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1.  Heterochromatin Formation Promotes Longevity and Represses Ribosomal RNA Synthesis 
PLoS Genetics  2012;8(1):e1002473.
Organismal aging is influenced by a multitude of intrinsic and extrinsic factors, and heterochromatin loss has been proposed to be one of the causes of aging. However, the role of heterochromatin in animal aging has been controversial. Here we show that heterochromatin formation prolongs lifespan and controls ribosomal RNA synthesis in Drosophila. Animals with decreased heterochromatin levels exhibit a dramatic shortening of lifespan, whereas increasing heterochromatin prolongs lifespan. The changes in lifespan are associated with changes in muscle integrity. Furthermore, we show that heterochromatin levels decrease with normal aging and that heterochromatin formation is essential for silencing rRNA transcription. Loss of epigenetic silencing and loss of stability of the rDNA locus have previously been implicated in aging of yeast. Taken together, these results suggest that epigenetic preservation of genome stability, especially at the rDNA locus, and repression of unnecessary rRNA synthesis, might be an evolutionarily conserved mechanism for prolonging lifespan.
Author Summary
Aging is characterized by a progressive decline in vitality and tissue function, leading to the demise of the organism. Many models have been proposed to explain the aging phenomenon. Among the many competing and/or overlapping models is the heterochromatin loss model of aging, which posits that heterochromatin domains (which are set up early in embryogenesis) are gradually lost with aging, resulting in de-repression of silenced genes and aberrant gene expression patterns associated with old age. In this paper, we genetically tested the role of heterochromatin in Drosophila aging. We find that heterochromatin levels indeed affect animal lifespan and that heterochromatin represses, among other things, rRNA transcription. Loss of heterochromatin thus leads to an increase in rRNA transcription, a rate-limiting step in ribosome biogenesis and protein synthesis. We suggest that the biological functions of heterochromatin formation include controlling rRNA transcription, which might play an important role in general protein synthesis and animal longevity.
doi:10.1371/journal.pgen.1002473
PMCID: PMC3266895  PMID: 22291607
2.  Centromere-Independent Accumulation of Cohesin at Ectopic Heterochromatin Sites Induces Chromosome Stretching during Anaphase 
PLoS Biology  2014;12(10):e1001962.
Live imaging of cells carrying rearranged chromosomes shows that misplaced heterochromatin is sufficient to induce ectopic cohesion and chromosome stretching during mitosis, and may compromise genetic stability.
Pericentric heterochromatin, while often considered as “junk” DNA, plays important functions in chromosome biology. It contributes to sister chromatid cohesion, a process mediated by the cohesin complex that ensures proper genome segregation during nuclear division. Long stretches of heterochromatin are almost exclusively placed at centromere-proximal regions but it remains unclear if there is functional (or mechanistic) importance in linking the sites of sister chromatid cohesion to the chromosomal regions that mediate spindle attachment (the centromere). Using engineered chromosomes in Drosophila melanogaster, we demonstrate that cohesin enrichment is dictated by the presence of heterochromatin rather than centromere proximity. This preferential accumulation is caused by an enrichment of the cohesin-loading factor (Nipped-B/NIPBL/Scc2) at dense heterochromatic regions. As a result, chromosome translocations containing ectopic pericentric heterochromatin embedded in euchromatin display additional cohesin-dependent constrictions. These ectopic cohesion sites, placed away from the centromere, disjoin abnormally during anaphase and chromosomes exhibit a significant increase in length during anaphase (termed chromatin stretching). These results provide evidence that long stretches of heterochromatin distant from the centromere, as often found in many cancers, are sufficient to induce abnormal accumulation of cohesin at these sites and thereby compromise the fidelity of chromosome segregation.
Author Summary
During cell division, chromosomes acquire their characteristic X-shaped morphology by having well-resolved chromosome arms while still remaining connected at the heterochromatic regions around the centromere. This connection is mediated by the cohesin complex, a “molecular glue” that keeps the two DNA molecules stuck together and ensures that the chromosomes are properly segregated. However, it is unclear how important it is for efficient chromosome segregation that these cohesive forces are specifically positioned near the centromere. In this study, we tested several strains of the fruit fly Drosophila melanogaster carrying chromosomal rearrangements in which long stretches of heterochromatin from near the centromere have been misplaced within distant euchromatic regions. We find that such inappropriately located heterochromatin is enough to promote increased levels of cohesin complex loading and the formation of additional constrictions, regardless of proximity to the centromere. Importantly, we further show that as cell division proceeds and the sister chromatids move to opposite poles of the cell, the presence of ectopic heterochromatin (and hence ectopic cohesion) leads to significant chromosome stretching due to impaired resolution of the ectopic cohesion sites. These results highlight the possibility that chromosome rearrangements involving heterochromatin regions near the centromeres, often seen in many cancers, can induce additional errors in cell division and thereby compromise genetic stability.
doi:10.1371/journal.pbio.1001962
PMCID: PMC4188515  PMID: 25290697
3.  Remodeling of heterochromatin induced by heavy metals in extreme old age 
Age  2010;33(3):433-438.
The levels of chromosome instability and heat absorption of chromatin have been studied in cultured lymphocytes derived from blood of 80–93- and 18–30-year-old individuals, under the effect of heavy metal Cu(II) and Cd(II) salts. The analysis of the results obtained indicates that 50 μM Cu(II) induced a significantly higher level of cells with chromosome aberrations in old donors (13.8 ± 1.5% vs control, 3.8 ± 1.7%), whereas treatment with 100 μM Cd(II) did not induce any changes in the background index. Analysis of the lymphocyte melting curves showed that Cu(II) ions caused more effective condensation of heterochromatin in old healthy individuals compared with young donors, which was expressed by the increase of the Tm of elderly chromatin by ~3°C compared with the norm. Treatment of lymphocyte chromatin of old individuals with 100 μM Cd(II) caused decondensation (deheterochromatinization) of both the facultative and constitutive domains of heterochromatin. The deheterochromatinization Tm was decreased by ~3–3.5°C compared with the Tm observed for young individuals. Thus, the chromatin of cultured lymphocytes from the old-aged individuals underwent modification under the influence of copper and cadmium salts. Cu(II) caused additional heterochromatinization of heterochromatin, and Cd(II) caused deheterochromatinization of facultative and constitutive heterochromatin. Our data may be important as new information on the remodeling of constitutive and facultative heterochromatin induced by heavy metals in aging, aging pathology, and pathology linked with metal ions.
doi:10.1007/s11357-010-9185-1
PMCID: PMC3168599  PMID: 20865337
Aberration; Aging; Cadmium; Chromosome; Copper; Heterochromatin; Heterochromatinization; Microcalorimetry
4.  Molecular Landscape of Modified Histones in Drosophila Heterochromatic Genes and Euchromatin-Heterochromatin Transition Zones 
PLoS Genetics  2008;4(1):e16.
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.
Author Summary
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.
doi:10.1371/journal.pgen.0040016
PMCID: PMC2211541  PMID: 18208336
5.  Heterochromatin Instability in Cancer: From the Barr Body to Satellites and the Nuclear Periphery 
Seminars in cancer biology  2012;23(2):99-108.
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.
doi:10.1016/j.semcancer.2012.06.008
PMCID: PMC3500402  PMID: 22722067
heterochromatin; XIST; cancer; satellite DNA; epigenetics; BRCA1
6.  Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis 
eLife  2014;3:e02996.
Despite the well-established role of heterochromatin in protecting chromosomal integrity during meiosis and mitosis, the contribution and extent of heterochromatic histone posttranslational modifications (PTMs) remain poorly defined. Here, we gained novel functional insight about heterochromatic PTMs by analyzing histone H3 purified from the heterochromatic germline micronucleus of the model organism Tetrahymena thermophila. Mass spectrometric sequencing of micronuclear H3 identified H3K23 trimethylation (H3K23me3), a previously uncharacterized PTM. H3K23me3 became particularly enriched during meiotic leptotene and zygotene in germline chromatin of Tetrahymena and C. elegans. Loss of H3K23me3 in Tetrahymena through deletion of the methyltransferase Ezl3p caused mislocalization of meiosis-induced DNA double-strand breaks (DSBs) to heterochromatin, and a decrease in progeny viability. These results show that an evolutionarily conserved developmental pathway regulates H3K23me3 during meiosis, and our studies in Tetrahymena suggest this pathway may function to protect heterochromatin from DSBs.
DOI: http://dx.doi.org/10.7554/eLife.02996.001
eLife digest
Inside the nucleus of a cell, the DNA is wound around histone proteins. This forms a structure called chromatin that allows the long DNA strands to fit inside the cell. Variations in chromatin structure also help the cell to control the functional properties of DNA. For example, a large proportion of chromatin in the cell is in the form of heterochromatin, which is very densely packed, and is associated with many roles such as gene silencing and keeping DNA intact during reproduction.
Many animals and plants have two copies of each DNA molecule: one inherited from the mother, and one from the father of the organism. Reproductive cells undergo a process called recombination when they form, where the matching copies of each DNA molecule break in a number of places and rejoin to form a new ‘blend’ of their mother's and their father's DNA, which is passed on to their own offspring. In contrast, most heterochromatin is inherited without recombining, preserving it in an unaltered form. This is important since recombination in heterochromatin can create genetic abnormalities.
Adding small chemical modifications—such as methyl groups—to the histone proteins at the core of the chromatin can change how the DNA is packed. However, the histone modifications that yield different chromatin structures, and the effect of these modifications, are not very well understood.
Papazyan et al. have taken advantage of a distinct feature of the protozoan Tetrahymena thermophila: a single-celled organism that divides its chromatin into two different nuclei. The smaller micronuclei contain only heterochromatin, and Papazyan et al. discovered that the histone H3 protein in the micronuclei is modified by methyl groups at a specific site that had not been studied before. Furthermore, this protozoan makes more of these modifications when it reproduces. An enzyme called Ezl3p adds these methyl groups, and without this enzyme T. thermophila reproduces more slowly and has offspring that are less likely to survive and more likely to be infertile. Papazyan et al. provide evidence that these characteristics arise because the cells without the histone modification are unable to prevent DNA breaks from occurring in heterochromatin during recombination.
The same histone modification also occurs when the microscopic worm Caenorhabditis elegans reproduces, suggesting that this method of DNA protection has been conserved throughout evolution. Papazyan et al. propose that the histone modification may prevent another enzyme that induces DNA breaks from accessing the heterochromatin in reproductive cells; but more work is required to support this hypothesis.
These findings reveal the importance of a new histone modification during reproduction, and could provide new directions for infertility research.
DOI: http://dx.doi.org/10.7554/eLife.02996.002
doi:10.7554/eLife.02996
PMCID: PMC4141274  PMID: 25161194
Tetrahymena thermophila; histones; chromatin; methylation; meiosis; DNA damage; C. elegans; other
7.  Histone Hyperacetylation in Mitosis Prevents Sister Chromatid Separation and Produces Chromosome Segregation Defects 
Molecular Biology of the Cell  2003;14(9):3821-3833.
Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-δ on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.
doi:10.1091/mbc.E03-01-0860
PMCID: PMC196571  PMID: 12972566
8.  Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster 
BMC Genomics  2011;12:205.
Background
In recent years, substantial progress has been made in understanding the organization of sequences in heterochromatin regions containing single-copy genes and transposable elements. However, the sequence and organization of tandem repeat DNA sequences, which are by far the majority fraction of D. melanogaster heterochromatin, are little understood.
Results
This paper reports that the heterochromatin, as well as containing long tandem arrays of pentanucleotide satellites (AAGAG, AAGAC, AATAT, AATAC and AACAC), is also enriched in other simple sequence repeats (SSRs) such as A, AC, AG, AAG, ACT, GATA and GACA. Non-denaturing FISH (ND-FISH) showed these SSRs to localize to the chromocentre of polytene chromosomes, and was used to map them on mitotic chromosomes. Different distributions were detected ranging from single heterochromatic clusters to complex combinations on different chromosomes. ND-FISH performed on extended DNA fibres, along with Southern blotting, showed the complex organization of these heterochromatin sequences in long tracts, and revealed subclusters of SSRs (several kilobase in length) flanked by other DNA sequences. The chromosomal characterization of C, AAC, AGG, AAT, CCG, ACG, AGC, ATC and ACC provided further detailed information on the SSR content of D. melanogaster at the whole genome level.
Conclusion
These data clearly show the variation in the abundance of different SSR motifs and reveal their non-random distribution within and between chromosomes. The greater representation of certain SSRs in D. melanogaster heterochromatin suggests that its complexity may be greater than previously thought.
doi:10.1186/1471-2164-12-205
PMCID: PMC3114746  PMID: 21521504
9.  Decelerating the Aging Process 
Diseases whose incidence and prevalence are increased in the elderly and whose cytopathology, hormones, and immunogenesis differ, generally are included in the field of geriatrics. These conditions may be precipitated or accelerated in quantity or type by a wide variety of genetic and environmental factors. Chronological and progressive deterioration of selected cells, organs, and tissues, and their functions may occur without major specific pathology. These processes are referred to as senescence and its study, is gerontology.
Geriatrics includes senility and diseases of the elderly. Terms associated with gerontology include benign agism or senescence, as a normal consequence of the aging processes common to all biological forms of life.
The age period, 65 to 85 years, does not necessarily imply senility, but the normal chronological aging of an individual in an industrialized, urbanized society. This paper emphasizes recommendations for deceleration of the normal aging process.
PMCID: PMC2537227  PMID: 423284
10.  High-Resolution Mapping Reveals Links of HP1 with Active and Inactive Chromatin Components 
PLoS Genetics  2007;3(3):e38.
Heterochromatin protein 1 (HP1) is commonly seen as a key factor of repressive heterochromatin, even though a few genes are known to require HP1-chromatin for their expression. To obtain insight into the targeting of HP1 and its interplay with other chromatin components, we have mapped HP1-binding sites on Chromosomes 2 and 4 in Drosophila Kc cells using high-density oligonucleotide arrays and the DNA adenine methyltransferase identification (DamID) technique. The resulting high-resolution maps show that HP1 forms large domains in pericentric regions, but is targeted to single genes on chromosome arms. Intriguingly, HP1 shows a striking preference for exon-dense genes on chromosome arms. Furthermore, HP1 binds along entire transcription units, except for 5′ regions. Comparison with expression data shows that most of these genes are actively transcribed. HP1 target genes are also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4 (H3K4me2), which are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually observed along entire transcription units, is limited to the 5′ ends of HP1-bound genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. Additionally, we observed that HP1-chromatin and Polycomb-chromatin are nonoverlapping, but often closely juxtaposed, suggesting an interplay between both types of chromatin. These results demonstrate that HP1-chromatin is transcriptionally active and has extensive links with several other chromatin components.
Author Summary
In each of our cells, a variety of proteins helps to organize the very long DNA fibers into a more compacted structure termed chromatin. Several different types of chromatin exist. Some types of chromatin package DNA rather loosely and thereby allow the genes to be active. Other types, often referred to as heterochromatin, are thought to package the DNA into a condensed structure that prevents the genes from being active. Thus, the different types of chromatin together determine the “gene expression programs” of cells. To understand how this works, it is necessary to identify the genes that are packaged by a particular type of chromatin and to reveal how various chromatin proteins work together to achieve this. Here we present highly detailed maps of the DNA sequences that are packaged by a heterochromatin protein named HP1. The results show that HP1 preferentially binds along the genes themselves and much less to intergenic regions. Contrary to what was previously thought, most genes packaged by HP1 are active. Finally, the data suggest that HP1 may compete with other types of chromatin proteins. These results contribute to our fundamental understanding of the roles of chromatin packaging in gene regulation.
doi:10.1371/journal.pgen.0030038
PMCID: PMC1808074  PMID: 17335352
11.  Ring-Like Distribution of Constitutive Heterochromatin in Bovine Senescent Cells 
PLoS ONE  2011;6(11):e26844.
Background
Cells that reach “Hayflick limit” of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining.
Methods
We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH).
Results
Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli.
Conclusions
Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.
doi:10.1371/journal.pone.0026844
PMCID: PMC3223162  PMID: 22132080
12.  Centromeric heterochromatin assembly in fission yeast—balancing transcription, RNA interference and chromatin modification 
Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of centromeric heterochromatin in fission yeast that integrates our current understanding of the competing forces of transcription, replication, and RNA decay that influence its assembly and propagation.
doi:10.1007/s10577-012-9288-x
PMCID: PMC3580186  PMID: 22733402
Centromere; Heterochromatin; RNAi; Non-coding RNA
13.  Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes 
Genome Biology  2004;5(11):R89.
An assay of the formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha satellite DNA revealed that only a small amount of heterochromatin may be required for centromere function and that replication late in S phase is not a requirement for centromere function.
Background
Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.
Results
Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.
Conclusions
Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.
doi:10.1186/gb-2004-5-11-r89
PMCID: PMC545780  PMID: 15535865
14.  High- and Low-mobility Populations of HP1 in Heterochromatin of Mammalian CellsD⃞ 
Molecular Biology of the Cell  2004;15(6):2819-2833.
Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50–80%) are highly mobile (recovery halftime: t1/2 ≈ 0.9 s; diffusion coefficient: D ≈ 0.6–0.7 μm2 s-1). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t1/2 ≈ 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5–7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8–14% of GFP-HP1α, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1α in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle.
doi:10.1091/mbc.E03-11-0827
PMCID: PMC420105  PMID: 15064352
15.  Molecular Dissection of Formation of Senescence-Associated Heterochromatin Foci▿ †  
Molecular and Cellular Biology  2007;27(6):2343-2358.
Senescence is characterized by an irreversible cell proliferation arrest. Specialized domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), are thought to contribute to the irreversible cell cycle exit in many senescent cells by repressing the expression of proliferation-promoting genes such as cyclin A. SAHF contain known heterochromatin-forming proteins, such as heterochromatin protein 1 (HP1) and the histone H2A variant macroH2A, and other specialized chromatin proteins, such as HMGA proteins. Previously, we showed that a complex of histone chaperones, histone repressor A (HIRA) and antisilencing function 1a (ASF1a), plays a key role in the formation of SAHF. Here we have further dissected the series of events that contribute to SAHF formation. We show that each chromosome condenses into a single SAHF focus. Chromosome condensation depends on the ability of ASF1a to physically interact with its deposition substrate, histone H3, in addition to its cochaperone, HIRA. In cells entering senescence, HP1γ, but not the related proteins HP1α and HP1β, becomes phosphorylated on serine 93. This phosphorylation is required for efficient incorporation of HP1γ into SAHF. Remarkably, however, a dramatic reduction in the amount of chromatin-bound HP1 proteins does not detectably affect chromosome condensation into SAHF. Moreover, abundant HP1 proteins are not required for the accumulation in SAHF of histone H3 methylated on lysine 9, the recruitment of macroH2A proteins, nor other hallmarks of senescence, such as the expression of senescence-associated β-galactosidase activity and senescence-associated cell cycle exit. Based on our results, we propose a stepwise model for the formation of SAHF.
doi:10.1128/MCB.02019-06
PMCID: PMC1820509  PMID: 17242207
16.  DNA Methylation and Normal Chromosome Behavior in Neurospora Depend on Five Components of a Histone Methyltransferase Complex, DCDC 
PLoS Genetics  2010;6(11):e1001196.
Methylation of DNA and of Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, and fungi. In Neurospora crassa, methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 (HP1) and the DIM-2 DNA methyltransferase. We report genetic, proteomic, and biochemical investigations into how DIM-5 is controlled. These studies revealed DCDC, a previously unknown protein complex including DIM-5, DIM-7, DIM-9, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora.
Author Summary
Eukaryotic genomes are composed of distinct structural and functional domains marked by various covalent modifications of histone proteins and, in some organisms, by methylation of cytosine bases in DNA. Gene-rich euchromatin exists in a relatively open conformation, facilitating DNA transactions such as transcription, whereas the gene-poor heterochromatin is more condensed and is a poor substrate for DNA–based transactions. Heterochromatin promotes genome stability by silencing transposons and may be essential for proper centromere function. DNA methylation is a common feature of heterochromatin in eukaryotes, including the filamentous fungus Neurospora crassa, which has served as a model system to elucidate the control of DNA methylation. All DNA methylation in Neurospora depends on histone H3 lysine-9 (H3K9) methylation, which is recognized by a complex of HP1 (Heterochromatin Protein 1) and the DNA methyltransferase, DIM-2. An important open question is what controls the H3K9 methyltransferase, DIM-5. We report the genetic and proteomic identification of a DIM-5 protein complex, DCDC, and demonstrate that it includes five proteins essential for H3K9 methylation, DNA methylation, proper chromosome segregation, and resistance to DNA damaging agents. In addition, we report molecular and genetic analyses revealing a hierarchy of protein interactions within DCDC.
doi:10.1371/journal.pgen.1001196
PMCID: PMC2973830  PMID: 21079689
17.  The Chromatin Remodelling Factor dATRX Is Involved in Heterochromatin Formation 
PLoS ONE  2008;3(5):e2099.
Despite extensive study of heterochromatin, relatively little is known about the mechanisms by which such a structure forms. We show that the Drosophila homologue of the human α-thalassemia and mental retardation X-linked protein (dATRX), is important in the formation or maintenance of heterochromatin through modification of position effect variegation. We further show that there are two isoforms of the dATRX protein, the longer of which interacts directly with heterochromatin protein 1 (dHP-1) through a CxVxL motif both in vitro and in vivo. These two proteins co-localise at heterochromatin in a manner dependent on this motif. Consistent with this observation, the long isoform of the dATRX protein localises primarily to the heterochromatin at the chromocentre on salivary gland polytene chromosomes, whereas the short isoform binds to many sites along the chromosome arms. We suggest that the establishment of a regular nucleosomal organisation may be common to heterochromatin and transcriptionally repressed chromatin in other locations, and may require the action of ATP dependent chromatin remodelling factors.
doi:10.1371/journal.pone.0002099
PMCID: PMC2324200  PMID: 18461125
18.  Integration of High-Resolution Physical and Genetic Map Reveals Differential Recombination Frequency between Chromosomes and the Genome Assembling Quality in Cucumber 
PLoS ONE  2013;8(5):e62676.
Cucumber is an important model crop and the first species sequenced in Cucurbitaceae family. Compared to the fast increasing genetic and genomics resources, the molecular cytogenetic researches in cucumber are still very limited, which results in directly the shortage of relation between plenty of physical sequences or genetic data and chromosome structure. We mapped twenty-three fosmids anchored by SSR markers from LG-3, the longest linkage group, and LG-4, the shortest linkage group on pachytene chromosomes 3 and 4, using uorescence in situ hybridization (FISH). Integrated molecular cytogenetic maps of chromosomes 3 and 4 were constructed. Except for three SSR markers located on heterochromatin region, the cytological order of markers was concordant with those on the linkage maps. Distinct structural differences between chromosomes 3 and 4 were revealed by the high resolution pachytene chromosomes. The extreme difference of genetic length between LG-3 and LG-4 was mainly attributed to the difference of overall recombination frequency. The significant differentiation of heterochromatin contents in chromosomes 3 and 4 might have a direct correlation with recombination frequency. Meanwhile, the uneven distribution of recombination frequency along chromosome 4 was observed, and recombination frequency of the long arm was nearly 3.5 times higher than that of the short arm. The severe suppression of recombination was exhibited in centromeric and heterochromatin domains of chromosome 4. Whereas a close correlation between the gene density and recombination frequency was observed in chromosome 4, no significant correlation was observed between them along chromosome 3. The comparison between cytogenetic and sequence maps revealed a large gap on the pericentromeric heterochromatin region of sequence map of chromosome 4. These results showed that integrated molecular cytogenetic maps can provide important information for the study of genetic and genomics in cucumber.
doi:10.1371/journal.pone.0062676
PMCID: PMC3646037  PMID: 23671621
19.  The JmjC domain protein Epe1 prevents unregulated assembly and disassembly of heterochromatin 
The EMBO Journal  2007;26(22):4670-4682.
Heterochromatin normally has prescribed chromosomal positions and must not encroach on adjacent regions. We demonstrate that the fission yeast protein Epe1 stabilises silent chromatin, preventing the oscillation of heterochromatin domains. Epe1 loss leads to two contrasting phenotypes: alleviation of silencing within heterochromatin and expansion of silent chromatin into neighbouring euchromatin. Thus, we propose that Epe1 regulates heterochromatin assembly and disassembly, thereby affecting heterochromatin integrity, centromere function and chromosome segregation fidelity. Epe1 regulates the extent of heterochromatin domains at the level of chromatin, not via the RNAi pathway. Analysis of an ectopically silenced site suggests that heterochromatin oscillation occurs in the absence of heterochromatin boundaries. Epe1 requires predicted iron- and 2-oxyglutarate (2-OG)-binding residues for in vivo function, indicating that it is probably a 2-OG/Fe(II)-dependent dioxygenase. We suggest that, rather than being a histone demethylase, Epe1 may be a protein hydroxylase that affects the stability of a heterochromatin protein, or protein–protein interaction, to regulate the extent of heterochromatin domains. Thus, Epe1 ensures that heterochromatin is restricted to the domains to which it is targeted by RNAi.
doi:10.1038/sj.emboj.7601892
PMCID: PMC2048757  PMID: 17948055
centromere; Epe1; fission yeast; heterochromatin; JmjC domain
20.  The Analysis of Mutant Alleles of Different Strength Reveals Multiple Functions of Topoisomerase 2 in Regulation of Drosophila Chromosome Structure 
PLoS Genetics  2014;10(10):e1004739.
Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.
Author Summary
Type II topoisomerases (Topo II) are enzymes that disentangle DNA molecules during essential cellular processes such as DNA replication, chromosome condensation and mitotic cell division. Topo II is a major component of mitotic chromosomes and it is a well known target for cancer chemotherapy. Topo II inhibitors block the Topo II enzymatic activity leading to extensive DNA damage, which ultimately kills the cancer cell. Thus, investigating the role of Topo II in the assembly and structural maintenance of chromosomes is not only relevant to understand chromosome biology but might also have a translational impact on cancer therapy. Here we used Drosophila as model system to analyze the effect of Topo II depletion on chromosome stability. We show that the chromosomal phenotypes of mutant flies vary with the amount of residual Topo II, ranging from site-specific chromosome breaks, variations in chromosome number (aneuploidy and poliploidy) and dramatic defects in chromosome morphology. The chromosomal phenotypes observed in flies recapitulate all phenotypes seen in Topo II-depleted vertebrate chromosomes, reconciling the phenotypic discrepancies reported in previous studies. In addition, our finding that the Topo II dependent phenotypes vary with the residual amount of the enzyme provides useful information on the possible outcome of cancer therapy with Topo II inhibitors.
doi:10.1371/journal.pgen.1004739
PMCID: PMC4207652  PMID: 25340516
21.  Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions 
PLoS Genetics  2009;5(5):e1000478.
The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.
Author Summary
Chromosomes are non-randomly positioned inside cells, and this organization is relevant for genome regulation. Spatial clustering of heterochromatic loci provides a striking example of nuclear compartmentalization. In S. cerevisiae, the presence of heterochromatic sub-nuclear domains has been well established, but their mechanisms of formation are not fully understood. Here, we analyzed the DNA elements and protein complexes that are critical for formation of heterochromatic clusters. We focused on heterochromatic regions on chromosome III—the two telomeres, as well as the silent mating type loci HML and HMR, located on the left and right end of the chromosome, respectively. We employed live cell 3-D imaging and chromosome conformation capture (3C) and found that these loci specifically interact most prominently near silencer elements that flank the loci. Analysis of a panel of mutants showed that complexes involved in silencing are also involved in long-range interactions. Interestingly, we find that heterochromatic interactions are mechanistically distinct from silencing and independent of tethering to the nuclear periphery. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin, and point to a step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions between heterochromatic loci.
doi:10.1371/journal.pgen.1000478
PMCID: PMC2673037  PMID: 19424429
22.  Changed Genome Heterochromatinization Upon Prolonged Activation of the Raf/ERK Signaling Pathway 
PLoS ONE  2010;5(10):e13322.
The Raf/ERK (Extracellular Signal Regulated Kinase) signal transduction pathway controls numerous cellular processes, including growth, differentiation, cellular transformation and senescence. ERK activation is thought to involve complex spatial and temporal regulation, to achieve a high degree of specificity, though precisely how this is achieved remains to be confirmed. We report here that prolonged activation of a conditional form of c-Raf-1 (BXB-ER) leads to profound changes in the level and distribution of a heterochromatic histone mark. In mouse fibroblasts, the heterochromatic trimethylation of lysine 9 in histone H3 (H3K9Me3) is normally confined to pericentromeric regions. However, following ERK activation a genome-wide redistribution of H3K9Me3 correlates with loss of the histone modification from chromocentres and the appearance of numerous punctuate sites throughout the interphase nucleus. These epigenetic changes during interphase correlate with altered chromosome structure during mitosis, where robust H3K9Me3 signals appear within telomeric heterochromatin. This pattern of heterochromatinization is distinct from previously described oncogene induced senescence associated heterochromatin foci (SAHF), which are excluded from telomeres. The H3K9Me3 histone mark is known to bind the major heterochromatin protein HP1 and we show that the alterations in the distribution of this histone epistate correlate with redistribution of HP1β throughout the nucleus. Interestingly while ERK activation is fully reversible, the observed chromatin changes induced by epigenetic modifications are not reversible once established. We describe for the first time a link from prolonged ERK activation to stable changes in genome organization through redistribution of heterochromatic domains involving the telomeres. These epigenetic changes provide a possible mechanism through which prolonged activation of Raf/ERK can lead to growth arrest or the induction of differentiation, senescence and cancer.
doi:10.1371/journal.pone.0013322
PMCID: PMC2953519  PMID: 20967285
23.  Chromosomal G-dark Bands Determine the Spatial Organization of Centromeric Heterochromatin in the Nucleus 
Molecular Biology of the Cell  2001;12(11):3563-3572.
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.
PMCID: PMC60276  PMID: 11694589
24.  High-resolution mapping of heterochromatin redistribution in a Drosophila position-effect variegation model 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1756-8935-2-1
PMCID: PMC2644302  PMID: 19178722
25.  Heading off with the herd: How cancer cells might maneuver supernumerary centrosomes for directional migration 
Cancer metastasis reviews  2013;32(0):269-287.
The complicity of centrosomes in carcinogenesis is unmistakable. Mounting evidence clearly implicates a robust correlation between centrosome amplification (CA) and malignant transformation in diverse tissue types. Furthermore, CA has been suggested as a marker of cancer aggressiveness, in particular the invasive phenotype, in breast and prostate cancers. One means by which CA promotes malignancy is through induction of transient spindle multipolarity during mitosis, which predisposes the cell to karyotypic changes arising from low-grade chromosome mis-segregation. It is well recognized that during cell migration in interphase, centrosome-mediated nucleation of a radial microtubule array is crucial for establishing a polarized Golgi apparatus, without which directionality is precluded. The question of how cancer cells maneuver their supernumerary centrosomes to achieve directionality during cell migration is virtually uncharted territory. Given CA is a hallmark of cancers and has been correlated with cancer aggressiveness, malignant cells are presumably competent in managing their centrosome surfeit during directional migration, although the cellular logistics of this process remain unexplored. Thus, another key angle worth pondering is whether an overabundance of centrosomes confers some advantage on cancer cells in terms of their migratory and invasive capabilities. Recent studies have uncovered a remarkable strategy that cancer cells employ to deal with the problem of excess centrosomes and ensure bipolar mitoses, viz., centrosome clustering. This review aims to change the narrative by exploring how an increased centrosome complement may, via aneuploidy-independent modulation of the microtubule cytoskeleton, enhance directional migration and invasion of malignant cells. We postulate that CA imbues cancer cells with cytoskeletal advantages that enhance cell polarization, Golgi-dependent vesicular trafficking, stromal invasion and other aspects of metastatic progression. We also propose that centrosome declustering may represent a novel, cancer cell-specific anti-metastatic strategy, as cancer cells may rely on centrosome clustering during migration as they do in mitosis. Elucidation of these details offers an exciting avenue for future research, as does investigating how CA may promote metastasis through enhanced directional migration.
doi:10.1007/s10555-012-9413-5
PMCID: PMC3581755  PMID: 23114845

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