Telomere-associated Gene Research Articles

Telomere-related prognostic signature for survival assessments in lung adenocarcinoma.

Telomere-related genes (TRGs) are important in many different types of cancers. However, there is a lack of research on the relationship between their expression and prognosis in lung adenocarcinoma (LUAD) patients. This study is to investigate the prognostic value of TRGs in LUAD and to develop a TRG signature that can predict patient survival. A total of 2,086 TRGs were obtained from a database of genes involved in telomere maintenance (TelNet), while the clinical information and tumor RNA expression profiles of 513 LUAD patients were acquired from The Cancer Genome Atlas (TCGA) database. Statistical methodologies, such as least absolute shrinkage and selection operator (LASSO)-Cox, were employed to construct a prognostic model with predictive capabilities. We analyzed 1,339 telomere-associated differentially expressed genes and identified a ten-gene predictive signature for LUAD. This signature exhibited effective prognostic classification capabilities across multiple datasets, including GSE3141 (58 samples), GSE8894 (63 samples), GSE50081 (127 samples), and GSE72094 (398 samples). Furthermore, we screened tumor-sensitive drugs targeting this signature. High telomere levels were associated with reduced survival in lung cancer patients who underwent surgery. Compared to the traditional TNM (tumor node metastasis classification) grading method, our telomere-associated gene panel demonstrated superior prediction accuracy. Notably, patients in the high-risk group, defined by the telomere-associated signature, exhibited improved responses to immunotherapy, suggesting potential benefits for this subgroup of patients. This study presents a comprehensive molecular signature comprising TRGs, which holds potential for functional and therapeutic investigations. Additionally, it serves as an integrated tool to identify crucial molecules for immunotherapy in lung cancer.

Identification of telomere-associated gene signatures to predict prognosis and drug sensitivity in glioma

ObjectiveGliomas are a heterogeneous group of brain tumors with distinct biological and clinical properties, leading to significant mortality and morbidity. Emerging evidence shows telomere maintenance has implicated in glioma susceptibility and prognosis. In this study, we comprehensively analyzed gene signatures related to telomere maintenance in glioma and their predictive values for predicting the prognosis and drug sensitivity in glioma. MethodsWe initially identified telomere-related genes differentially expressed between low-grade glioma (LGG) and glioblastoma (GBM) and accordingly developed a risk model by univariate and multivariate Cox analysis to assess the expressions of telomere-related genes across the risk groups. Finally, to assess these genes in immune function the anti-tumor medications often used in the clinical treatment of glioma, we computed immune cell infiltration analysis and drug sensitivity analysis. ResultsThe consensus clustering analysis identified 20 telomere-related genes which split LGG patients into two distinct subtypes. The patient survival, the expressions of key telomere-related DEGs, and immune cell infiltration significantly differed between Cluster 1 and Cluster 2. The LASSO risk model [riskScore=(0.086)*HOXA7+(0.242)*WEE1+(0.247)*IGF2BP3+(0.052)*DUSP10] showed significant differences regarding the 1-, 3-, 5-year overall survival, immune cell infiltration, and drug sensitivity between high- and low-risk groups. The predictive nomogram constructed to quantify the survival probability of each sample at 1, 3, and 5 years was consistent with the actual patient survival. ConclusionOur comprehensive characterization of telomere-associated gene signatures in glioma reveals their possible roles in the development, tumor microenvironment, and prognosis. The study provides some suggestive relationships between four telomere-related genes (HOXA7, WEE1, IGF2BP3, and DUSP10) and glioma prognosis.

Telomere-Associated Gene Signatures Correlate with Prognosis, Tumor Microenvironment, and Chemosensitivity in Breast Cancer.

BACKGROUND Telomeres are strongly associated with cancer, as their shortening over time is associated with an increased risk of tumor growth and progression. However, the prognostic value of telomere-related genes (TRGs) in breast cancer has not been systematically elucidated. MATERIAL AND METHODS The transcriptome and clinical data of breast cancer were downloaded from TCGA and GEO databases, and prognostic TRGs were identified by differential expression analysis and univariate and multivariate Cox regression analyses. Gene set enrichment analysis (GSEA) of different risk groups was performed. Molecular subtypes of breast cancer were constructed by consensus clustering analysis, and the differences in immune infiltration and chemotherapy sensitivity between subtypes were analyzed. RESULTS Differential expression analysis revealed 86 significantly differentially expressed TRGs in breast cancer, of which 43 were significantly associated with breast cancer prognosis. A predictive risk signature consisting of 6 tumor-related genes (TRGs) was developed, which can accurately stratify patients with breast cancer into 2 distinct groups with significantly different prognoses. Significantly different risk scores were found among different racial groups, treatment groups, and pathological features groups. GSEA results showed that patients in the low-risk group had activated immune responses and repressed cilium-related biological processes. Consistent clustering analysis based on these 6 TRGs obtained 2 molecular models with significant prognosis differences, which revealed distinct immune infiltration and chemotherapy sensitivity. CONCLUSIONS This study conducted a systematic investigation of the expression pattern of TRGs in breast cancer and its prognostic and clustering implications, thereby offering a reference for utilizing it to predict prognosis and evaluate treatment response.

Architectural groups of a subtelomeric gene family evolve along distinct paths in Candida albicans.

Subtelomeres are dynamic genomic regions shaped by elevated rates of recombination, mutation, and gene birth/death. These processes contribute to formation of lineage-specific gene family expansions that commonly occupy subtelomeres across eukaryotes. Investigating the evolution of subtelomeric gene families is complicated by the presence of repetitive DNA and high sequence similarity among gene family members that prevents accurate assembly from whole genome sequences. Here, we investigated the evolution of the telomere-associated (TLO) gene family in Candida albicans using 189 complete coding sequences retrieved from 23 genetically diverse strains across the species. Tlo genes conformed to the 3 major architectural groups (α/β/γ) previously defined in the genome reference strain but significantly differed in the degree of within-group diversity. One group, Tloβ, was always found at the same chromosome arm with strong sequence similarity among all strains. In contrast, diverse Tloα sequences have proliferated among chromosome arms. Tloγ genes formed 7 primary clades that included each of the previously identified Tloγ genes from the genome reference strain with 3 Tloγ genes always found on the same chromosome arm among strains. Architectural groups displayed regions of high conservation that resolved newly identified functional motifs, providing insight into potential regulatory mechanisms that distinguish groups. Thus, by resolving intraspecies subtelomeric gene variation, it is possible to identify previously unknown gene family complexity that may underpin adaptive functional variation.

Telomere associated gene expression as well as TERT protein level and telomerase activity\xa0are altered in the ovarian follicles of aged mice

Telomeres cap the ends of eukaryotic chromosomes to maintain genomic stability and integrity during an organism’s lifespan. The length of telomeres inevitably shortens due to DNA replication, genotoxic agents, and biological aging. A limited number of cell types, e.g., stem cells, germline cells, and early embryos can elongate shortened telomeres via the enzymatic action of telomerase, which is composed of telomerase reverse transcriptase (TERT) and telomerase RNA component (Terc). Additionally, telomere-associated proteins including telomeric repeat binding factor 1 (TRF1) and 2 (TRF2), as well as protection of telomeres 1a (POT1a), bind to telomeres to maintain their structural integrity and length. During ovarian aging in mammals, telomeres progressively shorten, accompanied by fertility loss; however, the molecular mechanism underlying this attrition during follicle development remains unclear. In this study, the primary, secondary, preantral, and antral follicles were obtained either from 6-week-old adult (n = 19) or 52-week-old aged (n = 12) mice. We revealed that the Tert, Terc, Trf1, Trf2, and Pot1a gene expression (P < 0.001) and TERT protein (P < 0.01) levels significantly decreased in certain ovarian follicles of the aged group when compared to those of the adult group. Also, telomerase activity exhibited remarkable changes in the follicles of both groups. Consequently, altered telomere-associated gene expression and reduced TERT protein levels in the follicles of aged mice may be a determinant of telomere shortening during ovarian aging, and infertility appearing in the later decades of reproductive lifespan. Further investigations are required to determine the molecular mechanisms underlying these alterations in the follicles during ovarian aging.

Functional diversification accompanies gene family expansion of MED2 homologs in Candida albicans.

Gene duplication facilitates functional diversification and provides greater phenotypic flexibility to an organism. Expanded gene families arise through repeated gene duplication but the extent of functional divergence that accompanies each paralogous gene is generally unexplored because of the difficulty in isolating the effects of single family members. The telomere-associated (TLO) gene family is a remarkable example of gene family expansion, with 14 members in the more pathogenic Candida albicans relative to two TLO genes in the closely-related species C. dubliniensis. TLO genes encode interchangeable Med2 subunits of the major transcriptional regulatory complex Mediator. To identify biological functions associated with each C. albicans TLO, expression of individual family members was regulated using a Tet-ON system and the strains were assessed across a range of phenotypes involved in growth and virulence traits. All TLOs affected multiple phenotypes and a single phenotype was often affected by multiple TLOs, including simple phenotypes such as cell aggregation and complex phenotypes such as virulence in a Galleria mellonella model of infection. No phenotype was regulated by all TLOs, suggesting neofunctionalization or subfunctionalization of ancestral properties among different family members. Importantly, regulation of three phenotypes could be mapped to individual polymorphic sites among the TLO genes, including an indel correlated with two phenotypes, growth in sucrose and macrophage killing. Different selective pressures have operated on the TLO sequence, with the 5’ conserved Med2 domain experiencing purifying selection and the gene/clade-specific 3’ end undergoing extensive positive selection that may contribute to the impact of individual TLOs on phenotypic variability. Therefore, expansion of the TLO gene family has conferred unique regulatory properties to each paralog such that it influences a range of phenotypes. We posit that the genetic diversity associated with this expansion contributed to C. albicans success as a commensal and opportunistic pathogen.

Cell Type-Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry.

Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described. Therefore, a novel combination of quantitative fluorescence in situ hybridization, tissue elution, and immunofluorescence staining techniques was developed. Caveolin-1 (type 1 pneumocytes), pro-surfactant protein C (type 2 pneumocytes), club cell-10 (club cells), and alpha smooth muscle actin (smooth muscle cells) markers were used to identify cell types. To visualize all the different probes, restaining the tissue by heat-mediated slide elution is essential. Fluorescent signals of telomeres and DNA double-strand breaks were quantified using the Telometer plugin of ImageJ. As example, we analyzed lung tissue from a familial pulmonary fibrosis patient with a mutation in the telomere-associated gene poly(A)-specific ribonuclease ( PARN). The protocol displays a novel opportunity to directly quantitatively link DNA double-strand breaks to telomere length in specific FFPE cells.

Altered expression of telomere-associated genes in leukocytes among BRCA1 and BRCA2 carriers.

Telomere dysfunction resulting from telomere shortening and deregulation of shelterin components has been linked to the pathogenesis of age-related disorders, including cancer. Recent evidence suggests that BRCA1/2 (BRCA1 and BRCA2) tumor suppressor gene products play an important role in telomere maintenance. Although telomere shortening has been reported in BRCA1/2 carriers, the direct effects of BRCA1/2 haploinsufficiency on telomere maintenance and predisposition to cancer development are not completely understood. In this study, we assessed the telomere-associated and telomere-proximal gene expression profiles in peripheral blood leukocytes from patients with a BRCA1 or BRCA2 mutation, compared to samples from sporadic and familial breast cancer individuals. We found that 25 genes, including TINF2 gene (a negative regulator of telomere length), were significantly differentially expressed in BRCA1 carriers. Leukocyte telomere length analysis revealed that BRCA1/2 carriers had relatively shorter telomeres than healthy controls. Further, affected BRCA1/2 carriers were well differentiated from unaffected BRCA1/2 carriers by the expression of telomere-proximal genes. Our results link BRCA1/2 haploinsufficiency to changes in telomere length, telomere-associated as well as telomere-proximal gene expression. Thus, this work supports the effect of BRCA1/2 haploinsufficiency in the biology underlying telomere dysfunction in cancer development. Future studies evaluating these findings will require a large study population.

Association between genetic polymorphism of telomere-associated gene ACYP2 and the risk of HAPE among the Chinese Han population: A Case-control study.

High-altitude pulmonary edema (HAPE) is a hypoxia-induced, life-threatening, pulmonary edema, which is characterized by exaggerated pulmonary hypertension caused by stress failure. ACYP2 was found to associated with telomere length, the aim of this study was to identify whether ACYP2 polymorphisms increase or decrease HAPE risk in the Chinese Han individuals.In present study, we have genotyped 7 single-nucleotide polymorphisms (SNPs) in ACYP2 to determine the haplotypes in a case–control study with 265 HAPE patients and 303 healthy individuals. Genotypes were determined using the Sequenom MassARRAY method. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression with adjustment for gender and age. We found 3 SNPs yielded significant evidence for association with HAPE risk which had not been investigated before. Rs6713088 was found to have a 1.85- and 1.30-fold increased risk of HAPE in the recessive and additive model. The GT of rs843752 also conferred an increased risk of HAPE (GT/TT: OR = 1.51, 95% CI: 1.05–2.16, P = 0.026) and the genotype frequency distributions of rs843752 had significant difference between cases and controls. The CC genotype of rs17045754 had a protect effect on HAPE patients, and it was found to have a 0.29-fold reduced risk of HAPE in the recessive model.Although additional, larger population-based studies are needed to confirm these findings, our study shed light on the association between ACYP2 variant and HAPE risk in Han Chinese population for the first time.

Association of polymorphisms in the telomere-related gene ACYP2 with lung cancer risk in the Chinese Han population.

Single nucleotide polymorphisms (SNPs) in the telomere-associated gene ACYP2 are associated with increased lung cancer risk. We explored the correlation between ACYP2 SNPs and lung cancer susceptibility in the Chinese Han population. A total of 554 lung cancer patients and 603 healthy controls were included in this study. Thirteen SNPs in ACYP2 were selected. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using unconditional logistic regression analysis. Multivariate logistic regression analysis was used assess the correlation between SNPs and lung cancer. We found that rs1682111 was associated with increased lung cancer risk in the recessive model (crude, OR=1.50, 95%CI: 1.04-2.16, p=0.029; adjusted for age, OR=1.55, 95%CI: 1.04-2.30, p=0.029), as was rs11896604 in the codominant model (crude, OR=0.65, 95%CI: 0.33-1.28, p=0.045; adjusted for age, OR=0.74, 95%CI: 0.36-1.53, p=0.049) and over-dominant model (crude, OR=1.30, 95%CI: 1.02-1.66, p=0.032; adjusted for age, OR=1.37, 95%CI: 1.05-1.78, p=0.020). Finally, rs843720 was associated with increased lung cancer risk in the recessive model (crude, OR=1.43, 95%CI: 1.02-2.02, p=0.040; adjusted for age, OR=1.48, 95%CI: 1.02-2.15, p=0.040). Thus three SNPs in ACYP2 (rs1682111, rs11896604 and rs843720) associate with lung cancer in the Chinese Han population.

Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the activation domain of ‘free’ Tlo protein competes with DNA bound transcription factors for targets that regulate key aspects of C. albicans cell physiology.

Candida albicans.

Candida albicans.

POT1 deficiency alters telomere length and telomere-associated gene expression in human gastric cancer cells

Telomeres are the end structures of linear chromosomes in eukaryotic cells. The integrity of a telomere is essential for the overall stability of the chromosome. The human protection of telomeres 1 (hPOT1) protein, a single-stranded telomeric DNA binding protein, plays an important role in telomere protection and telomere length regulation. Here, we show that the loss of hPOT1 by RNA interference in BGC823 (poorly differentiated human gastric adenocarcinoma) cells leads to an increase in multinucleated giant cells, a decrease in cell proliferation and colony formation, induction of senescence and apoptosis, shortened telomere length, upregulation of the TRF1 gene and downregulation of the TRF2, tankyrase1 and hTERT genes. These results suggest that the loss of hPOT1 results in a decrease in the viability of BGC823 cells; hPOT1 regulates telomere length positively and has an influence on the expression of other telomere-associated genes in the cells.

Silent chromatin at the middle and ends: lessons from yeasts

Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species.

Increased expression of telomere-related proteins correlates with resistance to radiation in human laryngeal cancer cell lines

Telomere-associated proteins function as survival factors in telomere maintenance, which are major contributors to radiosensitivity in human cancers. The aim of this study was to investigate the association of telomere-associated gene expression and radiation resistance in human larynx squamous carcinoma. The changes of telomere-associated gene expressions and bionomical characteristics that occur in two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were explored in the present study. Based on previous research, elevated POT1 and TPP1 expressions were detected by reverse transcription-PCR and Western blotting in Hep-2R cell lines. Furthermore, Hep-2R cells showed increased recovery ratio accompanied by a reduction of cell arrested in G2/S phase, suggesting that the radioresistance of Hep-2R cells was due to a faster growth in which telomere length had recently been demonstrated to be a powerful prognostic marker. These results manifest that radioresistant Hep-2R cell lines showed certain changes in gene expression and bionomical profiles that are different from the profile changes of the more-sensitive Hep-2 cell lines, and that evaluation of telomere-associated genes may be a prognostic marker for response to radiotherapy in larynx squamous cell carcinoma (LSCC).

Molecular Regulation of Histone H3 Trimethylation by COMPASS and the Regulation of Gene Expression

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.

Telomeric Heterochromatin Propagation and Histone Acetylation Control Mutually Exclusive Expression of Antigenic Variation Genes in Malaria Parasites

Malaria parasites use antigenic variation to avoid immune clearance and increase the duration of infection in the human host. Variation at the surface of P. falciparum-infected erythrocytes is mediated by the differential control of a family of surface antigens encoded by var genes. Switching of var gene expression occurs in situ, mostly from telomere-associated loci, without detectable DNA alterations, suggesting that it is controlled by chromatin structure. We have identified chromatin modifications at telomeres that spread far into telomere-proximal regions, including var gene loci (>50 kb). One type of modification is mediated by a protein homologous to yeast Sir2 called PfSir2, which forms a chromosomal gradient of heterochromatin structure and histone hypoacetylation. Upon activation of a specific telomere-associated var gene, PfSir2 is removed from the promoter region and acetylation of histone occurs. Our data demonstrate that mutually exclusive transcription of var genes is linked to the dynamic remodeling of chromatin.

The Paf1 Complex Is Required for Histone H3 Methylation by COMPASS and Dot1p: Linking Transcriptional Elongation to Histone Methylation

Methylation of histone proteins is one of their many modifications that affect chromatin structure and regulate gene expression. Methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively, is required for silencing of expression of genes located near chromosome telomeres in yeast. We report that the Paf1 protein complex, which is associated with the elongating RNA polymerase II, is required for methylation of lysines 4 and 79 of histone H3 and for silencing of expression of a telomere-associated gene. We show that the Paf1 complex is required for recruitment of the COMPASS methyltransferase to RNA polymerase II and that the subunits of these complexes interact physically and genetically. Collectively, our results suggest that the Paf1 complex is required for histone H3 methylation, therefore linking transcriptional elongation to chromatin methylation.

Myosin-like proteins 1 and 2 are not required for silencing or telomere anchoring, but act in the Tel1 pathway of telomere length control

The positioning of chromosomal domains in interphase nuclei is thought to facilitate transcriptional repression in yeast. It has been reported that two large coiled-coil proteins of the nuclear envelope, myosin-like proteins 1 and 2, play direct roles in anchoring yeast telomeres to the nuclear periphery, thereby creating a subcompartment enriched for Sir proteins. We have created strains containing complete deletions of mlp1 and mlp2 genes, as well as the double null strain, and find no evidence for the disruption of telomere anchoring at the nuclear periphery in these cells. We also detect no disruption of telomere-associated gene silencing. We confirm, on the other hand, that mlp mutants are particularly sensitive to DNA-damaging agents, such as bleomycin. Moreover, we show that rather than having short telomeres as in yKu-deficient strains, the mlp1 mlp2 strains have extended telomeres, resembling phenotypes of mutations in rif1. Whereas the mlp1 mlp2 mutations act on a pathway of telomere length regulation different from that of yKu70, the effects of the tel1 deletion are epistatic to the mlp mutations, suggesting that the Mlp proteins restrict telomere length in wild-type cells by influencing the Rif–Tel1 pathway of telomerase regulation.

SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres

Heritable inactivation of genes occurs in specific chromosomal domains located at the silent mating type loci and at telomeres of S. cerevisiae. The SIR genes (for silent information regulators) are trans-acting factors required for this repression mechanism. We show here that the SIR3 and SIR4 gene products have a subnuclear localization similar to the telomere-associated RAP1 protein, which is found primarily in foci at the nuclear periphery of fixed yeast spheroplasts. In strains deficient for either SIR3 or SIR4, telomeres lose their perinuclear localization, as monitored by RAP1 immunofluorescence. The length of the telomeric repeat shortens in sir3 and sir4 mutant strains, and the mitotic stability of chromosome V is reduced. These data suggest that SIR3 and SIR4 are required for both the integrity and subnuclear localization of yeast telomeres, the loss of which correlates with loss of telomere-associated gene repression.