Showing papers on "Heterochromatin published in 2007"

The epigenetic regulation of mammalian telomeres

Abstract: Increasing evidence indicates that chromatin modifications are important regulators of mammalian telomeres. Telomeres provide well studied paradigms of heterochromatin formation in yeast and flies, and recent studies have shown that mammalian telomeres and subtelomeric regions are also enriched in epigenetic marks that are characteristic of heterochromatin. Furthermore, the abrogation of master epigenetic regulators, such as histone methyltransferases and DNA methyltransferases, correlates with loss of telomere-length control, and telomere shortening to a critical length affects the epigenetic status of telomeres and subtelomeres. These links between epigenetic status and telomere-length regulation provide important new avenues for understanding processes such as cancer development and ageing, which are characterized by telomere-length defects.

Transcription and RNA interference in the formation of heterochromatin

Abstract: Transcription in heterochromatin seems to be an oxymoron--surely the 'silenced' form of chromatin should not be transcribed. But there have been frequent reports of low-level transcription in heterochromatic regions, and several hundred genes are found in these regions in Drosophila. Most strikingly, recent investigations implicate RNA interference mechanisms in targeting and maintaining heterochromatin, and these mechanisms are inherently dependent on transcription. Silencing of chromatin might involve trans-acting sources of the crucial small RNAs that carry out RNA interference, but in some cases, transcription of the region to be silenced seems to be required--an apparent contradiction.

An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster

Abstract: Heterochromatin, representing the silenced state of transcription, consists largely of transposon-enriched and highly repetitive sequences. Implicated in heterochromatin formation and transcriptional silencing in Drosophila are Piwi (P-element induced wimpy testis) and repeat-associated small interfering RNAs (rasiRNAs). Despite this, the role of Piwi in rasiRNA expression and heterochromatic silencing remains unknown. Here we report the identification and characterization of 12,903 Piwi-interacting RNAs (piRNAs) in Drosophila, showing that rasiRNAs represent a subset of piRNAs. We also show that Piwi promotes euchromatic histone modifications and piRNA transcription in subtelomeric heterochromatin (also known as telomere-associated sequence, or TAS), on the right arm of chromosome 3 (3R-TAS). Piwi binds to 3R-TAS and a piRNA uniquely mapped to 3R-TAS (3R-TAS1 piRNA). In piwi mutants, 3R-TAS loses euchromatic histone modifications yet accumulates heterochromatic histone modifications and Heterochromatin Protein 1a (HP1a). Furthermore, the expression of both the 3R-TAS1 piRNA and a white reporter gene in 3R-TAS becomes suppressed. A P element inserted 128 base pairs downstream of the 3R-TAS1 piRNA coding sequence restores the euchromatic histone modifications of 3R-TAS and the expression of 3R-TAS1 piRNA in piwi mutants, as well as partly rescuing their defects in germline stem-cell maintenance. These observations suggest that Piwi promotes the euchromatic character of 3R-TAS heterochromatin and its transcriptional activity, opposite to the known roles of Piwi and the RNA-mediated interference pathway in epigenetic silencing. This activating function is probably achieved through interaction with at least 3R-TAS1 piRNA and is essential for germline stem-cell maintenance.

Molecular Dissection of Formation of Senescence-Associated Heterochromatin Foci

Abstract: Senescence was initially described as a stable cell proliferation arrest resulting from the progression of primary human fibroblasts through a finite number of population doublings in vitro (35). However, activated oncogenes, oxidative stress, DNA damage, and drug-like inhibitors of specific enzymatic activities also induce senescence (14, 37, 82). In addition, senescence occurs in other cell types, such as primary human epithelial cells. In vivo, senescence is an important tumor suppression mechanism that restrains the proliferation of cells that harbor activated oncogenes (12, 16, 17, 51). Also, by limiting the self-renewal capacity of adult tissue stem cells, senescence is thought to contribute to tissue aging of many multicellular adult animals (38, 42, 53). Senescent cells are typically characterized by a large flat morphology and the expression of a senescence-associated β-galactosidase (SA β-gal) activity of unknown function (16, 21). In the nucleus of senescent cells, the chromatin undergoes dramatic remodeling through the formation of domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) (56, 57, 86). Cytologically, SAHF appear as compacted punctate DAPI (4,6-diamidino-2-phenylindole)-stained foci of DNA in senescent cell nuclei. The formation of SAHF is also reflected in a general increase in the resistance of nuclear chromatin to digestion by nucleases (57). SAHF contain modifications and associated proteins characteristic of transcriptionally silent heterochromatin, such as methylated lysine 9 of histone H3 (H3K9Me), heterochromatin protein 1 (HP1), and the histone H2A variant macroH2A. In addition, Narita et al. recently showed that high-mobility group A (HMGA) proteins, a family of abundant non-histone chromatin proteins, are essential structural components of SAHF (56). Proliferation-promoting genes, such as E2F target genes (e.g., cyclin A), are recruited into SAHF, dependent on the pRB tumor suppressor protein, thereby irreversibly silencing expression of those genes. Recently, we showed that two chromatin regulators, histone repressor A (HIRA) and antisilencing function 1a (ASF1a), drive the formation of SAHF in human cells (86). HIRA and ASF1a are the human orthologs of proteins known to create transcriptionally silent heterochromatin in yeasts, flies, and plants (9, 29, 39, 54, 63, 70-73, 78). In Saccharomyces cerevisiae, Hir1 and Hir2 are required for heterochromatin-mediated silencing of histone genes, telomeres, and mating loci, and the formation of pericentromeric chromatin structure (39, 70-73). Likewise, yeast Asf1p is required for heterochromatin-mediated silencing of telomeres, mating loci, and histone genes (40, 50, 70, 73, 75, 78) but also mediates nucleosome disassembly (2, 3, 68). Both HIRA and ASF1a bind to histones and exhibit histone chaperone activity in vitro (28, 64, 70, 78, 79). The HIRA/ASF1a-containing complex preferentially deposits the histone variant histone H3.3 into nucleosomes (46, 65, 76). Canonical human histone H3.1 and histone H3.3 differ in their primary amino acid sequences by only five amino acids. However, histone H3.1 is expressed periodically in the S phase of the cell cycle and is incorporated into chromatin during replication-coupled chromatin assembly (5, 36, 76). In contrast, histone H3.3 is expressed throughout the cell cycle and is incorporated into chromatin by the HIRA/ASF1a complex in a DNA replication- and repair-independent manner (5, 36, 76). Consistent with their partially overlapping biological and biochemical properties, yeast Asf1p and Hir proteins physically interact, and this interaction is necessary for telomeric silencing (19, 70). Likewise, the formation of SAHF in human cells by HIRA and ASF1a depends upon a physical interaction between these two proteins (76, 77, 86). A previous careful kinetic analysis of SAHF formation from our laboratory indicated that formation of SAHF is likely a multistep process (87). In the earliest defined step, the histone chaperone proteins HIRA and HP1 are both recruited to a specific subnuclear organelle, the acute promyelocytic leukemia (PML) nuclear body (10, 67). Most human cells contain 20 to 30 PML nuclear bodies, which are typically 0.1 to 1 μm in diameter and are enriched in the protein PML, as well as many other nuclear regulatory proteins (10, 67). PML bodies have been previously implicated in various cellular processes, including tumor suppression and cellular senescence (20, 23, 61). At a molecular level, they have been proposed as sites of assembly of macromolecular regulatory complexes and protein modification (24, 31, 61). After HIRA's translocation into PML bodies, chromatin condensation occurs, as defined by the appearance of DAPI-stained foci. Finally, H3K9Me accumulates, and HP1 and macroH2A proteins are recruited to SAHF. In this study, we set out to understand the series of events that contribute to the formation of SAHF in more detail and, in particular, to identify the molecular requirements for the different steps that were previously temporally defined. Here we report that during SAHF formation, each chromosome condenses into a single DAPI focus. Chromosome condensation mediated by the histone chaperone ASF1a depends on its binding to histone H3, as well as HIRA. Interestingly, HP1γ, but not HP1α and HP1β, is phosphorylated on serine 93 in senescent cells. This phosphorylation is not required for the protein's localization to PML bodies, but is required for its binding to SAHF. Remarkably, a large reduction in the amount of chromatin-bound HP1 proteins does not affect chromosome condensation, recruitment of histone variant macroH2A to SAHF, expression of SA β-gal, or senescence-associated cell cycle exit. Based on these data, we propose a multistep model of dependent and independent steps that culminate in the formation of mature SAHF.

Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres

Abstract: Mammalian telomeres have epigenetic marks of constitutive heterochromatin. Here, we study the impact of telomere length on the maintenance of heterochromatin domains at telomeres. Telomerase-deficient Terc−/− mice with short telomeres show decreased trimethylation of histone 3 at Lys9 (H3K9) and histone 4 at Lys20 (H4K20) in telomeric and subtelomeric chromatin as well as decreased CBX3 binding accompanied by increased H3 and H4 acetylation at these regions. Subtelomeric DNA methylation is also decreased in conjunction with telomere shortening in Terc−/− mice. In contrast, telomere repeat factors 1 and 2 show normal binding to telomeres independent of telomere length. These results indicate that loss of telomeric repeats leads to a change in the architecture of telomeric and subtelomeric chromatin consisting of loss of heterochromatic features leading to a more 'open' chromatin state. These observations highlight the importance of telomere repeats in the establishment of constitutive heterochromatin at mammalian telomeres and subtelomeres and point to histone modifications as important in counting telomere repeats.

Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis.

Abstract: During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants.

Sequence Finishing and Mapping of Drosophila melanogaster Heterochromatin

Abstract: Genome sequences for most metazoans and plants are incomplete because of the presence of repeated DNA in the heterochromatin. The heterochromatic regions of Drosophila melanogaster contain 20 million bases (Mb) of sequence amenable to mapping, sequence assembly, and finishing. We describe the generation of 15 Mb of finished or improved heterochromatic sequence with the use of available clone resources and assembly methods. We also constructed a bacterial artificial chromosome–based physical map that spans 13 Mb of the pericentromeric heterochromatin and a cytogenetic map that positions 11 Mb in specific chromosomal locations. We have approached a complete assembly and mapping of the nonsatellite component of Drosophila heterochromatin. The strategy we describe is also applicable to generating substantially more information about heterochromatin in other species, including humans.

Transcription and RNAi in heterochromatic gene silencing.

Abstract: Recent findings have challenged the longstanding belief that heterochromatin is an inert and transcriptionally inactive structure. Studies in organisms ranging from fission yeast to animals have found that noncoding RNAs transcribed from heterochromatic DNA repeats function in the assembly and function of heterochromatin. In this review, we discuss the roles of RNA and RNA turnover in mechanisms that mediate heterochromatin assembly and keep heterochromatic domains silent.

Gene regulation through nuclear organization

Abstract: The nucleus is a highly heterogeneous structure, containing various 'landmarks' such as the nuclear envelope and regions of euchromatin or dense heterochromatin. At a morphological level, regions of the genome that are permissive or repressive to gene expression have been associated with these architectural features. However, gene position within the nucleus can be both a cause and a consequence of transcriptional regulation. New results indicate that the spatial distribution of genes within the nucleus contributes to transcriptional control. In some cases, position seems to ensure maximal expression of a gene. In others, it ensures a heritable state of repression or correlates with a developmentally determined program of tissue-specific gene expression. In this review, we highlight mechanistic links between gene position, repression and transcription. Recent findings suggest that architectural features have multiple functions that depend upon organization into dedicated subcompartments enriched for distinct enzymatic machinery.

RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena.

Abstract: Methylated H3K27 is an important mark for Polycomb group (PcG) protein-mediated transcriptional gene silencing (TGS) in multicellular eukaryotes. Here a Drosophila E(z) homolog, EZL1, is characterized in the ciliated protozoan Tetrahymena thermophila and is shown to be responsible for H3K27 methylation associated with developmentally regulated heterochromatin formation and DNA elimination. Importantly, Ezl1p-catalyzed H3K27 methylation occurs in an RNA interference (RNAi)-dependent manner. H3K27 methylation also regulates H3K9 methylation in these processes. Furthermore, an “effector” of programmed DNA elimination, the chromodomain protein Pdd1p, is shown to bind both K27- and K9-methylated H3. These studies provide a framework for an RNAi-dependent, Polycomb group protein-mediated heterochromatin formation pathway in Tetrahymena and underscore the connection between the two highly conserved machineries in eukaryotes.

Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals.

Abstract: Double-strand break (DSB) damage in yeast and mammalian cells induces the rapid ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3 related)-dependent phosphorylation of histone H2AX (γ-H2AX). In budding yeast, a single endonuclease-induced DSB triggers γ-H2AX modification of 50 kb on either side of the DSB. The extent of γ-H2AX spreading does not depend on the chromosomal sequences. DNA resection after DSB formation causes the slow, progressive loss of γ-H2AX from single-stranded DNA and, after several hours, the Mec1 (ATR)-dependent spreading of γ-H2AX to more distant regions. Heterochromatic sequences are only weakly modified upon insertion of a 3-kb silent HMR locus into a γ-H2AX–covered region. The presence of heterochromatin does not stop the phosphorylation of chromatin more distant from the DSB. In mouse embryo fibroblasts, γ-H2AX distribution shows that γ-H2AX foci increase in size as chromatin becomes more accessible. In yeast, we see a high level of constitutive γ-H2AX in telomere regions in the absence of any exogenous DNA damage, suggesting that yeast chromosome ends are transiently detected as DSBs.

Heterochromatin-like regions as ecological niches for avirulence genes in the Leptosphaeria maculans genome : Map-based cloning of AvrLm6

Abstract: Map-based cloning of avirulence genes of the AvrLm1-2-6 cluster was recently undertaken in Leptosphaeria maculans and led to the identification of AvrLm1. The ensuing chromosome walk toward AvrLm6 resulted in the delineation of a 562-kb bacterial artificial chromosome (BAC) clone contig in an avirulent isolate. Following sequencing of the contig and sequence comparison with a virulent isolate, four AvrLm6 candidate genes were identified. Complementation of the virulent isolate with the four candidates was performed and one gene was found to fully restore the avirulent phenotype on Rlm6 oilseed rape genotypes. AvrLm6 was found to be located in the same genome context as AvrLm1, because it is a solo gene surrounded by 85 and 48 kb of degenerated repeats on its 5′ and 3′ sides, respectively. AvrLm6 is an orphan gene encoding a small, potentially secreted, cysteine-rich protein. Comparison of AvrLm1 and AvrLm6 expressions by quantitative reverse-transcription polymerase chain reaction revealed that both genes...

The Release 5.1 annotation of Drosophila melanogaster heterochromatin.

Abstract: 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.

Epigenetic Regulation of Tumor Necrosis Factor Alpha

Abstract: Tumor necrosis factor alpha (TNF-α) is a potent cytokine which regulates inflammation via the induction of adhesion molecules and chemokine expression. Its expression is known to be regulated in a complex manner with transcription, message turnover, message splicing, translation, and protein cleavage from the cell surface all being independently regulated. This study examined both cell lines and primary cells to understand the developmental regulation of epigenetic changes at the TNF-α locus. We demonstrate that epigenetic modifications of the TNF-α locus occur both developmentally and in response to acute stimulation and, importantly, that they actively regulate expression. DNA demethylates early in development, beginning with the hematopoietic stem cell. The TNF-α locus migrates from heterochromatin to euchromatin in a progressive fashion, reaching euchromatin slightly later in differentiation. Finally, histone modifications characteristic of a transcriptionally competent gene occur with myeloid differentiation and progress with differentiation. Additional histone modifications characteristic of active gene expression are acquired with stimulation. In each case, manipulation of these epigenetic variables altered the ability of the cell to express TNF-α. These studies demonstrate the importance of epigenetic regulation in the control of TNF-α expression. These findings may have relevance for inflammatory disorders in which TNF-α is overproduced.

Monoubiquitylation of H2A.Z Distinguishes Its Association with Euchromatin or Facultative Heterochromatin

Abstract: H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z- or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.

Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast

Abstract: DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2′-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.

Genome‐wide analysis of Agrobacterium T‐DNA integration sites in the Arabidopsis genome generated under non‐selective conditions

Abstract: Previous work from numerous laboratories has suggested that integration of Agrobacterium tumefaciens T-DNA into the plant genome occurs preferentially in promoter or transcriptionally active regions. However, all of these studies were conducted on plants recovered from selective conditions requiring the expression of transgenes. The conclusions of these studies may therefore have been biased because of the selection of transformants. In this study, we investigated T-DNA integration sites in the Arabidopsis genome by analyzing T-DNA/plant DNA junctions generated under non-selective conditions. We found a relatively high frequency of T-DNA insertions in heterochromatic regions, including centromeres, telomeres and rDNA repeats. These T-DNA insertion regions are disfavored under selective conditions. The frequency with which T-DNA insertions mapped to exon, intron, 5' upstream and 3' downstream regions closely resembled their respective proportions in the Arabidopsis genome. Transcriptional profiling indicated that expression levels of T-DNA pre-integration target sites recovered using selective conditions were significantly higher than those of random Arabidopsis sequences, whereas expression levels of genomic sequences targeted by T-DNA under non-selective conditions were similar to those of random Arabidopsis sequences. T-DNA target sites identified using non-selective conditions did not correlate with DNA methylation status, suggesting that T-DNA integration occurs without regard to DNA methylation. Our results indicate that T-DNA integration may occur more randomly than previously indicated, and that selection pressure may shift the recovery of T-DNA insertions into gene-rich or transcriptionally active regions of chromatin.

Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection.

Abstract: Histone acetylation is important in regulating DNA accessibility. Multifunctional Sin3 proteins bind histone deacetylases (HDACs) to assemble silencing complexes that selectively target chromatin. We show that, in fission yeast, an essential HDAC, Clr6, exists in two distinct Sin3 core complexes. Complex I contains an essential Sin3 homolog, Pst1, and other factors, and predominantly targets gene promoters. Complex II contains a nonessential Sin3 homolog, Pst2, and several conserved proteins. It preferentially targets transcribed chromosomal regions and centromere cores. Defects in complex II abrogate global protective functions of chromatin, causing increased accessibility of DNA to genotoxic agents and widespread antisense transcripts that are processed by the exosome. Notably, the two Clr6 complexes differentially repress forward and reverse centromeric repeat transcripts, suggesting that these complexes regulate transcription in heterochromatin and euchromatin in similar manners, including suppression of spurious transcripts from cryptic start sites.