complex that contains Mga and Max, and thus the complex can bind not only to the E2F-binding site but also to Myc- and Brachyury-binding sites. Moreover, the complex contains chromatin modifiers such as a novel histone methyltransferase that modifies lysine 9 of histone H3, HP1gamma, and Polycomb group (PcG) proteins. The E2F-6 complex preferentially occupies target promoters in G0 cells rather than in G1 cells. These data suggest that these chromatin modifiers contribute to silencing of E2F- and Myc-responsive genes in quiescent cells.