Telomeres are specialized structures at the ends of linear chromosomes that protect them from degradation and fusion. It’s replication is a complex process that involves both DNA polymerases and a specialized enzyme called telomerase which is a ribonucleoprotein complex that synthesizes telomeric DNA by using an internal RNA template. However, telomerase alone cannot fully replicate the telomeric DNA, and requires the cooperation of other factors, such as shelterin, CST, and DNA repair proteins. Telomere replication is tightly regulated by various mechanisms, such as cell cycle checkpoints, telomere length homeostasis, and telomere position effect and dysregulation of it can lead to genomic instability, cellular senescence, and cancer. Therefore, understanding the molecular details of telomere replication is crucial for elucidating the role of telomeres in aging and disease.

The science of telomeres and telomerase has made tremendous progress in recent decades. In this review, we consider it first in a historical context (the Carrel-Hayflick-Olovnikov-Blackburn chain of discoveries) and then review current knowledge of telomere structure and dynamics in norm and pathology. Central to the review are the consequences of telomere shortening, including telomere position effects, DNA damage signaling, and increased genetic instability. Cell senescence and the role of telomere length in its development are discussed separately. Therapeutic aspects and risks of telomerase and other telomere lengthening methods are also discussed.

Abstract Study question Whether the activation of DUX4, a key inducer in the process of zygotic genome activation (ZGA), is associated with telomere length. Summary answer Telomeres regulate the expression of DUX4/Dux through chromatin remodeling and are thereby involved in ZGA. What is known already In human early embryos, the expression of DUX4 is activated as a key inducer in the initial stage of ZGA, and it, in turn, activates hundreds of genes in the cleavage-stage embryo. Human DUX4 is localized to the subtelomeric region 4q35.2 with a D4Z4 repeat of approximately 10 to 100 units that encodes a homeodomain transcription factor. DUX4 expression is inversely proportional to the telomere length in myoblasts/myotubes derived from FSHD patients Study design, size, duration We characterize the dynamics of telomeres during preimplantation development, and assessed the relationship between the expression of DUX4/Dux and telomere length in preimplantation embryos and human embryonic stem cells. Participants/materials, setting, methods All sperm and immature oocytes were collected after obtaining written informed consent from the donor couples. Telomere length in gametes and early embryos by telomere-specific quantitative fluorescence in situ hybridization (Q-FISH). Main results and the role of chance Zygotic genome activation (ZGA) is initiated once the genome chromatin state is organized in the newly formed zygote. While telomeres are specialized chromatin structures at the ends of chromosomes and are reset during early embryogenesis, the details and significance of telomere changes in preimplantation embryos remain unclear. We demonstrated that the telomere length was shortened in the minor ZGA stage and significantly elongated in the major ZGA stage of human and mouse embryos. Expression of the ZGA pioneer factor DUX4/Dux was negatively correlated with the telomere length. ATAC-sequencing suggested that the chromatin accessibility peaks on the DUX4 promoter region (i.e., the subtelomere of chromosome 4q) were transiently augmented in human minor ZGA. Reduction of telomeric heterochromatin H3K9me3 in the telomeric region also synergistically activated DUX4 expression with p53 in hESCs. We propose herein that telomeres regulate the expression of DUX4/Dux through chromatin remodeling and are thereby involved in ZGA. Limitations, reasons for caution Since the mouse Dux gene is not located at the end of the chromosome, the classical telomere position effect (TPE) pathway may not involve in its activation in early embryos. 3D analysis as presented by Hi-C may be able to make a greater breakthrough in confirming the relationship. Wider implications of the findings We herein provided detailed data on changes in the telomere length during ZGA in human and mouse preimplantation embryos, explored the possibility that the TPE affects regulation of DUX4/Dux gene expression in embryos, and suggest that telomere chromatin remodeling is involved in the ZGA process. Trial registration number not applicable

ABSTRACT Telomeres are terminal structures that define the ends of linear chromosomes. They harbour specialized ribonucleoprotein complexes which play a major role in genome integrity by preventing unscheduled DNA damage repair events. Genes located adjacent to telomere repeat sequences are repressed by a phenomenon called telomere position effect (TPE) via epigenetic silencing. RNA surveillance pathways post-transcriptionally regulate any leaky transcripts arising from the telomeres. Recently, multiple non-coding RNA species originate from telomere ends, namely, TERRA (telomeric repeat‐containing RNA), ARRET, sub-telomeric XUTs and sub-telomeric CUTs have been identified. In this study, we report a role for the transcription termination complex (Rtt103-Rai1-Rat1) in regulating the abundance of the sub-telomeric transcripts in a transcription-dependent manner. We show that the Rtt103 mutants have elevated levels of TERRA and other sub-telomeric transcripts that are usually silenced. Our study suggests that Rtt103 potentially recruits the exonuclease, Rat1 in a RNA polymerase II dependent manner to degrade these transcripts and regulate their levels in the cell.

Telomeres are repetitive nucleotide sequences at the ends of each chromosome. It has been hypothesized that telomere attrition evolved as a tumor suppressor mechanism in large long-lived species. Long telomeres can silence genes millions of bases away through a looping mechanism called telomere position effect over long distances (TPE-OLD). The function of this silencing mechanism is unknown. We determined a set of 2322 genes with high positional conservation across replicatively aging species that includes known and candidate TPE-OLD genes that may mitigate potentially harmful effects of replicative aging. Notably, we identified PPP2R2C as a tumor suppressor gene, whose up-regulation by TPE-OLD in aged human fibroblasts leads to dephosphorylation of p70S6 kinase and mammalian target of rapamycin suppression. A mechanistic link between telomeres and a tumor suppressor mechanism supports the hypothesis that replicative aging fulfills a tumor suppressor function and motivates previously unknown antitumor and antiaging strategies.

Telomerase is a ribonucleoprotein enzyme complex that maintains telomeres at the ends of linear eukaryotic chromosomes. Telomerase minimally requires a telomerase reverse transcriptase (TERT) protein that uses a non-coding telomerase RNA as a template to extend 3ʹ ends of lagging strands. In addition to binding TERT and providing a template, telomerase RNA plays additional roles in binding accessory proteins and contributing to catalysis. Telomerase RNAs are rapidly evolving in length, sequence and structure, making phylogenetic comparisons challenging and limiting our understanding of telomerase structure-function relationships. Much of our current grasp of telomerase RNA function results from of loss-of-function mutations. In order to better understand telomerase RNA function, we devised a novel screening strategy to identify gain-of-function alleles in the budding yeast telomerase RNA. Briefly, our screen utilizes a counter-selectable marker, URA3, in a subtelomeric region that is sensitive to telomere position effect. When telomeres are long, the URA3 gene will be silenced to a greater extent, allowing better growth of yeast on media containing the chemical 5-fluoroorotic acid (5-FOA). To identify novel mutations, we used error-prone PCR to generate a library of random mutations in a miniaturized version of telomerase RNA, Mini-T(460). A pilot screen of our mutant library has isolated 5 new gain-of-function alleles that allow increased growth of yeast in the presence of 5-FOA. We are currently scaling up our screen to identify additional gain-of-function alleles. Simultaneously, we are investigating the mechanism by which these telomerase RNA alleles lead to longer telomeres. We hypothesize that these mutations could act through one or more mechanisms, including increasing RNA abundance, RNA stability, catalysis, repeat-addition processivity, and/or protein binding. Together, our work will significantly increase understanding of the structure and function of telomerase RNA.

Telomeres are repetitive non-coding nucleotide sequences (TTAGGGn) capping the ends of chromosomes. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1–10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Here, we describe an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. We examined two human genes, ISG15 and TERT, for which TPE-OLD has been described before. For both genes, we found less interaction with the telomere on the same chromosome in old cells compared to young cells; and experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.

Any theory suggesting an adaptive meaning for aging implicitly postulates the existence of specific mechanisms, genetically determined and modulated, causing progressive decline of an organism. According to the subtelomere-telomere theory, each telomere is covered by a hood formed in the first cell of an organism having a size preserved at each subsequent duplication. Telomere shortening, which is quantitatively different for each cell type according to the telomerase regulation, causes the hood to slide on the subtelomere repressing it by the telomeric position effect. At this point, the theory postulates existence of subtelomeric regulatory sequences, whose progressive transcriptional repression by the hood should cause cellular alterations that would be the likely determinant of aging manifestations. However, sequences with characteristics of these hypothetical sequences have already been described and documented. They are the [sub]TElomeric Repeat-containing RNA (TERRA) sequences. The repression of TERRA sequences causes progressively: (i) down- or up-regulation of many other regulatory sequences; (ii) increase in the probability of activation of cell senescence program (blockage of the ability to replicate and very significant alterations of the cellular functions). When cell senescence program has not been triggered and the repression is partial, there is a partial alteration of the cellular functions that is easily reversible by telomerase activation. Location of the extremely important sequences in chromosomal parts that are most vulnerable to repression by the telomeric hood is evolutionarily unjustifiable if aging is not considered adaptive: this location must be necessarily adaptive with the specific function of determining aging of the cell and consequently of the whole organism.

LINE 1 COPY NUMBER DECREASES AND TELOMERE LENGTH INCREASES WITH AGING IN SPERM CELLS

BackgroundDeletions removing 100s–1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the...

Genome distance between growth-regulating genes and telomeres is correlated with morpho-physiological traits in mammals

Heterochromatin in Saccharomyces cerevisiae is found at the telomeres and silent mating type loci. Many sub-telomeric loci are naturally silenced by this mechanism. In addition, when euchromatic genes are placed proximal to telomeric repeats they are subjected to heritable gene silencing that is referred to as telomere position effect. Establishment and maintenance of TPE is dependent on the assembly of silent information regulator proteins at these loci. Here we show that dosage of SUMO isopeptidase, Ulp1, is important for regulation of TPE. Moderate elevation of Ulp1 reduces silencing of both, the euchromatic gene placed proximal to telomeric repeats and the sub-telomeric genes that are silenced by TPE. We further demonstrate that this loss in silencing is due to reduced recruitment of one of the silent information regulators, Sir3p. We show that SUMO peptidase, Ulp1, regulates telomeric position effect by regulating the recruitment of Sir proteins.

The eukaryotic genome is divided into chromosomal domains of heterochromatin and euchromatin. Transcriptionally silent heterochromatin is found at subtelomeric regions, leading to the telomeric position effect (TPE) in yeast, fly, and human. Heterochromatin generally initiates and spreads from defined loci, and diverse mechanisms prevent the ectopic spread of heterochromatin into euchromatin. Here, we overexpressed the silencing factor Sir3 at varying levels in yeast and found that Sir3 spreads into extended silent domains (ESDs), eventually reaching saturation at subtelomeres. We observed the spread of Sir3 into subtelomeric domains associated with specific histone marks in wild-type cells, and stopping at zones of histone mark transitions including H3K79 trimethylation levels. Our study shows that the conserved H3K79 methyltransferase Dot1 is essential in restricting Sir3 spread beyond ESDs, thus ensuring viability upon overexpression of Sir3. Last, our analyses of published data demonstrate how ESDs unveil uncharacterized discrete domains isolating structural and functional subtelomeric features from the rest of the genome. Our work offers a new approach on how to separate subtelomeres from the core chromosome.

Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase. In the budding yeast Saccharomyces cerevisiae, TERRA accumulation is repressed by telomeric silencing and the Rat1 exonuclease. On the other hand, telomere shortening promotes expression of TERRA. So far, little is known about the biological processes that induce TERRA expression in yeast. Understanding the dynamics of TERRA expression and localization is essential to define its function in telomere biology. Here, we aim to study the dynamics of TERRA expression during yeast cell growth. Using live-cell imaging, RNA-FISH and quantitative RT-PCR, we show that TERRA expression is induced as yeast cells undergo diauxic shift, a lag phase during which yeast cells switch their metabolism from anaerobic fermentation to oxidative respiration. This induction is transient as TERRA levels decrease during post-diauxic shift. The increased expression of TERRA is not due to the shortening of telomeres or increased stability of this transcript. Surprisingly, this induction is coincident with a cytoplasmic accumulation of TERRA molecules. Our results suggest that TERRA transcripts may play extranuclear functions with important implications in telomere biology and add a novel layer of complexity in the interplay between telomere biology, metabolism and stress response.

ABSTRACTThe methylotrophic yeast Komagataella phaffii (Pichia pastoris) is homothallic and has been reported to switch mating type by an ancient inversion mechanism. Two mating-type (MAT) loci include homologs of the MATa and MATα transcription factor genes, with the expression from one locus downregulated by telomere position effects. However, not much is known about mating gene regulation, since the mixture of mating types complicates detailed investigations. In this study, we developed K. phaffii strains with stable mating types by deletion of the inverted-repeat region required for mating-type switching. These heterothallic strains retain their ability to mate with cells of the opposite mating type and were used to further elucidate mating gene regulation. Functional analysis of MAT mutant strains revealed the essential role of MATa2 and MATα1 in diploid cell formation. Disruption of MATa1 or MATα2 did not affect mating; however, in diploid cells, both genes are required for sporulation and the repression of shmoo formation. The heterothallic strains generated in this study allowed the first detailed characterization of mating gene regulation in K. phaffii. They will be a valuable tool for further studies investigating cell-type-specific behavior and will enable in-depth genetic analyses and strain hybridization in this industrially relevant yeast species.

Long-range telomere regulation of gene expression: Telomere looping and telomere position effect over long distances (TPE-OLD)

Telomeres at the ends of chromosomes safeguard genome integrity and stability in human nucleated cells. However, telomere repeats shed off during cell proliferation and other stress responses. Our recent studies show that telomere attrition induces not only epithelial stem cell senescence but also low-grade inflammation in the lungs. The senescence-associated low-grade inflammation (SALI) is characteristic of alveolar stem cell replicative senescence, increased proinflammatory and anti-inflammatory cytokines, infiltrated immune cells, and spillover effects. To date, the mechanisms underlying SALI remain unclear. Investigations demonstrate that senescent epithelial stem cells with telomere erosion are not the source of secreted cytokines, containing no significant increase in expression of the genes coding for increased cytokines, suggesting an alternative senescence-associated secretory phenotype (A-SASP). Given that telomere loss results in significant alterations in the genomes and accumulations of the cleaved telomeric DNA in the cells and milieu externe, we conclude that telomere position effects (TPEs) on gene expression and damage-associated molecular patterns (DAMPs) in antigen presentation are involved in A-SASP and SALI in response to telomere damage in mammals.

Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal.

DNA is organized into complex three-dimensional chromatin structures, but how this spatial organization regulates gene expression remains a central question. These DNA/chromatin looping structures can range in size from 10–20 kb (enhancers/repressors) to many megabases during intra- and inter-chromosomal interactions. Recently, the influence of telomere length on chromatin organization prior to senescence has revealed the existence of long-distance chromatin loops that dictate the expression of genes located up to 10 Mb from the telomeres (Telomere Position Effect–Over Long Distances [TPE-OLD]). Here, we demonstrate the existence of a telomere loop at the 4q35 locus involving the sorbin and SH3 domain-containing protein 2 gene, SORBS2, a skeletal muscle protein using a modification of the chromosome conformation capture method. The loop reveals a cis-acting mechanism modifying SORBS2 transcription. The expression of this gene is altered by TPE-OLD in myoblasts from patients affected with the age-associated genetic disease, facioscapulohumeral muscular dystrophy (FSHD1A, MIM 158900). SORBS2 is expressed in FSHD myoblasts with short telomeres, while not detectable in FSHD myoblasts with long telomeres or in healthy myoblasts regardless of telomere length. This indicates that TPE-OLD may modify the regulation of the 4q35 locus in a pathogenic context. Upon differentiation, both FSHD and healthy myotubes express SORBS2, suggesting that SORBS2 is normally up-regulated by maturation/differentiation of skeletal muscle and is misregulated by TPE-OLD-dependent variegation in FSHD myoblasts. These findings provide additional insights for the complexity and age-related symptoms of FSHD.

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.