Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing

The expression of short hairpin RNAs in several organisms silences gene expression by targeted mRNA degradation. This RNA interference (RNAi) pathway can also affect the genome, as DNA methylation arises at loci homologous to the target RNA in plants. We demonstrate in fission yeast that expression of a synthetic hairpin RNA is sufficient to silence the homologous locus in trans and causes the assembly of a patch of silent Swi6 chromatin with cohesin. This requires components of the RNAi machinery and Clr4 histone methyltransferase for small interfering RNA generation. A similar process represses several meiotic genes through nearby retrotransposon long terminal repeats (LTRs). These analyses directly implicate interspersed LTRs in regulating gene expression during cellular differentiation.     

RNAi-independent heterochromatin nucleation by the stress-activated ATF/CREB family proteins

At the silent mating-type interval of fission yeast, the RNA interference (RNAi) machinery cooperates with cenH, a DNA element homologous to centromeric repeats, to initiate heterochromatin formation. However, in RNAi mutants, heterochromatin assembly can still occur at low efficiency. Here, we report that Atf1 and Pcr1, two ATF/CREB family proteins, act in a parallel mechanism to the RNAi pathway for heterochromatin nucleation. Deletion of atf1 or pcr1 alone has little effect on silencing at the mating-type region, but when combined with RNAi mutants, double mutants fail to nucleate heterochromatin assembly. Moreover, deletion of atf1 or pcr1 in combination with cenH deletion causes loss of silencing and heterochromatin formation. Furthermore, Atf1 and Pcr1 bind to the mating-type region and target histone H3 lysine-9 methylation and the Swi6 protein essential for heterochromatin assembly. These analyses link ATF/CREB family proteins, involved in cellular response to environmental stresses, to nucleation of constitutive heterochromatin.     

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.     

Distinct cohesin complexes organize meiotic chromosome domains

Meiotic cohesin complexes at centromeres behave differently from those along chromosome arms, but the basis for these differences has remained elusive. The fission yeast cohesin molecule Rec8 largely replaces its mitotic counterpart, Rad21/Scc1, along the entire chromosome during meiosis. Here we show that Rec8 complexes along chromosome arms contain Rec11, whereas those in the vicinity of centromeres have a different partner subunit, Psc3. The arm associated Rec8-Rec11 complexes are critical for meiotic recombination. The Rec8-Psc3 complexes comprise two different types of assemblies. First, pericentromeric Rec8-Psc3 complexes depend on histone methylation-directed heterochromatin for their localization and are required for cohesion during meiosis II. Second, central core Rec8-Psc3 complexes form independently of heterochromatin and are presumably required for establishing monopolar attachment at meiosis I. These findings define distinct modes of assembly and functions for cohesin complexes at different regions along chromosomes.     

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.     

The Release 5.1 annotation of Drosophila melanogaster heterochromatin

The repetitive DNA that constitutes most of the heterochromatic regions of metazoan genomes has hindered the comprehensive analysis of gene content and other functions. We have generated a detailed computational and manual annotation of 24 megabases of heterochromatic sequence in the Release 5 Drosophila melanogaster genome sequence. The heterochromatin contains a minimum of 230 to 254 protein-coding genes, which are conserved in other Drosophilids and more diverged species, as well as 32 pseudogenes and 13 noncoding RNAs. Improved methods revealed that more than 77% of this heterochromatin sequence, including introns and intergenic regions, is composed of fragmented and nested transposable elements and other repeated DNAs. Drosophila heterochromatin contains "islands" of highly conserved genes embedded in these "oceans" of complex repeats, which may require special expression and splicing mechanisms.     

Genomic islands and the ecology and evolution of Prochlorococcus

Prochlorococcus ecotypes are a useful system for exploring the origin and function of diversity among closely related microbes. The genetic variability between phenotypically distinct strains that differ by less that 1% in 16S ribosomal RNA sequences occurs mostly in genomic islands. Island genes appear to have been acquired in part by phage-mediated lateral gene transfer, and some are differentially expressed under light and nutrient stress. Furthermore, genome fragments directly recovered from ocean ecosystems indicate that these islands are variable among cooccurring Prochlorococcus cells. Genomic islands in this free-living photoautotroph share features with pathogenicity islands of parasitic bacteria, suggesting a general mechanism for niche differentiation in microbial species.     

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.     

DNA replication-independent silencing in S. cerevisiae

In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G(2)/M phase, which has been widely assumed to be DNA replication. To determine whether DNA replication through a silent mating-type locus, HMRa, is required for silencing to be established, we monitored heterochromatin formation at HMRa on a chromosome and on a nonreplicating extrachromosomal cassette as cells passed through S phase. Cells that passed through S phase established silencing at both the chromosomal HMRa locus and the extrachromosomal HMRa locus with equal efficiency. Thus, in contrast to the prevailing view, the establishment of silencing occurred in the absence of passage of the DNA replication fork through or near the HMR locus, but retained a cell cycle dependence.     

siRNA介导的染色质基因沉默

探讨了siRNA在染色质调控中的作用,指出siRNA可使靶DNA发生甲基化并参与异染色质的形成以抑制基因表达。     

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.     

Virus-specific splicing inhibitor in extracts from cells infected with HIV-1

Human immunodeficiency virus type 1 (HIV-1), in contrast with most other retroviruses, encodes trans-regulatory proteins for virus gene expression. It is shown in this study, by means of an in vitro splicing system, that nuclear extracts obtained from cells infected with HIV-1 contain a factor (or factors) that specifically inhibits splicing of a synthetic SP6/HIV pre-messenger RNA (pre-mRNA)-containing donor and acceptor splice sites in the coding region for the envelope protein. It is also shown that the SP6/HIV pre-mRNA is not capable of assembly in a ribonucleoprotein complex, spliceosome, in extracts from infected cells. These findings raise the possibility that specific inhibition of pre-mRNA splicing in the envelope protein coding region by HIV-1 trans-regulatory factors might be one control mechanism for efficient production of structural viral proteins and virion assembly.     

Gene body-specific methylation on the active X chromosome

Differential DNA methylation is important for the epigenetic regulation of gene expression. Allele-specific methylation of the inactive X chromosome has been demonstrated at promoter CpG islands, but the overall pattern of methylation on the active X(Xa) and inactive X (Xi) chromosomes is unknown. We performed allele-specific analysis of more than 1000 informative loci along the human X chromosome. The Xa displays more than two times as much allele-specific methylation as Xi. This methylation is concentrated at gene bodies, affecting multiple neighboring CpGs. Before X inactivation, all of these Xa gene body-methylated sites are biallelically methylated. Thus, a bipartite methylation-demethylation program results in Xa-specific hypomethylation at gene promoters and hypermethylation at gene bodies. These results suggest a relationship between global methylation and expression potentiality.