Native Telomeres Research Articles

Telomeric transposons are pervasive in linear bacterial genomes.

Eukaryotes have linear DNA and their telomeres are hotspots for transposons, which in some cases took over telomere maintenance. While many bacteria also have linear chromosomes and plasmids, no transposons were known to target bacterial telomeres. Here we show several families of independently evolved telomeric transposons in cyanobacteria and Streptomyces . While these elements have one specific transposon end sequence with the second boundary being the telomere, we can show they move using two transposon ends. Telomeres are transiently bridged by the telomere maintenance systems, providing a duplex substrate for mobilization of the element and the associated telomere. We identify multiple instances where telomeric transposons have replaced native telomeres, making the host cell dependent on the new telomere system for genome maintenance. This work indicates how telomeric transposons can promote gene transfer both between and within genomes, significantly influencing the evolutionary dynamics of linear genomes.

Replication Control of Human Telomere G-Quadruplex DNA by G-Quadruplex Ligands Dependent on Solution Environment.

The human telomere region is known to contain guanine-rich repeats and form a guanine-quadruplex (G4) structure. As telomeres play a role in the regulation of cancer progression, ligands that specifically bind and stabilize G4 have potential therapeutic applications. However, as the human telomere sequence can form G4 with various topologies due to direct interaction by ligands and indirect interaction by the solution environment, it is of great interest to study the topology-dependent control of replication by ligands. In the present study, a DNA replication assay of a template with a human telomere G4 sequence in the presence of various ligands was performed. Cyclic naphthalene diimides (cNDI1 and cNDI2) efficiently increased the replication stall of the template DNA at G4 with an anti-parallel topology. This inhibition was stability-dependent and topology-selective, as the replication of templates with hybrid or parallel G4 structures was not affected by the cNDI and cNDI2. Moreover, the G4 ligand fisetin repressed replication with selectivity for anti-parallel and hybrid G4 structures without stabilization. Finally, the method used, referred to as quantitative study of topology-dependent replication (QSTR), was adopted to evaluate the correlation between the replication kinetics and the stability of G4. Compared to previous results obtained using a modified human telomere sequence, the relationship between the stability of G4 and the effect on the topology-dependent replication varied. Our results suggest that native human telomere G4 is more flexible than the modified sequence for interacting with ligands. These findings indicate that the modification of the human telomeric sequence forces G4 to rigidly form a specific structure of G4, which can restrict the change in topology-dependent replication by some ligands.

Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata

Adherence, an important virulence factor, is mediated by the EPA (Epithelial Adhesin) genes in the opportunistic pathogen Candida glabrata Expression of adhesin-encoding genes requires tight regulation to respond to harsh environmental conditions within the host. The majority of EPA genes are localized in subtelomeric regions regulated by subtelomeric silencing, which depends mainly on Rap1 and the Sir proteins. In vitro adhesion to epithelial cells is primarily mediated by Epa1. EPA1 forms a cluster with EPA2 and EPA3 in the right telomere of chromosome E (E-R). This telomere contains a cis-acting regulatory element, the protosilencer Sil2126 between EPA3 and the telomere. Interestingly, Sil2126 is only active in the context of its native telomere. Replacement of the intergenic regions between EPA genes in E-R revealed that cis-acting elements between EPA2 and EPA3 are required for Sil2126 activity when placed 32 kb away from the telomere (Sil@-32kb). Sil2126 contains several putative binding sites for Rap1 and Abf1, and its activity depends on these proteins. Indeed, Sil2126 binds Rap1 and Abf1 at its native position and also when inserted at -32 kb, a silencing-free environment in the parental strain. In addition, we found that Sil@-32kb and Sil2126 at its native position can physically interact with the intergenic regions between EPA1-EPA2 and EPA2-EPA3 respectively, by chromosome conformation capture assays. We speculate that Rap1 and Abf1 bound to Sil2126 can recruit the Silent Information Regulator complex, and together mediate silencing in this region, probably through the formation of a chromatin loop.

Rif1 phosphorylation site analysis in telomere length regulation and the response to damaged telomeres

Telomeres, the ends of eukaryotic chromosomes, consist of repetitive DNA sequences and their bound proteins that protect the end from the DNA damage response. Short telomeres with fewer repeats are preferentially elongated by telomerase. Tel1, the yeast homolog of human ATM kinase, is preferentially recruited to short telomeres and Tel1 kinase activity is required for telomere elongation. Rif1, a telomere-binding protein, negatively regulates telomere length by forming a complex with two other telomere binding proteins, Rap1 and Rif2, to block telomerase recruitment. Rif1 has 14 SQ/TQ consensus phosphorylation sites for ATM kinases, including 6 in a SQ/TQ Cluster Domain (SCD) similar to other DNA damage response proteins. These 14 sites were analyzed as N-terminal, SCD and C-terminal domains. Mutating some sites to non-phosphorylatable residues increased telomere length in cells lacking Tel1 while a different set of phosphomimetic mutants increased telomere length in cells lacking Rif2, suggesting that Rif1 phosphorylation has both positive and negative effects on length regulation. While these mutations did not alter the sensitivity to DNA damaging agents, inducing telomere-specific damage by growing cells lacking YKU70 at high temperature revealed a role for the SCD. Mass spectrometry of Rif1 from wild type cells or those induced for telomere-specific DNA damage revealed increased phosphorylation in cells with telomere damage at an ATM consensus site in the SCD, S1351, and non-ATM sites S181 and S1637. A phosphomimetic rif1-S1351E mutation caused an increase in telomere length at synthetic telomeres but not natural telomeres. These results indicate that the Rif1 SCD can modulate Rif1 function. As all Rif1 orthologs have one or more SCD domains, these results for yeast Rif1 have implications for the regulation of Rif1 function in humans and other organisms.

Telomere repeats induce domains of H3K27 methylation in Neurospora.

Development in higher organisms requires selective gene silencing, directed in part by di-/trimethylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain (~225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation.

Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu-Sir4 interaction.

Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y’-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y’-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu–Sir4 interaction, but Sir2 and Sir3 independent.

The Chromatin and Transcriptional Landscape of Native Saccharomyces cerevisiae Telomeres and Subtelomeric Domains.

Saccharomyces cerevisiae telomeres have been a paradigm for studying telomere position effects on gene expression. Telomere position effect was first described in yeast by its effect on the expression of reporter genes inserted adjacent to truncated telomeres. The reporter genes showed variable silencing that depended on the Sir2/3/4 complex. Later studies examining subtelomeric reporter genes inserted at natural telomeres hinted that telomere position effects were less pervasive than previously thought. Additionally, more recent data using the sensitive technology of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq) revealed a discrete and noncontinuous pattern of coenrichment for all three Sir proteins at a few telomeres, calling the generality of these conclusions into question. Here we combined the ChIP-Seq of the Sir proteins with RNA sequencing (RNA-Seq) of messenger RNAs (mRNAs) in wild-type and in SIR2, SIR3, and SIR4 deletion mutants to characterize the chromatin and transcriptional landscape of all native S. cerevisiae telomeres at the highest achievable resolution. Most S. cerevisiae chromosomes had subtelomeric genes that were expressed, with only ∼6% of subtelomeric genes silenced in a SIR-dependent manner. In addition, we uncovered 29 genes with previously unknown cell-type-specific patterns of expression. These detailed data provided a comprehensive assessment of the chromatin and transcriptional landscape of the subtelomeric domains of a eukaryotic genome.

Rad59-facilitated acquisition of Y' elements by short telomeres delays the onset of senescence.

Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1–3-like tracts present between subtelomeric X and Y′ elements, which is followed by BIR-mediated non-reciprocal translocation of Y′ element and terminal TG1–3 repeats from the donor end onto the shortened telomere. We found that choice of the Y′ donor was not random, since both engineered telomere VII-L and native VI-R acquired Y′ elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y′ translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1–3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y′ translocation events taking place during presenescence are genetically separable from Rad51-dependent Y′ amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y′ translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.

Correction: A Human Artificial Chromosome Recapitulates the Metabolism of Native Telomeres in Mammalian Cells

Correction: A Human Artificial Chromosome Recapitulates the Metabolism of Native Telomeres in Mammalian Cells

A Human Artificial Chromosome Recapitulates the Metabolism of Native Telomeres in Mammalian Cells

Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.

Length-dependent processing of telomeres in the absence of telomerase

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5′-3′ resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5′-3′ resection at the shortest telomeres.

The N-terminus and Tudor domains of Sgf29 are important for its heterochromatin boundary formation function

Eukaryotic chromosomes are organized into heterochromatin and euchromatin domains. Heterochromatin domains are transcriptionally repressed and prevented from spreading into neighbouring genes by chromatin boundaries. Previously, we identified 55 boundary-related genes in Saccharomyces cerevisiae. In this study, we describe the characterization of one of these boundary genes, named SGF29, which was previously reported as a component of the SAGA, SLIK, ADA and HAT-A2 complex. A domain analysis of Sgf29 identified two minimal regions that can function as individual boundaries. The N-terminal minimal region comprising amino acids 1-12, which has not been defined as a functional domain, showed stronger boundary formation ability than the C-terminal minimal region comprising amino acids 110-255, which contains Tudor domains. Together with Ada2, Ada3 and Sgf29, which are all components of SAGA, Gcn5 acetylates multiple lysine residues on nucleosomal histone H3, which is associated with an open chromatin structure. However, the results presented in this study suggest that the boundary formation ability of the Sgf29 minimal regions is independent of Gcn5. An in vivo analysis also revealed that Sgf29 and Gcn5 perform distinct functions at native telomere boundary regions on the chromosome.

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3'-GT-overhangs that extend beyond the complementary 5'-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G2 phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5'-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5'-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.

Coordinated Interactions of Multiple POT1-TPP1 Proteins with Telomere DNA

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5'-TTAGGG-3' in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.

Deregulated telomere transcription causes replication-dependent telomere shortening and promotes cellular senescence

Telomeres are transcribed into non-coding TElomeric Repeat containing RNAs (TERRA). We have employed a transcriptionally inducible telomere to investigate how telomere transcription affects telomere function in Saccharomyces cerevisiae. We report that telomere shortening resulting from high levels of telomere transcription stems from a DNA replication-dependent loss of telomere tracts, which can occur independent of both telomerase inhibition and homologous recombination. We show that in order for telomere loss to occur, transcription must pass through the telomere tract itself producing a TERRA molecule. We demonstrate that increased telomere transcription of a single telomere leads to a premature cellular senescence in the absence of a telomere maintenance mechanism (telomerase and homology directed repair). Similar rapid senescence and telomere shortening are also seen in sir2Δ cells with compromised telomere maintenance, where TERRA levels are increased at natural telomeres. These data suggest that telomere transcription must be tightly controlled to prevent telomere loss and early onset senescence.

Mutually Exclusive Binding of Telomerase RNA and DNA by Ku Alters Telomerase Recruitment Model

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both invitro and invivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:

Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP

Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.

Shelterin-Like Proteins and Yku Inhibit Nucleolytic Processing of Saccharomyces cerevisiae Telomeres

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. Here we investigate the role of key telomeric proteins in protecting budding yeast telomeres from degradation. We show that the Saccharomyces cerevisiae shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres, where Yku plays the major role in preventing initiation, whereas Rif1, Rif2, and Rap1 act primarily by limiting extensive resection. In fact, exonucleolytic degradation of a de novo telomere is more efficient in yku70Δ than in rif2Δ G1 cells, but generation of ssDNA in Yku-lacking cells is limited to DNA regions close to the telomere tip. This limited processing is due to the inhibitory action of Rap1, Rif1, and Rif2, as their inactivation allows extensive telomere resection not only in wild-type but also in yku70Δ G1 cells. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases.

Repressive and non-repressive chromatin at native telomeres in Saccharomyces cerevisiae

BackgroundIn Saccharomyces cerevisiae genes that are located close to a telomere can become transcriptionally repressed by an epigenetic process known as telomere position effect. There is large variation in the level of the telomere position effect among telomeres, with many native ends exhibiting little repression.ResultsChromatin analysis, using microccocal nuclease and indirect end labelling, reveals distinct patterns for ends with different silencing states. Differences were observed in the promoter accessibility of a subtelomeric reporter gene and a characteristic array of phased nucleosomes was observed on the centromere proximal side of core X at a repressive end. The silent information regulator proteins 2 - 4, the yKu heterodimer and the subtelomeric core X element are all required for the maintenance of the chromatin structure of repressive ends. However, gene deletions of particular histone modification proteins can eliminate the silencing without the disruption of this chromatin structure.ConclusionOur data identifies chromatin features that correlate with the silencing state and indicate that an array of phased nucleosomes is not sufficient for full repression.