Subtelomeres are patchworks of evolutionary conserved sequence blocks and harbor the transcriptional start sites for telomere repeat containing RNAs (TERRA). Recent studies suggest that the interplay between telomeres and subtelomeric chromatin is required for maintaining telomere function. To further characterize chromatin remodeling of subtelomeres in relation to telomere shortening and cellular senescence, we systematically quantified histone modifications and DNA methylation at the subtelomeres of chromosomes 7q and 11q in primary human WI-38 fibroblasts. Upon senescence, both subtelomeres were characterized by a decrease in markers of constitutive heterochromatin, suggesting relative chromatin relaxation. However, we did not find increased levels of markers of euchromatin or derepression of the 7q VIPR2 gene. The repressed state of the subtelomeres was maintained upon senescence, which could be attributed to a rise in levels of facultative heterochromatin markers at both subtelomeres. While senescence-induced subtelomeric chromatin remodeling was similar for both chromosomes, chromatin remodeling at TERRA promoters displayed chromosome-specific patterns. At the 7q TERRA promoter, chromatin structure was co-regulated with the more proximal subtelomere. In contrast, the 11q TERRA promoter, which was previously shown to be bound by CCCTC-binding factor CTCF, displayed lower levels of markers of constitutive heterochromatin that did not change upon senescence, whereas levels of markers of facultative heterochromatin decreased upon senescence. In line with the chromatin state data, transcription of 11q TERRA but not 7q TERRA was detected. Our study provides a detailed description of human subtelomeric chromatin dynamics and shows distinct regulation of the TERRA promoters of 7q and 11q upon cellular senescence.
TERRA transcripts; cellular aging; chromatin remodeling; histone modifications; senescence; subtelomere
Constitutive heterochromatin is enriched in repetitive sequences and histone H3-methylated-at-lysine 9. Both components contribute to heterochromatin's ability to silence euchromatic genes. However, heterochromatin also harbors hundreds of expressed genes in organisms such as Drosophila. Recent studies have provided a detailed picture of sequence organization of D. melanogaster heterochromatin, but how histone modifications are associated with heterochromatic sequences at high resolution has not been described. Here, distributions of modified histones in the vicinity of heterochromatic genes of normal embryos and embryos homozygous for a chromosome rearrangement were characterized using chromatin immunoprecipitation and genome tiling arrays. We found that H3-di-methylated-at-lysine 9 (H3K9me2) was depleted at the 5′ ends but enriched throughout transcribed regions of heterochromatic genes. The profile was distinct from that of euchromatic genes and suggests that heterochromatic genes are integrated into, rather than insulated from, the H3K9me2-enriched domain. Moreover, the profile was only subtly affected by a Su(var)3–9 null mutation, implicating a histone methyltransferase other than SU(VAR)3–9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos. On a chromosomal scale, we observed a sharp transition to the H3K9me2 domain, which coincided with increased retrotransposon density in the euchromatin-heterochromatin (eu-het) transition zones on the long chromosome arms. Thus, a certain density of retrotransposons, rather than specific boundary elements, may demarcate Drosophila pericentric heterochromatin. We also demonstrate that a chromosome rearrangement that created a new eu-het junction altered H3K9me2 distribution and induced new euchromatic sites of enrichment as far as several megabases away from the breakpoint. Taken together, the findings argue against simple classification of H3K9me as the definitive signature of silenced genes, and clarify roles of histone modifications and repetitive DNAs in heterochromatin. The results are also relevant for understanding the effects of chromosome aberrations and the megabase scale over which epigenetic position effects can operate in multicellular organisms.
The chromosomal domain “heterochromatin” was first defined at the cytological level by its deeply staining appearance compared to more lightly stained domains called “euchromatin.” Abnormal juxtaposition of these two domains by chromosome rearrangements results in silencing of the nearby euchromatic genes. This effect is mediated by heterochromatin-enriched chromosomal proteins and led to the prevalent view of heterochromatin as incompatible with gene expression. Paradoxically, some expressed genes reside within heterochromatin. In this study, we examined how heterochromatic genes fit into a genomic context known for silencing effects. We found that Drosophila heterochromatic genes are integrated into the domain enriched in the modified histone H3K9me2, suggesting that the effect of this protein on gene expression is context-dependent. We also investigated the molecular nature of euchromatin-heterochromatin transition zones in the normal and rearranged chromosomes. The results provide insights into the functions of repetitive DNAs and H3K9me2 in heterochromatin and document the long distance over which a heterochromatic breakpoint can affect the molecular landscape of a chromosomal region. These findings have implications for understanding the consequences of chromosome abnormalities in organisms, including humans.
Chromatin is highly dynamic and subject to extensive remodeling under many physiological conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We find increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In skeletal muscle we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, post mitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to further investigate the progressive epigenetic changes that occur during aging.
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.
Chromatin is the support of our genetic information. It is composed of numerous repeated units called nucleosomes, in which DNA wraps around a core of histone proteins. Modifications in the composition and biochemical properties of nucleosomes play major roles in the regulation of genome function. Such modifications are termed “epigenetic” when they are inherited across cell divisions and confer new information to chromatin, in addition to the genetic information provided by DNA. It is usually believed that during genome replication, the basic chromatin assembly machinery builds up “naïve” nucleosomes, and, in a subsequent step, nucleosomes are selectively modified by a series of enzymes to acquire epigenetic information. Here, the authors studied the role of a basic chromatin assembly factor (CAF-1) in mouse embryonic stem cells and early embryos. Surprisingly, they show that CAF-1 confers epigenetic information to specific genomic regions. In addition, this study revealed that CAF-1 is required for the proper spatial organization of chromosomes in the nucleus. This new knowledge may contribute to better understanding the role of chromatin in the maintenance of embryonic stem cell identity and plasticity.
Establishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.
Our results demonstrate that histone H3 tri-methylated at lysine 9 (H3K9me3), a hallmark of constitutive heterochromatin, as well as the chromatin remodeling protein ATRX remained associated with pericentric heterochromatin regions in spite of their extensive hypo-methylation. This suggests that in neonatal spermatogonia, chromosomal 5-methyl cytosine patterns are regulated independently of changes in histone methylation, potentially reflecting a crucial mechanism to maintain pericentric heterochromatin silencing. Furthermore, chromatin immunoprecipitation and fluorescence in situ hybridization, revealed that ATRX as well as H3K9me3 associate with Y chromosome-specific DNA sequences and decorate both arms of the Y chromosome, suggesting a possible role in heterochromatinization and the predominant transcriptional quiescence of this chromosome during spermatogenesis.
These results are consistent with a role for histone modifications and chromatin remodeling proteins such as ATRX in maintaining transcriptional repression at constitutive heterochromatin domains in the absence of 5-methyl cytosine and provide evidence suggesting that the establishment and/or maintenance of repressive histone and chromatin modifications at pericentric heterochromatin following genome-wide epigenetic reprogramming in the germ line may precede the establishment of chromosomal 5-methyl cytosine patterns as a genomic silencing strategy in neonatal spermatogonia.
Purpose: Higher order chromatin structure progressively changes with cell differentiation and seems to play an important role in DNA double-strand break (DSB) induction and repair (reviewed in ). We compared DNA damage in heterochromatin (Hc) upon the action of qualitatively different radiations. We also studied, how is the sensitivity to DSB induction, assembly of repair foci and processing of DSBs influenced by the differentiation-induced changes in chromatin structure and composition.
Materials and methods: Formation, localization (relative to higher-order chromatin domains) and mutual colocalization of γH2AX and p53BP1 repair foci have been studied together with DSB repair kinetics in spatially fixed human skin fibroblast and differently differentiated white blood cells (WBC) irradiated with gamma rays, protons of different energies [2, 3], and 20Ne ions (submitted). Immunostaining and ImmunoFISH were used in combination with high-resolution confocal microscopy [2, 3] and living cell imaging .
Results: We found that less DSBs appear in Hc after irradiating cells with gamma rays and protons but not 20Ne ions (preliminary results). In addition, contrary to γ-irradiated human skin fibroblasts and lymphocytes, mature granulocytes neither express DSB repair proteins nor form functional repair foci . At least some DSB repair proteins (e.g. 53BP1) are expressed and γH2AX foci still occur in immature granulocytes and monocytes [2, 5]; however, the colocalization of γH2AX with 53BP1 is low and the majority of DSBs are not repaired. Despite this fact, γH2AX foci protrude from Hc into nuclear subcompartments with low chromatin density. Our living cell observations suggest that 53BP1 can penetrate into the interior of dense Hc domains only after their decondensation .
Conclusions: We show that Hc is less sensitive to DSB induction by gamma rays but not heavy ions; lower Hc hydratation and higher protein density (when compared with euchromatin) probably reduce formation of free radicals and increase their sequestration, respectively. This mechanism can protect cells against the indirect effect of ionizing radiation (marked for gamma rays and protons but not heavy ions). Hc features, however, preclude DSB repair, which is best illustrated by its absence in differentiated WBC but not their immature precursors. The protrusion of Hc-DSBs into low-density chromatin nuclear subdomains, however, appears also in differentiated WBC, so the process might simply follow physical forces (e.g. as suggested by M Durante's group).
There is no Clinical Trial Registration number.
DNA double-strand breaks (DSB); DSB repair; white blood cells differentiation; higher-order chromatin structure; ionizing radiations of different quality; ionizing radiation-induced repair foci (IRIF)
Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila melanogaster and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. Here we report that, as observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, we found that only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrated that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation.
Heterochromatin is defined as regions of compact chromatin that persist throughout the cell cycle (Heitz, 1928). The earliest cytological observations of heterochromatin were followed by ribonucleotide labeling experiments that showed it to be transcriptionally inert relative to the more typical euchromatic regions that decondense during interphase. Genetic studies of rearrangements that place euchromatic genes next to blocks of heterochromatin also pointed out the repressive nature of heterochromatin (Grigliatti, 1991; and references therein). The discovery of the heterochromatin-enriched protein heterochromatin protein 1 (HP1)* by Elgin and co-workers in the mid-1980s suggested that the distinct cytological features of this chromatin may be related to its unique nucleoprotein composition (James and Elgin, 1986; James et al., 1989). HP1 immunostaining on polytene chromosomes from Drosophila larval salivary glands was used to show enrichment of the protein in pericentric heterochromatin. Since that initial discovery, HP1 homologues have been found in species ranging from fission yeast to humans where it is associated with gene silencing (Eissenberg and Elgin, 2000; and references therein). A number of euchromatic sites of localization were also reported in this original study. It has been generally assumed that these sites might constitute euchromatic sites of transcriptional repression by HP1. Indeed, several genes located at one of these sites (cytological region 31) have increased transcript levels in mutants for HP1 (Hwang et al., 2001).
Purpose: Ionizing radiations of different qualities (e.g. high-LET and low-LET) might differently interact with structurally and functionally distinct higher order chromatin domains (discussed in [
1] and citations therein); this might be reflected by DNA double strand break (DSB) repair efficiency and the mechanism of how cancerogenous chromosomal translocations (CHT) form. Therefore, we compared the DSB repair kinetics and formation of γH2AX/p53BP1 repair clusters upon the action of γ-rays [
2, 3], protons (15 and 30 MeV) [
4], and 20Ne ions (preliminary data). Consequently, we discuss biological impacts of these clusters.
Material and methods: Immunostaining methods in combination with high-resolution confocal microscopy, performed on 3D-fixed normal human skin fibroblasts [
4], were used to study initial distributions of γH2AX and p53BP1 repair foci and their changes during the post-irradiation (PI) time, with a special concern on foci clustering. Irradiations with γ-rays, protons of different energies (15 and 30 MeV), and high-LET 20Ne ions was performed in IBP ASCR Brno (CR), NPI AVCR Řež (CR) and JINR Dubna (Russia), respectively.
Results: Upon irradiating cells with 20Ne ions, tracks of multiple clustered γH2AX and p53BP1 repair foci appeared immediately after the irradiation; these clusters, called here as the ‘primary clusters’, were rare in cells irradiated with γ-rays or protons (submitted). Though γH2AX/p53BP1 foci were positionally quite stable [
2], ‘secondary clusters’ occasionally appeared after all kinds of irradiation during about 30 min PI. The formation of secondary clusters usually appeared due to the heterochromatin decondensation at the sites of heterochromatic DNA double-strand breaks (hcDSBs), followed by their protrusion into a limited space of nuclear subdomains of low density-chromatin (discussed in [
1, 2, 5]).
Conclusions: Primary clusters appear in cell nuclei immediately PI as the consequence of highly localized energy deposition, while secondary clusters develop during (and because of) DSB repair. Primary DSB clusters probably represent the main cause of chromosomal translocations induced with high-LET radiations while secondary clusters seem to be more important for low-LET γ-rays and protons. Secondary clusters of primary clusters (higher-order clusters) observed for 20Ne ions might explain frequent formation of complex translocations upon the action of high-LET radiations. Finally, we suggest [
1, 2, 4] a model that describes the relationship between the higher order chromatin structure, DSB formation, repair and misrepair.
DNA double-strand breaks (DSB); DSB repair; chromosomal translocations; primary and secondary DSB clusters; higher order chromatin structure; ionizing radiation of different quality
Early embryonic lethality of interspecies hybrids in Drosophila can be caused by defects in mitotic segregation of paternal X chromatids carrying a critical domain of heterochromatic DNA.
Postzygotic reproductive barriers such as sterility and lethality of hybrids are important for establishing and maintaining reproductive isolation between species. Identifying the causal loci and discerning how they interfere with the development of hybrids is essential for understanding how hybrid incompatibilities (HIs) evolve, but little is known about the mechanisms of how HI genes cause hybrid dysfunctions. A previously discovered Drosophila melanogaster locus called Zhr causes lethality in F1 daughters from crosses between Drosophila simulans females and D. melanogaster males. Zhr maps to a heterochromatic region of the D. melanogaster X that contains 359-bp satellite repeats, suggesting either that Zhr is a rare protein-coding gene embedded within heterochromatin, or is a locus consisting of the noncoding repetitive DNA that forms heterochromatin. The latter possibility raises the question of how heterochromatic DNA can induce lethality in hybrids. Here we show that hybrid females die because of widespread mitotic defects induced by lagging chromatin at the time during early embryogenesis when heterochromatin is first established. The lagging chromatin is confined solely to the paternally inherited D. melanogaster X chromatids, and consists predominantly of DNA from the 359-bp satellite block. We further found that a rearranged X chromosome carrying a deletion of the entire 359-bp satellite block segregated normally, while a translocation of the 359-bp satellite block to the Y chromosome resulted in defective Y segregation in males, strongly suggesting that the 359-bp satellite block specifically and directly inhibits chromatid separation. In hybrids produced from wild-type parents, the 359-bp satellite block was highly stretched and abnormally enriched with Topoisomerase II throughout mitosis. The 359-bp satellite block is not present in D. simulans, suggesting that lethality is caused by the absence or divergence of factors in the D. simulans maternal cytoplasm that are required for heterochromatin formation of this species-specific satellite block. These findings demonstrate how divergence of noncoding repetitive sequences between species can directly cause reproductive isolation by altering chromosome segregation.
Speciation is most commonly understood to occur when two species can no longer reproduce with each other, and sterility and lethality of hybrids formed between different species are widely observed causes of such reproductive isolation. Several protein-coding genes have been previously discovered to cause hybrid sterility and lethality. We show here that first generation hybrid females in Drosophila die during early embryogenesis because of a failure in mitosis. However, we have discovered that this is not a general failure in mitosis, because only the paternally inherited X chromosome fails to segregate properly. Our analyses further demonstrate that this mitotic failure is caused by a large heterochromatic region of DNA (millions of base pairs) that contains many repetitive copies of short noncoding sequences that are normally transcriptionally quiescent. Interestingly, this block of heterochromatin is only found in the paternal species. We suggest that a failure of the maternal species to package this paternally inherited DNA region into heterochromatin leads to mitotic failure and hybrid lethality. If this is a general phenomenon it may explain other examples of hybrid lethality in which F1 females die but F1 males survive.
Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci.
An important question in virology is the mechanism(s) by which persistent viruses such as the herpesviruses and human immunodeficiency virus (HIV) establish a latent infection in specific types of cells. In the case of herpesviruses, herpes simplex virus (HSV) infection of epithelial cells results in a lytic infection, whereas latent infection is established in sensory neurons. Recent studies have shown the importance of chromatin structure in the regulation of latent infection for both HSV and HIV. For HSV, we have shown previously that the viral latency-associated transcript (LAT) promotes lytic gene silencing and the association of one heterochromatin marker, dimethylation of lysine 9 on histone H3 (H3K9me2), with viral lytic genes. In this study, we further defined the structure of latent viral chromatin by examining the heterochromatin markers on histones associated with the HSV latent genome. We detected the H3K9me2, H3K9me3, and H3K27me3 modifications, with H3K27me3, which is indicative of facultative heterochromatin, exhibiting the highest enrichment on all viral promoters tested. A modification associated with cellular centromeric heterochromatin, H4K20me3, was not detected. A mutant virus containing a 1.8-kbp deletion within the LAT region showed reduced levels of the facultative heterochromatin marker (H3K27me3) along with H3K9me3 during latency, whereas a viral mutant defective for the LAT promoter showed a specific reduction in H3K27me3. Cellular long, noncoding RNAs induce facultative heterochromatin, and this study shows that transcription of a viral noncoding RNA can also induce facultative heterochromatin to promote lytic gene silencing during latency.
Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.
DNA double-strand breaks (DSBs) generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair.
Methods and Materials
Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent) and heterochromatin (electron-dense) in cortical neurons of irradiated mouse brain.
While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer) are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥6 gold beads) may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥10 gold beads), occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage.
Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing severe chromatin disruptions. Imperfect restoration of chromatin configurations may leave DSB-induced epigenetic memory of damage with potentially pathological repercussions.
Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in Schizosaccharomyces pombe have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on Drosophila heterochromatin structure.
The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (dcr-2, ago1, ago2, piwi, Lip [D], aub and hls). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of white-mottled4h position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using Drosophila embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.
Our experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in Drosophila. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in Drosophila.
Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA–immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.
Heterochromatin Protein 1 (HP1a) is a very well known prototype protein of a general model for heterochromatin formation and epigenetic gene silencing in different species including humans. Here, we report our experiments showing that HP1a is also required for the positive regulation of more than one hundred euchromatic genes by its association with the corresponding RNA transcripts and by its interaction with heterogeneous nuclear ribonucleoproteins (hnRNPs) belonging to different classes. Importantly, we also found that all the tested hnRNP proteins bind to the heterochromatin and are dominant suppressors of position effect variegation, thus suggesting they also have a role in heterochromatin organization. Taken together, our data show novel and important functions, not only for HP1a, but also for hnRNPs, which were previously believed to participate only in RNA processing. These results shed new light on the epigenetic mechanisms of gene silencing and gene expression. They also establish a link between RNA transcript metabolism and heterochromatin formation and change several aspects of the canonical views about these apparently different processes.
In recent years it has been recognized that the development of cancer involves a series of not only genetic but epigenetic changes across the genome. At the same time, connections between epigenetic regulation, chromatin packaging, and overall nuclear architecture are increasingly appreciated. The cell-type specific organization of heterochromatin, established upon cell differentiation, is responsible for maintaining much of the genome in a repressed state, within a highly compartmentalized nucleus. This review focuses on recent evidence that in cancer the normal packaging and higher organization of heterochromatin is often compromised. Gross changes in nuclear morphology have long been a criterion for pathologic diagnosis of many cancers, but the specific nuclear components impacted, the mechanisms involved, and the implications for cancer progression have barely begun to emerge. We discuss recent findings regarding distinct heterochromatin types, including the inactive X chromosome, constitutive heterochromatin of peri/centric satellites, and the peripheral heterochromatic compartment (PHC). A theme developed here is that the higher-order organization of satellites and the peripheral heterochromatic compartment may be tightly linked, and that compromise of this organization may promote broad epigenomic imbalance in cancer. Recent studies into the potential role(s) of the breast cancer tumor suppressor, BRCA1, in maintaining heterochromatin will be highlighted. Many questions remain about this new area of cancer epigenetics, which is likely more important in cancer development and progression than widely appreciated. We propose that broad, stochastic compromise in heterochromatin maintenance would create a diversity of expression profiles, and thus a rich opportunity for one or more cells to emerge with a selective growth advantage and potential for neoplasia.
heterochromatin; XIST; cancer; satellite DNA; epigenetics; BRCA1
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.
NETosis, the process wherein neutrophils release highly decondensed chromatin called neutrophil extracellular traps (NETs), has gained much attention as an alternative means of killing bacteria. In vivo, NETs are induced by bacteria and pro-inflammatory cytokines. We have reported that peptidylarginine deiminase 4 (PAD4), an enzyme that converts Arg or monomethyl-Arg to citrulline in histones, is essential for NET formation. The areas of extensive chromatin decondensation along the NETs were rich in histone citrullination. Here, upon investigating the effect of global citrullination in cultured cells, we discovered that PAD4 overexpression in osteosarcoma U2OS cells induces extensive chromatin decondensation independent of apoptosis. The highly decondensed chromatin is released to the extracellular space and stained strongly by a histone citrulline-specific antibody. The structure of the decondensed chromatin is reminiscent of NETs but is unique in that it occurs without stimulation of cells with pro-inflammatory cytokines and bacteria. Furthermore, histone citrullination during chromatin decondensation can dissociate heterochromatin protein 1 beta (HP1β) thereby offering a new molecular mechanism for understanding how citrullination regulates chromatin function. Taken together, our study suggests that PAD4 mediated citrullination induces chromatin decondensation, implicating its essential role in NET formation under physiological conditions in neutrophils.
pad4; hypercitrullination; neutrophil extracellular traps; chromatin decondensation; heterochromatin protein 1; histone modifications
Posttranslational histone modifications and histone variants form a unique epigenetic landscape on mammalian chromosomes where the principal epigenetic heterochromatin markers, trimethylated histone H3(K9) and the histone H2A.Z, are inversely localized in relation to each other. Trimethylated H3(K9) marks pericentromeric constitutive heterochromatin and the male Y chromosome, while H2A.Z is dramatically reduced at these chromosomal locations. Inactivation of a lysosomal and nuclear protease, cathepsin L, causes a global redistribution of epigenetic markers. In cathepsin L knockout cells, the levels of trimethylated H3(K9) decrease dramatically, concomitant with its relocation away from heterochromatin, and H2A.Z becomes enriched at pericentromeric heterochromatin and the Y chromosome. This change is also associated with global relocation of heterochromatin protein HP1 and histone H3 methyltransferase Suv39h1 away from constitutive heterochromatin; however, it does not affect DNA methylation or chromosome segregation, phenotypes commonly associated with impaired histone H3(K9) methylation. Therefore, the key constitutive heterochromatin determinants can dynamically redistribute depending on physiological context but still maintain the essential function(s) of chromosomes. Thus, our data show that cathepsin L stabilizes epigenetic heterochromatin markers on pericentromeric heterochromatin and the Y chromosome through a novel mechanism that does not involve DNA methylation or affect heterochromatin structure and operates on both somatic and sex chromosomes.
The HIV-1 protein Vpr disrupts higher-order chromatin structure by altering histone modification and displacing important heterochromatin proteins, resulting in chromatid cohesion defects.
Although pericentromeric heterochromatin is essential for chromosome segregation, its role in humans remains controversial. Dissecting the function of HIV-1–encoded Vpr, we unraveled important properties of heterochromatin during chromosome segregation. In Vpr-expressing cells, hRad21, hSgo1, and hMis12, which are crucial for proper chromosome segregation, were displaced from the centromeres of mitotic chromosomes, resulting in premature chromatid separation (PCS). Interestingly, Vpr displaced heterochromatin protein 1-α (HP1-α) and HP1-γ from chromatin. RNA interference (RNAi) experiments revealed that down-regulation of HP1-α and/or HP1-γ induced PCS, concomitant with the displacement of hRad21. Notably, Vpr stimulated the acetylation of histone H3, whereas p300 RNAi attenuated the Vpr-induced displacement of HP1-α and PCS. Furthermore, Vpr bound to p300 that was present in insoluble regions of the nucleus, suggesting that Vpr aberrantly recruits the histone acetyltransferase activity of p300 to chromatin, displaces HP1-α, and causes chromatid cohesion defects. Our study reveals for the first time centromere cohesion impairment resulting from epigenetic disruption of higher-order structures of heterochromatin by a viral pathogen.
This study shows how young sex chromosomes have altered their chromatin structure in Drosophila, and what genomic changes have led to silencing of the Y, and hyper-transcription of the X.
Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.
Sex chromosomes differ from non-sex chromosomes (“autosomes”) at the genomic, transcriptomic, and epigenomic level, yet the X and Y share a common evolutionary origin. The Drosophila Y chromosome is gene-poor and associated with a compact and transcriptionally inactive form of genetic material called heterochromatin. The X, in contrast, is enriched for activating chromatin marks and is consequently hyper-transcribed, a process thought to be an adaptation to decay and silencing of genes on the Y, resulting in “dosage compensation.” How sex chromosomes have altered their chromatin structure, and what genomic changes led to this dramatically different epigenetic makeup, however, has remained a mystery. By studying the genome, epigenome, and transcriptome of a species with a very recently evolved pair of sex chromosomes (the neo-X and neo-Y of a fruit fly, Drosophila miranda), we here recapitulate how both dosage compensation and heterochromatin formation evolve in Drosophila and establish several novel and important principles governing the evolution of chromatin structure. We dissect the evolutionary history of over 60 novel binding sites for the dosage compensation complex that evolved by natural selection on the neo-X within the last one million years. We show that the 21-bp consensus motifs for recruiting the dosage compensation complex were acquired by diverse molecular mechanisms along the neo-X, while the onset of heterochromatin formation is triggered by the accumulation of transposable elements, leading to silencing of adjacent neo-Y genes. We find that spreading of these chromatin modifications results in massive mis-expression of neo-sex linked genes, and that little correspondence exists between functional activity of genes on the neo-Y and whether they are dosage-compensated on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those that are heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities of the ancestral chromosome that formed the sex chromosome to evolve active versus repressive chromatin configurations. These findings have broad implications for current models of sex chromosome evolution.
Position-effect variegation (PEV) is the stochastic transcriptional silencing of a gene positioned adjacent to heterochromatin. white-mottled X-chromosomal inversions in Drosophila are classic PEV models that show variegation of the eye color gene white due to its relocation next to pericentric heterochromatin. It has been suggested that in these models the spreading of heterochromatin across the rearrangement breakpoint causes the silencing of white. However, the extent of this spreading and the precise pattern of heterochromatin redistribution have remained unclear. To obtain insight into the mechanism of PEV, we constructed high-resolution binding maps of Heterochromatin Protein 1 (HP1) on white-mottled chromosomes.
We find that HP1 invades euchromatin across the inversion breakpoints over ~175 kb and ~30 kb, causing de novo association of HP1 with 20 genes. However, HP1 binding levels in these regions show substantial local variation, and white is the most strongly bound gene. Remarkably, white is also the only gene that is detectably repressed by heterochromatin. Furthermore, we find that HP1 binding to the invaded region is particularly sensitive to the dosage of the histone methyltransferase Su(var)3-9, indicating that the de novo formed heterochromatin is less stable than naturally occurring constitutive heterochromatin.
Our molecular maps demonstrate that heterochromatin can invade a normally euchromatic region, yet the strength of HP1 binding and effects on gene expression are highly dependent on local context. Our data suggest that the white gene has an unusual intrinsic affinity for heterochromatin, which may cause this gene to be more sensitive to PEV than most other genes.
Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.
Centromeres are essential to chromatin structures, providing a binding platform for the mitotic spindle. Defects in centromere structure or function can lead to chromosome missegregation or chromosome breakage. This, in turn, can cause cancer in metazoans. Centromeres are defined by specialized chromatin that contains the histone H3 variant CENP-A (also called CenH3, or Cse4 in budding yeast), and transcription over centromeres is tightly controlled. Budding yeast centromeres are composed of a single nucleosome containing the essential Cse4. Loss of one of these specialized centromeric nucleosomes can lead to chromosome missegregation during mitosis followed by cell death. We provide evidence that energy-dependent chromatin remodeling factor Fun30 supports faithful chromosome segregation, especially when centromere structure is challenged by mutation of Cse4 or by forced transcription through centromeres, which disrupt centromere structure. We show that Fun30 binds to centromeres and that loss of Fun30 leads to various defects in centromere chromatin, suggesting a direct role for Fun30 in promoting normal centromere function. Our analysis shows that Fun30 affects nucleosome positioning at many genomic sites, including centromeres, and is required for normal occupancy of histone variant Htz1. In the absence of Fun30, we detect an increase in transcription through centromeres. We suggest that an important function of Fun30 is to limit transcription over centromeres.
Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric α-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric α-satellite DNA in human lymphoid cells synchronized at G0/G1 is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric α-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that “chromosomal environment” plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.