Subtelomeric Heterochromatin Research Articles

A mechanism for telomere-specific telomere length regulation.

Telomeric DNA, composed of short, direct repeats, is of crucial importance for chromosome stability. Due to intrinsic problems with replicating this DNA, the repeat tracts shorten at each cell division. Once repeat tracts become critically short, a telomeric stress signal induces cellular senescence and division arrest, which eventually may lead to devastating age-related degenerative diseases associated with dysfunctional telomers. Conversely, maintenance of telomere length by telomerase upregulation is a hallmark of cancer. Therefore, telomere length is a critical determinant of telomere function. How telomere length is established and molecular mechanisms for telomere-specific length regulation remained unknown. Here we show that subtelomeric chromatin is a determinant for how telomere equilibrium set-length is established in cis. The results demonstrate that telomerase recruitment mediated by the telomere-associated Sir4 protein is modulated on chromosome 3L in a telomere-specific way. Increased Sir4 abundance on subtelomeric heterochromatin of this specific telomere leads to telomere lengthening of only that telomere in cis, but not at other telomeres. Therefore, this work describes a mechanism for a how telomere-specific repeat tract length can be established. Further, our results will force the evaluation of telomere length away from a generalized view to a more telomere-specific consideration.

Организация некодирующих элементов в геномах птиц

The review focuses on non-coding elements in bird genomes. Like most living organisms, birdscarry a significant number of non-coding sequences in their genome. Technically, these sequences cannot beclassified as genes. They include DNA regions with structural functions and high level of conservatismas well as a considerable number of sequences, including repetitive fractions, whose functions are not yetknown. Apart from a general overview of tandem repeats in avian genomes, the paper provides a special focuson differences in copy number of individual repeats. Interestingly, this number directly correlates witha reduction in genome size in birds. Besides, the review provides a detailed description of how repetitivesequences are involved in structural and functional organisation of centromeric and telomeric regions witha special emphasis given to the organisation of pericentromeric and subtelomeric heterochromatin. It alsodescribes what makes these regions different from centromeric and telomeric sequences per se. The data onthe distribution of repeats in bird genomes and their specific organisation are compared with the level of theirconservatism and their role in maintaining structural and functional organisation of the genome.

The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states.

SUMMARYHeterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.

Ccq1-Raf2 interaction mediates CLRC recruitment to establish heterochromatin at telomeres.

Telomeres, highly ordered DNA-protein complexes at eukaryotic linear chromosome ends, are specialized heterochromatin loci conserved among eukaryotes. In Schizosaccharomyces pombe, the shelterin complex is important for subtelomeric heterochromatin establishment. Despite shelterin has been demonstrated to mediate the recruitment of the Snf2/histone deacetylase-containing repressor complex (SHREC) and the Clr4 methyltransferase complex (CLRC) to telomeres, the mechanism involved in telomeric heterochromatin assembly remains elusive due to the multiple functions of the shelterin complex. Here, we found that CLRC plays a dominant role in heterochromatin establishment at telomeres. In addition, we identified a series of amino acids in the shelterin subunit Ccq1 that are important for the specific interaction between Ccq1 and the CLRC subunit Raf2. Finally, we demonstrated that the Ccq1-Raf2 interaction is essential for the recruitment of CLRC to telomeres, that contributes to histone H3 lysine 9 methylation, nucleosome stability and the shelterin-chromatin association, promoting a positive feedback mechanism for the nucleation and spreading of heterochromatin at subtelomeres. Together, our findings provide a mechanistic understanding of subtelomeric heterochromatin assembly by shelterin-dependent CLRC recruitment to chromosomal ends.

Satellite DNA-Mediated Gene Expression Regulation: Physiological and Evolutionary Implication.

Satellite DNAs are tandemly repeated sequences organized in large clusters within (peri)centromeric and/or subtelomeric heterochromatin. However, in many species, satellite DNAs are not restricted to heterochromatin but are also dispersed as short arrays within euchromatin. Such genomic organization together with transcriptional activity seems to be a prerequisite for the gene-modulatory effect of satellite DNAs which was first demonstrated in the beetle Tribolium castaneum upon heat stress. Namely, enrichment of a silent histone mark at euchromatic repeats of a major beetle satellite DNA results in epigenetic silencing of neighboring genes. In addition, human satellite III transcripts induced by heat shock contribute to genome-wide gene silencing, providing protection against stress-induced cell death. Gene silencing mediated by satellite RNA was also shown to be fundamental for the early embryonic development of the mosquito Aedes aegypti. Apart from a physiological role during embryogenesis and heat stress response, activation of satellite DNAs in terms of transcription and proliferation can have an evolutionary impact. Spreading of satellite repeats throughout euchromatin promotes the variation of epigenetic landscapes and gene expression diversity, contributing to the evolution of gene regulatory networks and to genome adaptation in fluctuating environmental conditions.

How diverse is heterochromatin in the Caesalpinia group? Cytogenomic characterization of Erythrostemon hughesii Gagnon & G.P. Lewis (Leguminosae: Caesalpinioideae).

Cytogenomic characterization of Erythrostemon hughesii reveals a heterogeneity of repeats in its subtelomeric heterochromatin. Comparative analyses with other Caesalpinia group species reveal a significant reduction in the abundance of Ty3-gypsy/Chromovirus Tekay retrotransposons during its evolution. In numerically stable karyotypes, repetitive DNA variability is one of the main causes of genome and chromosome variation and evolution. Species from the Caesalpinia group (Leguminosae) are karyotypically characterized by 2n = 24, with small chromosomes and highly variable CMA+ heterochromatin banding patterns that correlate with environmental variables. Erythrostemon hughesii differs from other species of the group examined to date for having subtelomeric CMA+ bands; this contrasts with most species in the group which have proximal bands. Here we analyse the repeatome of E.hughesii using genome skimming and chromosomal mapping approaches to characterize the identity of the most abundant repetitive elements and their physical location. The repetitive fraction of E. hughesii comprises 28.73% of the genome. The most abundant elements were retrotransposons (RT) with long terminal repeats (LTR-RT; 9.76%) and satellite DNAs (7.83%). Within the LTR-RTs, the most abundant lineages were: Ty1/copia-Ale (1%), Ty3/gypsy CRM (0.88%) and Ty3/gypsy Athila (0.75%). Using fluorescent in situ hybridization four satellite DNAs and several LTR-RT elements were shown to be present in most subtelomeric CMA+ bands. These results highlight how the repeatome in E. hughesii, a species from Oaxaca state in Mexico, is clearly distinct from Northeast Brazilian species of the Caesalpinia group, mainly due to its high diversity of repeats in its subtelomeric heterochromatic bands and low amount of LTR-RT Ty3/gypsy-Tekay elements. Comparative sequence analysis of Tekay elements from different species is congruent with a clade-specific origin of this LTR-RT after the divergence of the Caesalpinia group. We hypothesize that repeat-rich heterochromatin may play a role in leading to faster genomic divergence between individuals, increasing speciation and diversification.

Intricate regulation on epigenetic stability of the subtelomeric heterochromatin and the centromeric chromatin in fission yeast.

In eukaryotes, the integrity of chromatin structure and organization is crucial to diverse key cellular processes from development to disease avoidance. To maintain the cell identity through mitotic cell generations, the genome (the genomic DNA sequence) as well as the epigenome (pertaining various forms of epigenetic information carriers, such as histone modifications, nucleosome positioning and the chromatin organization) is inherited with high fidelity. In comparison to the wealth of knowledge on genetic stability, we know much less on what may control the accuracy of epigenetic inheritance. In our recent work in the fission yeast Schizosaccharomyces pombe, by quantifying the epigenetic fidelity of CENP-A/Cnp1 or H3K9me2 nucleosome inheritance through cell divisions, we demonstrated that Ccp1, a homolog of histone chaperone Vps75 in budding yeast, participates in the modulation of centromeric nucleosomal epigenetic stability as well as proper heterochromatin organization. In this essay, we focus on discussing the uniquely high dynamicity of the subtelomeric heterochromatin regions and the complex mechanisms regulating epigenetic stability of centromeric chromatin.

Subtelomeric methylation distinguishes between subtypes of Immunodeficiency, Centromeric instability and Facial anomalies syndrome.

Human telomeres and adjacent subtelomeres are packaged as heterochromatin. Subtelomeric DNA undergoes methylation during development by DNA methyltransferase 3B (DNMT3B), including the CpG-rich promoters of the long non-coding RNA (TERRA) embedded in these regions. The factors that direct DNMT3B methylation to human subtelomeres and maintain this methylation throughout lifetime are yet unknown. The importance of subtelomeric methylation is manifested through the abnormal telomeric phenotype in Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome type 1 patients carrying mutations in DNMT3B. Patient cells demonstrate subtelomeric hypomethylation, accompanied by elevated TERRA transcription, accelerated telomere shortening and premature senescence of fibroblasts. ICF syndrome can arise due to mutations in at least three additional genes, ZBTB24 (ICF2), CDCA7 (ICF3) and HELLS (ICF4). While pericentromeric repeat hypomethylation is evident in all ICF syndrome subtypes, the status of subtelomeric DNA methylation had not been described for patients of subtypes 2-4. Here we explored the telomeric phenotype in cells derived from ICF2-4 patients with the aim to determine whether ZBTB24, CDCA7 and HELLS also play a role in establishing and/or maintaining human subtelomeric methylation. We found normal subtelomeric methylation in ICF2-4 and accordingly low TERRA levels and unperturbed telomere length. Moreover, depleting the ICF2-4-related proteins in normal fibroblasts did not influence subtelomeric methylation. Thus, these gene products are not involved in establishing or maintaining subtelomeric methylation. Our findings indicate that human subtelomeric heterochromatin has specialized methylation regulation and highlight the telomeric phenotype as a characteristic that distinguishes ICF1 from ICF2-4.

Changes in genome organization of parasite-specific gene families during the Plasmodium transmission stages

The development of malaria parasites throughout their various life cycle stages is coordinated by changes in gene expression. We previously showed that the three-dimensional organization of the Plasmodium falciparum genome is strongly associated with gene expression during its replication cycle inside red blood cells. Here, we analyze genome organization in the P. falciparum and P. vivax transmission stages. Major changes occur in the localization and interactions of genes involved in pathogenesis and immune evasion, host cell invasion, sexual differentiation, and master regulation of gene expression. Furthermore, we observe reorganization of subtelomeric heterochromatin around genes involved in host cell remodeling. Depletion of heterochromatin protein 1 (PfHP1) resulted in loss of interactions between virulence genes, confirming that PfHP1 is essential for maintenance of the repressive center. Our results suggest that the three-dimensional genome structure of human malaria parasites is strongly connected with transcriptional activity of specific gene families throughout the life cycle.

Epigenetic features of human telomeres.

Although subtelomeric regions in humans are heterochromatic, the epigenetic nature of human telomeres remains controversial. This controversy might have been influenced by the confounding effect of subtelomeric regions and interstitial telomeric sequences (ITSs) on telomeric chromatin structure analyses. In addition, different human cell lines might carry diverse epigenetic marks at telomeres. We have developed a reliable procedure to study the chromatin structure of human telomeres independently of subtelomeres and ITSs. This procedure is based on the statistical analysis of multiple ChIP-seq experiments. We have found that human telomeres are not enriched in the heterochromatic H3K9me3 mark in most of the common laboratory cell lines, including embryonic stem cells. Instead, they are labeled with H4K20me1 and H3K27ac, which might be established by p300. These results together with previously published data argue that subtelomeric heterochromatin might control human telomere functions. Interestingly, U2OS cells that exhibit alternative lengthening of telomeres have heterochromatic levels of H3K9me3 in their telomeres.

Subtelomeres constitute a safeguard for gene expression and chromosome homeostasis.

The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.

Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance.

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.

Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show thatMot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S.cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.

Combination of short-read, long-read, and optical mapping assemblies reveals large-scale tandem repeat arrays with population genetic implications.

Accurate and contiguous genome assembly is key to a comprehensive understanding of the processes shaping genomic diversity and evolution. Yet, it is frequently constrained by constitutive heterochromatin, usually characterized by highly repetitive DNA. As a key feature of genome architecture associated with centromeric and subtelomeric regions, it locally influences meiotic recombination. In this study, we assess the impact of large tandem repeat arrays on the recombination rate landscape in an avian speciation model, the Eurasian crow. We assembled two high-quality genome references using single-molecule real-time sequencing (long-read assembly [LR]) and single-molecule optical maps (optical map assembly [OM]). A three-way comparison including the published short-read assembly (SR) constructed for the same individual allowed assessing assembly properties and pinpointing misassemblies. By combining information from all three assemblies, we characterized 36 previously unidentified large repetitive regions in the proximity of sequence assembly breakpoints, the majority of which contained complex arrays of a 14-kb satellite repeat or its 1.2-kb subunit. Using whole-genome population resequencing data, we estimated the population-scaled recombination rate (ρ) and found it to be significantly reduced in these regions. These findings are consistent with an effect of low recombination in regions adjacent to centromeric or subtelomeric heterochromatin and add to our understanding of the processes generating widespread heterogeneity in genetic diversity and differentiation along the genome. By combining three different technologies, our results highlight the importance of adding a layer of information on genome structure that is inaccessible to each approach independently.

The expansion of heterochromatin blocks in rye reflects the co-amplification of tandem repeats and adjacent transposable elements.

BackgroundA prominent and distinctive feature of the rye (Secale cereale) chromosomes is the presence of massive blocks of subtelomeric heterochromatin, the size of which is correlated with the copy number of tandem arrays. The rapidity with which these regions have formed over the period of speciation remains unexplained.ResultsUsing a BAC library created from the short arm telosome of rye chromosome 1R we uncovered numerous arrays of the pSc200 and pSc250 tandem repeat families which are concentrated in subtelomeric heterochromatin and identified the adjacent DNA sequences. The arrays show significant heterogeneity in monomer organization. 454 reads were used to gain a representation of the expansion of these tandem repeats across the whole rye genome. The presence of multiple, relatively short monomer arrays, coupled with the mainly star-like topology of the monomer phylogenetic trees, was taken as indicative of a rapid expansion of the pSc200 and pSc250 arrays. The evolution of subtelomeric heterochromatin appears to have included a significant contribution of illegitimate recombination. The composition of transposable elements (TEs) within the regions flanking the pSc200 and pSc250 arrays differed markedly from that in the genome a whole. Solo-LTRs were strongly enriched, suggestive of a history of active ectopic exchange. Several DNA motifs were over-represented within the LTR sequences.ConclusionThe large blocks of subtelomeric heterochromatin have arisen from the combined activity of TEs and the expansion of the tandem repeats. The expansion was likely based on a highly complex network of recombination mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2667-5) contains supplementary material, which is available to authorized users.

The proper connection between shelterin components is required for telomeric heterochromatin assembly.

Telomeric regions contain prominent sites of heterochromatin, which is associated with unique histone modification profiles such as the methylation of histone H3 at Lys9 (H3K9me). In fission yeast, the conserved telomeric shelterin complex recruits the histone H3K9 methyltransferase complex CLRC to establish subtelomeric heterochromatin. Although many shelterin mutations affect subtelomeric heterochromatin assembly, the mechanism remains elusive due to the diverse functions of shelterin. Through affinity purification, we found that shelterin directly associates with CLRC through the Ccq1 subunit. Surprisingly, mutations that disrupt interactions between shelterin subunits compromise subtelomeric heterochromatin without affecting CLRC interaction with shelterin component Pot1, located at chromosome ends. We further discovered that telomeric repeats are refractory to heterochromatin spreading and that artificial restoration of shelterin connections or increased heterochromatin spreading rescued heterochromatin defects in these shelterin mutants. Thus, subtelomeric heterochromatin assembly requires both the recruitment of CLRC by shelterin to chromosome ends and the proper connection of shelterin components, which allows CLRC to skip telomeric repeats to internal regions.

Mapping the 'breaker' element of the gametocidal locus proximal to a block of sub-telomeric heterochromatin on the long arm of chromosome 4S(sh) of Aegilops sharonensis.

Key message The ‘breaker’ element ( GcB ) of the gametocidal locus derived from Aegilops sharonensis has been mapped to a region proximal to a block of sub-telomeric heterochromatin on chromosome 4S sh L. The production of alien chromosome addition lines allows the transfer of useful genetic variation into elite wheat varieties from related wild species. However, some wild relatives of wheat, particularly those within the Sitopsis section of the genus Aegilops, possess chromosomes that are transmitted preferentially to the offspring when addition lines are generated. Species within the Sitopsis group possess the S genome, and among these species, Aegilops sharonensis (2n = 14, SshSsh) carries the Ssh genome which is closely related to the D genome of hexaploid wheat. Some S genome chromosomes carry gametocidal loci, which induce severe chromosome breakage in gametes lacking the gametocidal chromosome, and hence, result in gamete abortion. The preferential transmission of gametocidal loci could be exploited in wheat breeding, because linking gametocidal loci with important agronomic traits in elite wheat varieties would ensure retention of these traits through successive generations. In this study, we have mapped the breaker element of the gametocidal locus derived from Ae. sharonensis to the region immediately proximal to a block of sub-telomeric heterochromatin on the long arm of chromosome 4Ssh.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-015-2489-x) contains supplementary material, which is available to authorized users.

Dominant Lethal Pathologies in Male Mice Engineered to Contain an X-Linked DUX4 Transgene

Facioscapulohumeral muscular dystrophy (FSHD) isan enigmatic disease associated with epigenetic alterations in the subtelomeric heterochromatin of the D4Z4 macrosatellite repeat. Each repeat unit encodes DUX4, a gene that is normally silent in most tissues. Besides muscular loss, most patients suffer retinal vascular telangiectasias. To generate an animal model, we introduced a doxycycline-inducible transgene encoding DUX4 and 3' genomic DNA into a euchromatic region of the mouse X chromosome. Without induction, DUX4 RNA was expressed at low levels in many tissues and animals displayed a variety of unexpected dominant leaky phenotypes, including male-specific lethality. Remarkably, rare live-born males expressed DUX4 RNA in the retina and presented a retinal vascular telangiectasia. By using doxycycline to induce DUX4 expression in satellite cells, we observed impaired myogenesis invitro and invivo. This mouse model, which shows pathologies due to FSHD-related D4Z4 sequences, is likely to be useful for testing anti-DUX4 therapies in FSHD.

ATM mediates adaptive epigenetic responses to mitochondrial stress (961.1)

Reactive oxygen species (ROS) are important signaling molecules that regulate numerous cellular processes. In contrast to their historic association with disease and accelerated aging, evidence in humans and model organisms suggests that ROS signaling can promote health and extend lifespan, yet ROS signaling pathways remain relatively unknown. We recently demonstrated that a mitochondrial ROS (mtROS) signal extends lifespan of the budding yeast Saccharomyces cerevisiae by activating conserved DNA damage response kinases that inactivate a jumonji‐family histone demethylase specifically at subtelomeric regions and repress gene expression. Here, we present evidence that this pathway is conserved in mammals. The DNA damage response kinase ATM, which dimerizes following ROS‐induced disulfide bond formation, also senses mtROS. Formation of the ATM dimer correlates with increased levels of H3K9me3, a histone modification characteristic of heterochromatin formation. Increased H3K9me3 following mtROS signaling is ATM‐dependent, and is associated with repressed telomeric and subtelomeric gene expression. Experiments are in progress to examine the role of jumonji‐family histone demethylases in the mammalian response to mtROS signaling. These results indicate that mtROS signaling via DNA damage response kinases and mtROS‐dependent subtelomeric heterochromatin formation are conserved from yeast to human and expand on the important roles of ATM in redox homeostasis, telomere regulation, and the DNA damage response.Grant Funding Source: NIH F31AG043242‐01

Heterochromatin Blocks Constituting the Entire Short Arms of Acrocentric Chromosomes of Azara's Owl Monkey: Formation Processes Inferred From Chromosomal Locations

Centromeres and telomeres of higher eukaryotes generally contain repetitive sequences, which often form pericentric or subtelomeric heterochromatin blocks. C-banding analysis of chromosomes of Azara's owl monkey, a primate species, showed that the short arms of acrocentric chromosomes consist mostly or solely of constitutive heterochromatin. The purpose of the present study was to determine which category, pericentric, or subtelomeric is most appropriate for this heterochromatin, and to infer its formation processes. We cloned and sequenced its DNA component, finding it to be a tandem repeat sequence comprising 187-bp repeat units, which we named OwlRep. Subsequent hybridization analyses revealed that OwlRep resides in the pericentric regions of a small number of metacentric chromosomes, in addition to the short arms of acrocentric chromosomes. Further, in the pericentric regions of the acrocentric chromosomes, OwlRep was observed on the short-arm side only. This distribution pattern of OwlRep among chromosomes can be simply and sufficiently explained by assuming (i) OwlRep was transferred from chromosome to chromosome by the interaction of pericentric heterochromatin, and (ii) it was amplified there as subtelomeric heterochromatin. OwlRep carries several direct and inverted repeats within its repeat units. This complex structure may lead to a higher frequency of chromosome scission and may thus be a factor in the unique distribution pattern among chromosomes. Neither OwlRep nor similar sequences were found in the genomes of the other New World monkey species we examined, suggesting that OwlRep underwent rapid amplification after the divergence of the owl monkey lineage from lineages of the other species.