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Showing papers on "Heterochromatin published in 2005"


Journal ArticleDOI
21 Oct 2005-Cell
TL;DR: In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread and enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci.

710 citations


Journal ArticleDOI
TL;DR: Analysis of composition and mode of assembly of SAHF indicates that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.

657 citations


Journal ArticleDOI
11 Mar 2005-Cell
TL;DR: In this article, the authors show that Pol IV helps produce siRNAs that target de novo cytosine methylation events required for facultative heter-chromatin formation and higher-order heter-romatin associations.

641 citations


Journal ArticleDOI
TL;DR: The data suggest that TFL2/LHP1 recognizes specifically H3K27me3 in vivo as part of a mechanism that represses the expression of many genes targeted by PRC2.
Abstract: TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) is the only Arabidopsis protein with overall sequence similarity to the HETEROCHROMATIN PROTEIN 1 (HP1) family of metazoans and S. pombe. TFL2/LHP1 represses transcription of numerous genes, including the flowering-time genes FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC), as well as the floral organ identity genes AGAMOUS (AG) and APETALA 3 (AP3). These genes are also regulated by proteins of the Polycomb repressive complex 2 (PRC2), and it has been proposed that TFL2/LHP1 represents a potential stabilizing factor of PRC2 activity. Here we show by chromatin immunoprecipitation and hybridization to an Arabidopsis Chromosome 4 tiling array (ChIP-chip) that TFL2/LHP1 associates with hundreds of small domains, almost all of which correspond to genes located within euchromatin. We investigated the chromatin marks to which TFL2/LHP1 binds and show that, in vitro, TFL2/LHP1 binds to histone H3 di- or tri-methylated at lysine 9 (H3K9me2 or H3K9me3), the marks recognized by HP1, and to histone H3 trimethylated at lysine 27 (H3K27me3), the mark deposited by PRC2. However, in vivo TFL2/LHP1 association with chromatin occurs almost exclusively and co-extensively with domains marked by H3K27me3, but not H3K9me2 or -3. Moreover, the distribution of H3K27me3 is unaffected in lhp1 mutant plants, indicating that unlike PRC2 components, TFL2/LHP1 is not involved in the deposition of this mark. Rather, our data suggest that TFL2/LHP1 recognizes specifically H3K27me3 in vivo as part of a mechanism that represses the expression of many genes targeted by PRC2.

577 citations


Journal ArticleDOI
TL;DR: The analyses reveal that transcriptomes of Arabidopsis seed are characterized by multiple regulatory mechanisms: epigenetic chromatin structures, chromosomal locations (e.g. co-regulated gene clusters) and cis-acting elements, which suggest that such regions might be transcriptionally silenced by methylation or heterochromatin structures.
Abstract: To reveal the transcriptomes of Arabidopsis seed, comprehensive expression analysis was performed using ATH1 GeneChips (Affymetrix, Santa Clara, CA, USA). In the dry seed, more than 12 000 stored mRNA species were detected, including all ontological categories. Statistical analysis revealed that promoters of highly expressed genes in wild-type dry seeds overrepresented abscisic acid-responsive elements (ABREs) containing the core motif ACGT. Although the coupling element and seed-specific enhancer RY motif alone were not prominently overrepresented in genes with high expression, the presence of these elements in combination with ABRE was associated with particularly high gene expression. The transcriptome of the imbibed seeds differed from that of the dry seed even at 6 h after seed imbibition. After imbibition many upregulated and downregulated genes were co-regulated in clusters of three to five genes. Genes for which expression was affected by the abi5 mutation tended to be located in clusters, suggesting that transactivation by ABI5 is not restricted to a single gene, but affects other proximal genes. Furthermore, cytosine methylation was observed not only in large silent retrotransposon clusters in centromeric regions, but also in non-centromeric silent gene clusters in the seed. These results suggest that such regions might be transcriptionally silenced by methylation or heterochromatin structures. Our analyses reveal that transcriptomes of Arabidopsis seed are characterized by multiple regulatory mechanisms: epigenetic chromatin structures, chromosomal locations (e.g. co-regulated gene clusters) and cis-acting elements.

526 citations


Journal ArticleDOI
TL;DR: In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences, so it may have limited impact on the implementation of some lineage-specific gene expression programs.
Abstract: The ribonuclease III enzyme Dicer is essential for the processing of micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability, and translation in a wide range of organisms. To provide a model system to explore the role of Dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional Dicer allele. Deletion of Dicer at an early stage of T cell development compromised the survival of αβ lineage cells, whereas the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin), and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one third of mammalian mRNAs are putative miRNA targets, Dicer seems to be dispensable for CD4/8 lineage commitment, a process in which epigenetic regulation of lineage choice has been well documented. Thus, although Dicer seems to be critical for the development of the early embryo, it may have limited impact on the implementation of some lineage-specific gene expression programs.

508 citations


Journal ArticleDOI
TL;DR: It is found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs and an important role for the RNAi machinery in maintaining genomic integrity is uncovered.
Abstract: The organization of eukaryotic genomes into distinct structural and functional domains is important for the regulation and transduction of genetic information. Here, we investigated heterochromatin and euchromatin profiles of the entire fission yeast genome and explored the role of RNA interference (RNAi) in genome organization. Histone H3 methylated at Lys4, which defines euchromatin, was not only distributed across most of the chromosomal landscape but was also present at the centromere core, the site of kinetochore assembly. In contrast, histone H3 methylated at Lys9 and its interacting protein Swi6/HP1, which define heterochromatin, coated extended domains associated with a variety of repeat elements and small islands corresponding to meiotic genes. Notably, RNAi components were distributed throughout all these heterochromatin domains, and their localization depended on Clr4/Suv39h histone methyltransferase. Sequencing of small interfering RNAs (siRNAs) associated with the RITS RNAi effector complex identified hot spots of siRNAs, which mapped to a diverse array of elements in these RNAi-heterochromatin domains. We found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs. Our analyses also uncover an important role for the RNAi machinery in maintaining genomic integrity.

484 citations


Journal ArticleDOI
08 Apr 2005-Cell
TL;DR: The data demonstrate that mutually exclusive transcription of var genes is linked to the dynamic remodeling of chromatin, and this work identifies chromatin modifications at telomeres that spread far into telomere-proximal regions, including var gene loci (>50 kb).

465 citations


Journal ArticleDOI
TL;DR: It is shown that Clr3, a fission yeast homolog of mammalian class II HDACs, acts in a distinct pathway parallel to RNAi-directed heterochromatin nucleation to recruit Clr4 and mediate H3K9 methylation at the silent mating-type region and centromeres and that it limits RNA polymerase II accessibility to naturally silenced repeats at heterochromaatin domains.

316 citations


Journal ArticleDOI
TL;DR: It is shown that a point mutation within the catalytic domain of Rdp1 abolished its RNA-dependent RNA polymerase activity and resulted in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering.
Abstract: In fission yeast, factors involved in the RNA interference (RNAi) pathway including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for heterochromatin assembly at centromeric repeats and the silent mating-type region. Previously, we have shown that RNA-induced initiation of transcriptional gene silencing (RITS) complex containing the Argonaute protein and small interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism to couple siRNA generation with heterochromatin formation. Here, we investigate the role of RNA-dependent RNA polymerase (Rdp1) and its polymerase activity in the assembly of heterochromatin. We find that Rdp1, similar to RITS, localizes to all known heterochromatic loci, and its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins. We show that a point mutation within the catalytic domain of Rdp1 abolished its RNA-dependent RNA polymerase activity and resulted in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering. Moreover, the RITS complex in the rdp1 mutant does not contain siRNAs, and is delocalized from centromeres. These results not only implicate Rdp1 as an essential component of a self-enforcing RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase activity in siRNA production necessary for heterochromatin formation.

311 citations


Journal ArticleDOI
TL;DR: The ‘functionalist’ perspective on repetitive DNA leads to new ways of thinking about the systemic organisation of cellular genomes and provides several novel possibilities involving repeat elements in evolutionarily significant genome reorganisation.
Abstract: There are clear theoretical reasons and many well-documented examples which show that repetitive DNA is essential for genome function. Generic repeated signals in the DNA are necessary to format expression of unique coding sequence files and to organise additional functions essential for genome replication and accurate transmission to progeny cells. Repetitive DNA sequence elements are also fundamental to the cooperative molecular interactions forming nucleoprotein complexes. Here, we review the surprising abundance of repetitive DNA in many genomes, describe its structural diversity, and discuss dozens of cases where the functional importance of repetitive elements has been studied in molecular detail. In particular, the fact that repeat elements serve either as initiators or boundaries for heterochromatin domains and provide a significant fraction of scaffolding/matrix attachment regions (S/MARs) suggests that the repetitive component of the genome plays a major architectonic role in higher order physical structuring. Employing an information science model, the ‘functionalist’ perspective on repetitive DNA leads to new ways of thinking about the systemic organisation of cellular genomes and provides several novel possibilities involving repeat elements in evolutionarily significant genome reorganisation. These ideas may facilitate the interpretation of comparisons between sequenced genomes, where the repetitive DNA component is often greater than the coding sequence component.

Journal ArticleDOI
TL;DR: It is shown that the DM1 insulator is maintained in a local heterochromatin context: an antisense transcript emanating from the adjacent SIX5 regulatory region extends into the insulator element and is converted into 21 nucleotide fragments with associated regional histone H3 lysine 9 (H3-K9) methylation and HP1gamma recruitment.

Journal ArticleDOI
TL;DR: Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.
Abstract: The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L-/- germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L-/- meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Journal ArticleDOI
15 Jul 2005-Science
TL;DR: A mutation in the second largest subunit of RNA polymerase II (RNAPII) resulted in the loss of heterochromatic histone modifications and in the accumulation of pericentromeric transcripts, accompanied by the Loss of siRNAs, which suggests that RNAPII couples percentromeric transcription with siRNA processing and heterochROMatin assembly.
Abstract: In Schizosaccharomyces pombe, the RNA interference (RNAi) machinery converts pericentromeric transcripts into small interfering RNAs (siRNAs) and is required for the assembly of pericentromeric heterochromatin. Here we describe a mutation in the second largest subunit of RNA polymerase II (RNAPII). Both wild-type and mutant RNAPII localized to the pericentromere. However, the mutation resulted in the loss of heterochromatic histone modifications and in the accumulation of pericentromeric transcripts, accompanied by the loss of siRNAs. This phenotype resembles mutants in RNAi and suggests that RNAPII couples pericentromeric transcription with siRNA processing and heterochromatin assembly.

Journal ArticleDOI
TL;DR: The results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle.
Abstract: The initiation of DNA replication in S phase requires the prior assembly of an origin recognition complex (ORC)-dependent pre-replicative complex on chromatin during G1 phase of the cell division cycle. In human cells, the Orc2 subunit localized to the nucleus as expected, but it also localized to centrosomes throughout the entire cell cycle. Furthermore, Orc2 was tightly bound to heterochromatin and heterochromatin protein 1α (HP1α) and HP1β in G1 and early S phase, but during late S, G2 and M phases tight chromatin association was restricted to centromeres. Depletion of Orc2 by siRNA caused multiple phenotypes. A population of cells showed an S-phase defect with little proliferating cell nuclear antigen (PCNA) on chromatin, although MCM proteins remained. Orc2 depletion also disrupted HP1 localization, but not histone-H3-lysine-9 methylation at prominent heterochromatic foci. Another subset of Orc2-depleted cells containing replicated DNA arrested with abnormally condensed chromosomes, failed chromosome congression and multiple centrosomes. These results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle.

01 Jan 2005
TL;DR: In this article, a mutation in the second largest subunit of RNA polymerase II (RNAPII) was described, which resulted in the loss of heterochromatic histone modifications and in the accumulation of pericentromeric transcripts.
Abstract: In Schizosaccharomyces pombe, the RNA interference (RNAi) machinery converts pericentromeric transcripts into small interfering RNAs (siRNAs) and is required for the assembly of pericentromeric heterochromatin. Here we describe a mutation in the second largest subunit of RNA polymerase II (RNAPII). Both wild-type and mutant RNAPII localized to the pericentromere. However, the mutation resulted in the loss of heterochromatic histone modifications and in the accumulation of pericentromeric transcripts, accompanied by the loss of siRNAs. This phenotype resembles mutants in RNAi and suggests that RNAPII couples pericentromeric transcription with siRNA processing and heterochromatin assembly.

Journal ArticleDOI
TL;DR: Ch chromatin immunoprecipitation is used to show that HSV lytic-gene promoters become complexed with modified histones associated with heterochromatin during the course of establishment of latent infection and contribute to the persistence of its genome in a quiescent form in sensory neurons.
Abstract: Herpes simplex virus (HSV) persists in its human host and evades the immune response by undergoing a latent infection in sensory neurons, from which it can reactivate periodically. HSV expresses >80 gene products during productive (“lytic”) infection, but only the latency-associated transcript (LAT) gene is expressed at abundant levels during latent infection. The LAT gene has been shown to repress lytic-gene expression in sensory neurons. In this study, we use chromatin immunoprecipitation to show that HSV lytic-gene promoters become complexed with modified histones associated with heterochromatin during the course of establishment of latent infection. Experiments comparing LAT-negative and LAT-positive viruses show that a function encoded by the LAT gene increases the amount of dimethyl lysine 9 form of histone H3 or heterochromatin and reduces the amount of dimethyl lysine 4 form of histone H3, a part of active chromatin, on viral lytic-gene promoters. Thus, HSV, and in particular the HSV LAT gene, may manipulate the cellular histone modification machinery to repress its lytic-gene expression and contribute to the persistence of its genome in a quiescent form in sensory neurons.

Journal ArticleDOI
TL;DR: It is shown that pericentric heterochromatin aggregates during myogenic differentiation, causing a dose-dependent clustering of chromocenters in the absence of differentiation, and this MeCP2- and MBD2-mediated chromatin reorganization may represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.
Abstract: Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.

Journal ArticleDOI
TL;DR: The unique evolutionary history and resulting genomic structure of the X chromosome as well as the current understanding of the factors and events involved in silencing an X chromosome in mammals are described.
Abstract: ▪ Abstract Mammalian X chromosome inactivation is one of the most striking examples of epigenetic gene regulation. Early in development one of the pair of ∼160-Mb X chromosomes is chosen to be silenced, and this silencing is then stably inherited through subsequent somatic cell divisions. Recent advances have revealed many of the chromatin changes that underlie this stable silencing of an entire chromosome. The key initiator of these changes is a functional RNA, XIST, which is transcribed from, and associates with, the inactive X chromosome, although the mechanism of association with the inactive X and recruitment of facultative heterochromatin remain to be elucidated. This review describes the unique evolutionary history and resulting genomic structure of the X chromosome as well as the current understanding of the factors and events involved in silencing an X chromosome in mammals.

Journal ArticleDOI
15 Jul 2005-Cell
TL;DR: There is increasing evidence that RNA-mediated chromatin modifications play an important role in epigenetic transcriptional gene silencing.

Journal ArticleDOI
TL;DR: Lineage-specific differences provide a glimpse into the developmental complexity of the epigenetic marks that ensure the inactive state of the inactive X.

Journal ArticleDOI
TL;DR: It is found that telomeric repeats are sufficient for the establishment of Swi6 (a fission-yeast HP1 homolog) heterochromatin, and the establishment requires Taz1, a telomere binding protein of the TRF family, as well as RNAi-RITS, which is commonly used at the constitutive heterochROMatin regions.

Journal ArticleDOI
TL;DR: Analysis of fission yeast analyses uncover a role for Cul4-based protein ubiquitination in regulating H3K9me and heterochromatin formation.
Abstract: In eukaryotes, heterochromatin mediates diverse processes including gene silencing and regulation of long-range chromatin interactions. The formation of heterochromatin involves a conserved array of histone modifications; in particular, methylation of histone H3 at Lys 9 (H3K9me) is essential for recruiting HP1/Swi6 proteins. In fission yeast, the Clr4 methyltransferase is responsible for H3K9me across all heterochromatic domains. However, the mechanism of Clr4 recruitment to these loci is poorly understood. We show that Clr4 associates with Cul4, a cullin family protein that serves as a scaffold for assembling ubiquitin ligases. Mutations in Cul4 result in defective localization of Clr4 and loss of silencing at heterochromatic loci. This is accompanied by a severe reduction in H3K9me and Swi6 levels, and accumulation of transcripts corresponding to naturally silenced repeat elements within heterochromatic domains. Moreover, heterochromatin defects in Cul4 mutants could not be rescued by expression of Cul4 protein lacking Nedd8 modification, which is essential for its ubiquitin ligase activity. Rik1, a protein related to DNA damage binding protein DDB1 and required for H3K9me, also interacts with Cul4, the association of which might serve to target Clr4 to heterochromatic loci. These analyses uncover a role for Cul4-based protein ubiquitination in regulating H3K9me and heterochromatin formation.

Journal ArticleDOI
TL;DR: Roles for transcribed repeats in the epigenetic inheritance and evolution of centromeres are proposed and Histone H3 lysine-9 dimethylation is associated with both classes of repeats.
Abstract: Centromeres interact with the spindle apparatus to enable chromosome disjunction and typically contain thousands of tandemly arranged satellite repeats interspersed with retrotransposons. While their role has been obscure, centromeric repeats are epigenetically modified and centromere specification has a strong epigenetic component. In the yeast Schizosaccharomyces pombe, long heterochromatic repeats are transcribed and contribute to centromere function via RNA interference (RNAi). In the higher plant Arabidopsis thaliana, as in mammalian cells, centromeric satellite repeats are short (180 base pairs), are found in thousands of tandem copies, and are methylated. We have found transcripts from both strands of canonical, bulk Arabidopsis repeats. At least one subfamily of 180–base pair repeats is transcribed from only one strand and regulated by RNAi and histone modification. A second subfamily of repeats is also silenced, but silencing is lost on both strands in mutants in the CpG DNA methyltransferase MET1, the histone deacetylase HDA6/SIL1, or the chromatin remodeling ATPase DDM1. This regulation is due to transcription from Athila2 retrotransposons, which integrate in both orientations relative to the repeats, and differs between strains of Arabidopsis. Silencing lost in met1 or hda6 is reestablished in backcrosses to wild-type, but silencing lost in RNAi mutants and ddm1 is not. Twenty-four–nucleotide small interfering RNAs from centromeric repeats are retained in met1 and hda6, but not in ddm1, and may have a role in this epigenetic inheritance. Histone H3 lysine-9 dimethylation is associated with both classes of repeats. We propose roles for transcribed repeats in the epigenetic inheritance and evolution of centromeres.

Journal ArticleDOI
TL;DR: It is demonstrated that the maintenance DNA methyltransferase DNMT1 is necessary for proper PcG body assembly independent of DNMT-associated histone deacetylase activity and suggested a highly regulated recruitment of PRC1 to chromatin.
Abstract: Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized. Polycomb repressive complex 2 (PRC2) is thought to be involved in the initiation of gene silencing, whereas Polycomb repressive complex 1 (PRC1) is implicated in the stable maintenance of gene repression. Here, we investigate the kinetic properties of the binding of one of the PRC1 core components, BMI1, with PcG bodies. PcG bodies are unique nuclear structures located on regions of pericentric heterochromatin, found to be the site of accumulation of PcG complexes in different cell lines. We report the presence of at least two kinetically different pools of BMI1, a highly dynamic and a less dynamic fraction, which may reflect BMI1 pools with different binding capacities to these stable heterochromatin domains. Interestingly, PRC2 members EED and EZH2 appear to be essential for BMI1 recruitment to the PcG bodies. Furthermore, we demonstrate that the maintenance DNA methyltransferase DNMT1 is necessary for proper PcG body assembly independent of DNMT-associated histone deacetylase activity. Together, these results provide new insights in the mechanism for regulation of chromatin silencing by PcG proteins and suggest a highly regulated recruitment of PRC1 to chromatin.

Journal ArticleDOI
TL;DR: Two novel proteins are identified, Raf1 and Raf2, which are found to be required for H3-K9 methylation and for transcriptional silencing within centromeric heterochromatin formation in fission yeast.
Abstract: Heterochromatin is critical for proper centromere and telomere function, and it plays a key role in the transcriptional silencing of specific genomic loci. In fission yeast, the Rik1 protein functions with the Clr4 histone methyltransferase at an early step in heterochromatin formation. Here, we use mass spectrometry and tandem affinity purification of a Rik1-TAP fusion protein to identify Rik1-associated proteins. These studies identify two novel proteins, Raf1 and Raf2, which we find are required for H3-K9 methylation and for transcriptional silencing within centromeric heterochromatin. We also find that subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4, and Rik1-TAP preparations exhibit robust E3 ubiquitin ligase activity. Furthermore, expression of a dominant-negative allele of the Pcu4 cullin subunit disrupts regulation of K4 methylation within heterochromatin. These studies provide evidence for a novel Rik1-associated E3 ubiquitin ligase that is required for heterochromatin formation.

Journal ArticleDOI
TL;DR: A affinity-purified antibodies against the N-terminal region of H2A.Z are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types to resolve an apparent dichotomy in its reported links to gene expression and preventing the spread of heterochromatin in yeast and mammals.
Abstract: The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a triacetylated and non-acetylated state, are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 5 0 end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an epigenetic marker for active genes, its N-terminal acetylation does not.

Journal ArticleDOI
TL;DR: It is suggested that early B lymphoid epigenetic changes generate differential structures that serve as the basis for allelic exclusion and are associated with heterochromatin protein-γ and Ikaros.
Abstract: To become accessible for rearrangement, the immunoglobulin kappa locus must undergo a series of epigenetic changes. This begins in pro-B cells with the relocation of both immunoglobulin kappa alleles from the periphery to the center of the nucleus. In pre-B cells, one allele became preferentially packaged into an active chromatin structure characterized by histone acetylation and methylation of histone H3 lysine 4, while the other allele was recruited to heterochromatin, where it was associated with heterochromatin protein-gamma and Ikaros. These events in cis made only one allele accessible to trans-acting factors, such as RelB, which mediated DNA demethylation, to facilitate rearrangement. These results suggest that early B lymphoid epigenetic changes generate differential structures that serve as the basis for allelic exclusion.

Journal ArticleDOI
TL;DR: The role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells is underline.
Abstract: During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.

Journal ArticleDOI
TL;DR: It is proposed that RNAi-mediated heterochromatin formation involves therecruitment of Clr4-Rik1-Cul4 complex through the putative nucleic acid binding domain of Rik1, which is associated with the fissionyeast E3 ubiquitin ligase Cullin4.
Abstract: The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast,H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNAInducedTranscriptional Silencing (RITS) complex. Here we report the purification of a novelcomplex that associates with the Clr4 methyltransferase. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fissionyeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4+), the ubiquitin-like protein, Ned8, andtwo previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complexcontains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24.Deletion of cul4+, cmc1+, cmc2+, and rad24+ results in a complete loss of silencing of a ura4+ reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each ofthe above del...