Telomere Addition Research Articles

Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.

Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), which predisposes to tumorigenesis. To interrogate this risk, we developed a two- color flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of cells following a DSB. After this initial increase, cells with LOH decrease due to a competitive disadvantage with parental cells, but if isolated, they stably propagate. Segmental loss from terminal deletions with de novo telomere addition and nonreciprocal translocations is observed as well as whole chromosome loss, especially following a centromeric DSB. LOH spans megabases distal from the DSB, but also frequently tens of megabases centromere-proximal. Inhibition of microhomology-mediated end joining massively increases LOH, which is synergistically increased with concomitant inhibition of canonical nonhomologous end joining. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end joining inhibition.

End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the human and pig parasitic nematode Ascaris to characterize the DSBs. Using END-seq, we demonstrate that DSBs are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3'-overhangs before the addition of neotelomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends may be due to the sequestration of the eliminated DNA into micronuclei, preventing neotelomere formation at their ends. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for PDE. Overall, our data indicate that telomere healing of DSBs is specific to the break sites responsible for nematode PDE.

Neotelomeres and telomere-spanning chromosomal arm fusions in cancer genomes revealed by long-read sequencing

Alterations in the structure and location of telomeres are pivotal in cancer genome evolution. Here, we applied both long-read and short-read genome sequencing to assess telomere repeat-containing structures in cancers and cancer cell lines. Using long-read genome sequences that span telomeric repeats, we defined four types of telomere repeat variations in cancer cells: neotelomeres where telomere addition heals chromosome breaks, chromosomal arm fusions spanning telomere repeats, fusions of neotelomeres, and peri-centromeric fusions with adjoined telomere and centromere repeats. These results provide a framework for the systematic study of telomeric repeats in cancer genomes, which could serve as a model for understanding the somatic evolution of other repetitive genomic elements.

A persistent variant telomere sequence in a human pedigree

The telomere sequence, TTAGGG, is conserved across all vertebrates and plays an essential role in suppressing the DNA damage response by binding a set of proteins termed shelterin. Changes in the telomere sequence impair shelterin binding, initiate a DNA damage response, and are toxic to cells. Here we identify a family with a variant in the telomere template sequence of telomerase, the enzyme responsible for telomere elongation, that led to a non-canonical telomere sequence. The variant is inherited across at least one generation and one family member reports no significant medical concerns despite ~9% of their telomeres converting to the novel sequence. The variant template disrupts telomerase repeat addition processivity and decreased the binding of the telomere-binding protein POT1. Despite these disruptions, the sequence is readily incorporated into cellular chromosomes. Incorporation of a variant sequence prevents POT1-mediated inhibition of telomerase suggesting that incorporation of a variant sequence may influence telomere addition. These findings demonstrate that telomeres can tolerate substantial degeneracy while remaining functional and provide insights as to how incorporation of a non-canonical telomere sequence might alter telomere length dynamics.

Diverse modes of chromosome terminal deletion in spontaneous canavanine-resistant Schizosaccharomyces pombe mutants.

Canavanine resistance has been used to analyze mutation rates in the fission yeast Schizosaccharomyces pombe . However, the genetic basis of canavanine resistance in this organism remains incompletely understood. Here, we performed whole genome sequencing on five spontaneously arising canavanine-resistant S. pombe mutants, including the can2-1 mutant isolated in the 1970s. This analysis revealed that three mutants, including can2-1 , experienced terminal deletions of the left arm of chromosome II, leading to the loss of multiple amino acid transporter genes. Interestingly, these three mutants underwent chromosome terminal deletion through distinct mechanisms, including homology-driven translocation, homology-independent chromosome fusion, and de novo telomere addition. Our findings shed new light on the genetic basis of canavanine resistance and mechanisms underlying chromosome terminal deletions in fission yeast.

Programmed chromosome fragmentation in ciliated protozoa: multiple means to chromosome ends.

SUMMARYCiliated protozoa undergo large-scale developmental rearrangement of their somatic genomes when forming a new transcriptionally active macronucleus during conjugation. This process includes the fragmentation of chromosomes derived from the germline, coupled with the efficient healing of the broken ends by de novo telomere addition. Here, we review what is known of developmental chromosome fragmentation in ciliates that have been well-studied at the molecular level (Tetrahymena, Paramecium, Euplotes, Stylonychia, and Oxytricha). These organisms differ substantially in the fidelity and precision of their fragmentation systems, as well as in the presence or absence of well-defined sequence elements that direct excision, suggesting that chromosome fragmentation systems have evolved multiple times and/or have been significantly altered during ciliate evolution. We propose a two-stage model for the evolution of the current ciliate systems, with both stages involving repetitive or transposable elements in the genome. The ancestral form of chromosome fragmentation is proposed to have been derived from the ciliate small RNA/chromatin modification process that removes transposons and other repetitive elements from the macronuclear genome during development. The evolution of this ancestral system is suggested to have potentiated its replacement in some ciliate lineages by subsequent fragmentation systems derived from mobile genetic elements.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition (dnTA) on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker's yeast, Saccharomyces cerevisiae, that act as hotspots of dnTA [termed Sites of Repair-associated Telomere Addition (SiRTAs)], but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

Telomere Power: How to Live Longer and Healthier

Did you know that your daily choices, including how much you exercise, what you eat, and even how you think, can change the basic units that are present in your cells? In this article, I will tell you about telomeres, which are the protective tips of DNA molecules—the molecules where the organism’s genes are stored. I will also introduce you to an enzyme that we discovered, called telomerase, which is responsible for the addition and maintenance of telomeres. After diving into the molecular details of telomeres and telomerase, I will reveal to you some very interesting connections between telomeres and human health. I hope, by the end of this article, you will be amazed by the way in which psychological, environmental, and societal factors can influence the fundamentals of our biology.

Hotspot of de novo telomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions.

Evolution is driven by the accumulation of competing mutations that influence survival. A broad form of genetic variation is the amplification or deletion of DNA (≥50 bp) referred to as copy number variation (CNV). In humans, CNV may be inconsequential, contribute to minor phenotypic differences, or cause conditions such as birth defects, neurodevelopmental disorders, and cancers. To identify mechanisms that drive CNV, we monitored the experimental evolution of Saccharomyces cerevisiae populations grown under sulfate-limiting conditions. Cells with increased copy number of the gene SUL1, which encodes a primary sulfate transporter, exhibit a fitness advantage. Previously, we reported interstitial inverted triplications of SUL1 as the dominant rearrangement in a haploid population. Here, in a diploid population, we find instead that small linear fragments containing SUL1 form and are sustained over several generations. Many of the linear fragments are stabilized by de novo telomere addition within a telomere-like sequence near SUL1 (within the SNF5 gene). Using an assay that monitors telomerase action following an induced chromosome break, we show that this region acts as a hotspot of de novo telomere addition and that required sequences map to a region of <250 base pairs. Consistent with previous work showing that association of the telomere-binding protein Cdc13 with internal sequences stimulates telomerase recruitment, mutation of a four-nucleotide motif predicted to associate with Cdc13 abolishes de novo telomere addition. Our study suggests that internal telomere-like sequences that stimulate de novo telomere addition can contribute to adaptation by promoting genomic plasticity.

Abstract 2407: Cis- and trans-acting factors affecting the location and frequency of de novo telomere addition in yeast

Abstract 2407: Cis- and trans-acting factors affecting the location and frequency of de novo telomere addition in yeast

The nematode Oscheius tipulae as a genetic model for programmed DNA elimination

Programmed DNA elimination (PDE) is a notable exception to the paradigm of genome integrity. In metazoa, PDE often occurs coincident with germline to somatic cell differentiation. During PDE, portions of genomic DNA are lost, resulting in reduced somatic genomes. Prior studies have described the sequences lost, as well as chromosome behavior, during metazoan PDE. However, a system for studying the mechanisms and consequences of PDE in metazoa is lacking. Here, we present a functional and genetic model for PDE in the free-living Rhabditidae nematode Oscheius tipulae, a family that also includes Caenorhabditis elegans. O.tipulae was recently suggested to eliminate DNA. Using staged embryos and DNA FISH, we showed that O.tipulae PDE occurs during embryogenesis at the 8-16 cell stages. We identified a conserved motif, named Sequence For Elimination (SFE), for all 12 break sites on the six chromosomes at the junctions of retained and eliminated DNA. SFE mutants exhibited a "fail-to-eliminate" phenotype only at the modified sites. END-seq revealed that breaks can occur at multiple positions within the SFE, with extensive end resection followed by telomere addition to both retained and eliminated ends. We identified many functional SFEs at the chromosome ends through END-seq in the wild-type embryos, genome sequencing of SFE mutants, and comparative genomics of 23 wild isolates. We suggest that these alternative SFEs provide flexibility in the sequences eliminated and a fail-safe mechanism for PDE. These studies establish O.tipulae as a new, attractive model for studying the mechanisms and consequences of PDE in a metazoan.

Abstract 2169: New telomeres and chromosomal arm fusions in cancer genomes revealed by long-read genome sequencing

Abstract Chromosomal rearrangements are widespread features of cancer genomes, and often lead to the generation of both larger and smaller chromosomes that are readily observed by cytogenetics analysis. The generation of these chromosomal alterations likely involves alterations in the structure and location of telomeres at chromosome ends, though these events have been difficult to assess due to the long and highly repetitive nature of telomeres. Here, we developed an analytic method (TeloFuse) to identify new telomeres and chromosomal arm fusions which are characterized by telomeric repeats at intra-chromosomal locations. We applied TeloFuse to identify telomere-containing fusions as well as novel sites of telomere addition using short-read genome sequencing data from 326 cancer cell lines followed by long-read genome sequencing and cytogenetic analysis of selected cell lines. We observed new telomeres at sites of terminal deletion in long read data (telomere length = 2-9 kb), and in 76/326 (23%) of cell line genomes with short read data. Telomere sequence-containing fusions at intra-chromosomal sites were detected in 75/326 (23%) of cancer cell lines and confirmed as chromosomal arm fusion events by long-read genome sequencing. Long-read genome sequencing further suggests that new telomeres have similar telomere length as telomeres found on normal chromosomal arms (median = 5kb). Conversely, telomeric repeats at sites of chromosomal arm fusions were found to be critically short (~200-500bp long). Extending our analysis to 97 lung adenocarcinoma patient samples from The Cancer Genome Atlas, we further found new telomeres in 17/97 (18%) and chromosomal arm fusions in 28/97 (29%) patients with short-read data. Notably, new telomeres and chromosomal arm fusion events were also found to disrupt protein-coding genes, suggesting that these events represent a poorly appreciated mode for gene inactivation in cancer. Our results suggest that the formation of new telomeres and chromosomal arm fusions are prevalent events during cancer genome evolution and are likely critical for the stabilization of new chromosomes in human cancers. More broadly, our findings also highlight the utility of long-read genome sequencing in delineating chromosomal-scale alterations that have been widely observed by cytogenetics analysis. Citation Format: Kar-Tong Tan, Michael K. Slevin, Max Garrity-Janger, Heng Li, Matthew Meyerson. New telomeres and chromosomal arm fusions in cancer genomes revealed by long-read genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2169.

Functional interaction between compound heterozygous TERT mutations causes severe telomere biology disorder

AbstractTelomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.

Flexibility of telomerase in binding the RNA template and DNA telomeric repeat

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.

The Bur1 cyclin-dependent kinase regulates telomere length in Saccharomyces cerevisiae.

Telomere length regulation is essential for cell viability in eukaryotes. While many pathways that affect telomere length are known, we do not yet have a complete understanding of the mechanism of length regulation. To identify new pathways that might regulate telomere length, we carried out a genetic screen in yeast and identified the cyclin‐dependent kinase complex Bur1/2 as a regulator of telomere length. Mutations in either BUR1 cyclin‐dependent kinase or the associated BUR2 cyclin resulted in short telomeres. This regulation did not function through the known role of BUR1 in regulating histone modification as bur1∆ set2∆ and bur2∆ set2∆ double mutants rescued cell growth but did not rescue the telomere shortening effects. We found that both bur1∆ and bur2∆ set2∆ were also defective in de novo telomere addition, and deletion of SET2 did also not rescue this elongation defect. The Bur1/2 cyclin‐dependent kinase regulates transcription of many genes. We found that TLC1 RNA levels were reduced in bur2∆ set2∆ mutants; however, overexpression of TLC1 restored the transcript levels but did not restore de novo telomere elongation or telomere length. These data suggest that the Bur1/2 kinase plays a role in telomere elongation separate from its role in transcription of telomerase components. Dissecting the role of the Bur1/2 kinase pathway at telomeres will help complete our understanding of the complex network of telomere length regulation.

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage

BackgroundThe main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells.ResultsBy mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed “non-telomeric binding sites” (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability.ConclusionsOur results provide a novel model of telomerase regulation in the cell cycle using internal regions as “parking spots” of Est2 but marking them as hotspots for telomere addition.

Mechanism of MRX inhibition by Rif2 at telomeres

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5’ end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5’ resection.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

When the Ends Justify the Means: Regulation of Telomere Addition at Double-Strand Breaks in Yeast.

Telomeres, repetitive sequences located at the ends of most eukaryotic chromosomes, provide a mechanism to replenish terminal sequences lost during DNA replication, limit nucleolytic resection, and protect chromosome ends from engaging in double-strand break (DSB) repair. The ribonucleoprotein telomerase contains an RNA subunit that serves as the template for the synthesis of telomeric DNA. While telomere elongation is typically primed by a 3′ overhang at existing chromosome ends, telomerase can act upon internal non-telomeric sequences. Such de novo telomere addition can be programmed (for example, during chromosome fragmentation in ciliated protozoa) or can occur spontaneously in response to a chromosome break. Telomerase action at a DSB can interfere with conservative mechanisms of DNA repair and results in loss of distal sequences but may prevent additional nucleolytic resection and/or chromosome rearrangement through formation of a functional telomere (termed “chromosome healing”). Here, we review studies of spontaneous and induced DSBs in the yeast Saccharomyces cerevisiae that shed light on mechanisms that negatively regulate de novo telomere addition, in particular how the cell prevents telomerase action at DSBs while facilitating elongation of critically short telomeres. Much of our understanding comes from the use of perfect artificial telomeric tracts to “seed” de novo telomere addition. However, endogenous sequences that are enriched in thymine and guanine nucleotides on one strand (TG-rich) but do not perfectly match the telomere consensus sequence can also stimulate unusually high frequencies of telomere formation following a DSB. These observations suggest that some internal sites may fully or partially escape mechanisms that normally negatively regulate de novo telomere addition.

Telomere length dynamics in response to DNA damage in malaria parasites

SummaryMalaria remains a major cause of morbidity and mortality in the developing world. Recent work has implicated chromosome end stability and the repair of DNA breaks through telomere healing as potent drivers of variant antigen diversification, thus associating basic mechanisms for maintaining genome integrity with aspects of host-parasite interactions. Here we applied long-read sequencing technology to precisely examine the dynamics of telomere addition and chromosome end stabilization in response to double-strand breaks within subtelomeric regions. We observed that the process of telomere healing induces the initial synthesis of telomere repeats well in excess of the minimal number required for end stability. However, once stabilized, these newly created telomeres appear to function normally, eventually returning to a length nearing that of intact chromosome ends. These results parallel recent observations in humans, suggesting an evolutionarily conserved mechanism for chromosome end repair.