Advance Publication by J-STAGE Genes & Genetic Systems

Received for publication: March 16, 2017 Accepted for publication: July 4, 2017 Published online: October 6, 2017

Telomere biology in aging and cancer: early history and perspectives Makoto T Hayashi* Department of Gene Mechanisms, Graduate School of Biostudies/The Hakubi Center for Advanced Research, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan *Corresponding author. Makoto T Hayashi Tel: 010-81-75-753-4196 Fax: 010-81-75-753-4197 E-mail: [email protected] Running head: History of telomere biology Key words: Telomere, Aging, Cancer, History

Abstract The ends of eukaryotic linear chromosomes are protected from undesired enzymatic activities by a nucleoprotein complex called the telomere. Expanding evidence indicates that telomeres have central functions in human aging and tumorigenesis. While it is undoubtedly important to follow current advances in telomere biology, it is also fruitful to be well informed in seminal historical studies for a comprehensive understanding of telomere biology, and for the anticipation of future directions. With this in mind, I here summarize the early history of telomere biology and current advances in the field, mostly focusing on mammalian studies relevant to aging and cancer.

Introduction All eukaryotes possess linear chromosomes, each of which must have two intrinsic ends. Natural end of chromosome is structurally indistinguishable from an internal broken end, which causes immediate activation of DNA damage checkpoint pathway. Once it’s activated, cell cycle is halted until the damage is repaired mainly through non-homologous end joining (NHEJ) or homologous recombination (HR) pathway. If the damage is excess and cannot be repaired in a certain time range, the cell chooses to commit suicide (e.g. apoptosis). Therefore, natural ends must be protected from activating these pathways and such protective function is performed by a special nucleo-protein complex called telomere. Telomere is composed of repetitive DNA, which has 3’ protruding end called 3’-overhang, and proteins that specifically recognize and assemble on the repetitive double and single strand sequence. In case of mammalian systems, the protein complex is called shelterin, loss of which renders chromosome ends vulnerable to enzymatic activities, resulting in activation of DNA damage checkpoint pathway. Loss of telomeric repeats also leads to failure of shelterin binding and end protection. Because DNA polymerase synthesizes DNA strictly from 5’ to 3’ orientation, the end of lagging strand cannot be fully replicated in each S phase of the cell cycle. On the other hand, the end of leading strand is processed to regenerate 3’-overhang following replication. Thus, both leading and lagging ends of telomere gradually shorten each cell cycle unless they are replenished by a special reverse transcriptase called telomerase. Telomerase is highly expressed in unicellular eukaryotes as well as reproductive cells in multicellular organisms, while its activity is hardly detectable in normal human somatic cells, which show gradual shortening of telomeres in vitro and in vivo. Therefore, telomeres in human soma eventually reach to a critical length after finite cell division and activate DNA damage checkpoint, which causes irreversible cell cycle arrest (i.e. replicative senescence) and underlies cellular aging. In the absence of functional checkpoint, cells keep dividing with ongoing telomere shortening, entering next stage called crisis. In crisis stage, chromosomes become unstable because of frequent end-to-end fusions, which are associated with massive cell

death through mitotic catastrophe. Eventually, rare survivors that acquire telomere maintenance pathway may emerge, resulting in tumor generation. It is also well documented that mutations in a variety of genes involved in telomere maintenance cause diseases related to human aging. Thus, telomere biology is of great importance to understanding of human aging and tumorigenesis. Recent expansion of molecular biological technology has greatly advanced the field, yet all of the current advances are based on seminal accomplishments by pioneers in the field. In this review, I will summarize an early history (~2000’s) of telomere biology with a focus on mammalian system to see milestone works in a new light, some of which are visualized in a chronological manner in Figures 1 and 2. Besides the early history, recent advances in the field and perspectives will also be briefly discussed. A brief history of telomere biology Beginning of the end story Everything has the beginning. The whole telomere biology had been started by two Nobel prize winners, Hermann Muller and Barbara McClintock more than seven decades ago. Muller had studied chromosome instability, such as translocation, induced by ionizing radiation in fly, which led him to find that natural chromosome ends never fuse to broken chromosome ends while broken ends often do so (Muller, 1938). He suggested the idea that natural chromosome ends are somehow protected against repair process, which connect two broken ends, and named such protective structure “telomere”. A similar phenomenon was independently found by McClintock through her cytological studies using ionized

maize

chromosomes

(McClintock,

1931).

She

proposed

a

Breakage-Fusion-Bridge (BFB) cycle hypothesis to explain chromosome instability: broken ends of two sister chromatids fuse together, which generate DNA bridge during anaphase in mitosis, eventually leading to breakage of the bridge. Such broken ends will fuse again in the next cell cycle, starting a multiple fusion and breakage cycle (McClintock, 1941). She could probably have seen how relevant the BFB cycle hypothesis is in cancer biology with her own eyes in her later years.

Their seminal studies had not been revisited until 1970’s, when James Watson and Alexey Olovnikov had proposed an “end replication problem” (Olovnikov, 1971, 1973; Watson, 1972). They had independently pointed out that unidirectionality of DNA polymerase should leave a gap at the very end of chromosome replicated by lagging strand synthesis. This means telomeres get shorter and shorter upon their replication. Such telomere shortening had been proposed as a molecular basis of replicative senescence (or Hayflick limit), an irreversible arrest of cellular growth following serial passages of human culture cells, which was first observed by Leonard Hayflick (Hayflick and Moorhead, 1961). Confirming this proposal molecularly, however, had awaited the next big bang in telomere biology, which had been accomplished by the 2009 Nobel Prize winners, Jack Szostak, Elizabeth Blackburn and Carol Greider. Thanks to the development of Sanger sequencing technology (Sanger and Coulson, 1975), Blackburn could successfully read telomeric sequences of Tetrahymena (Blackburn and Gall, 1978). This finding led Szostak to device artificial minichromosome capped with Tetrahymena telomeric sequences at the end, which was successfully maintained in yeast (Szostak and Blackburn, 1982): the first molecular evidence that telomeric DNA sequence possesses protective function. The end replication problem and the structural and dynamic properties of telomeres implied the presence of enzymatic activity that counteract telomere shortening. Such enzyme, a terminal transferase that utilizes RNA as a template, was indeed identified by Greider and Blackburn from Tetrahymena and named telomerase (Greider and Blackburn, 1985, 1987). Szostak had then developed a system to identify mutants of yeast that are defective for telomere maintenance and found that null mutants of a gene named EST1 (Ever Shorter Telomeres) show gradual telomere shortening and a senescence phenotype (Lundblad and Szostak, 1989). These series of the elegant studies pioneered a new era of telomere biology (Blackburn et al., 2006) (Fig. 1). Telomeric DNA meets protein After telomeric DNA sequence of Tetrahymena had been published, several studies inferred the existence of telomere binding proteins other than histone

(Blackburn and Chiou, 1981; Gottschling and Cech, 1984), and such proteins had soon been identified in the ciliated protozoan, Oxytricha nova (Gottschling and Zakian, 1986). Although the function of the telomeric proteins were unclear at that moment, it was proposed that “they act as stability elements or chromosome caps, thereby preventing fusion of telomeres” (Gottschling and Zakian, 1986). The first evidence that telomeric protein protects chromosome ends came from two independent studies using budding yeast, in which David Shore and Virginia Zakian groups analyzed the function of yeast telomere binding protein Rap1 (Conrad et al., 1990; Lustig et al., 1990). By this time, the concept of “checkpoints” that safeguard the order of cell cycle events from intrinsic and extrinsic insults was proposed by Leland Hartwell and Ted Weinert (Hartwell and Weinert, 1989). Among several cell cycle checkpoints, DNA damage checkpoint is highly relevant to telomere end protection, since chromosome ends resemble DNA ends caused by DNA double strand break (DSB), which causes delay in DNA synthesis and had been linked to increase of the “genome guardian”, p53 (Kastan et al., 1991). In the same year, Calvin Harley described that “when a certain length is reached on one or more telomeres in a dividing cell, a “checkpoint”, analogous to those regulating cell cycle events is signaled which evokes the Hayflick Limit: cells stop dividing” (Harley, 1991). Therefore, it was very tempting to speculate that telomeric proteins possess protective function to prevent checkpoint activation at the chromosome ends (de Lange, 1995). Determination of human telomeric DNA sequence (TTAGGG)n (Moyzis et al., 1988), which is conserved among vertebrate species (Meyne et al., 1989), allowed Titia de Lange and co-workers to perform electrophoretic mobility shift assays to identify a telomere binding activity (Zhong et al., 1992) and then clone telomeric repeat binding factor, which was named TRF (later renamed TRF1) for TTAGGG repeat binding factor, from HeLa cell extract (Chong et al., 1995). The protein was shown to be specifically localized to telomeric DNA in vivo, and overexpression of full-length and truncated dominant-negative TRF1 resulted in shortening and elongation of telomeric DNA, respectively, providing the first evidence that mammalian telomeric protein is also involved in telomere homeostasis (van Steensel and de Lange, 1997). In the meanwhile, Eric Gilson

group raised antibodies against DNA-binding motif of yeast telomere binding protein, Tbf1, to search similar proteins in human extract and identified two telomere binding proteins (Bilaud et al., 1996), one of which turned out to be TRF1 and the other was named TRF2 (Bilaud et al., 1997). TRF2 was independently identified on the basis of sequence similarity to the TRF1 by Titia de Lange group at the same time (Broccoli et al., 1997), and soon shown to be central in telomere protection: overexpression of truncated dominant-negative TRF2 caused chromosome end-to-end fusion in cultured human cells (van Steensel et al., 1998), and checkpoint kinase ATM (Ataxia Telangiectasia Mutated)-dependent p53 activation, resulting in apoptosis or senescence in cell-type dependent manner (Karlseder et al., 1999). These seminal studies paved the way to elucidate the molecular function of telomere binding proteins in variety of species. The next component of mammalian telomeric protein complex had been identified in 1999 by a yeast two-hybrid screen and named a TRF1-interacting nuclear protein, TIN2 (Kim et al., 1999). Searches for TRF2-interacting proteins by a yeast two-hybrid screen and nanoelectrospray tandem mass spectrometry independently identified Rap1, the mammalian ortholog of yeast Rap1, in the following year (Li et al., 2000; Zhu et al., 2000). Although TRF1 and TRF2 were identified as double-stranded DNA binding factors, the end of mammalian telomeres had been shown to possess protruding single-stranded TTAGGG 3’-overhang (or G-overhang) (Makarov et al., 1997), which had been originally found in hypotricha ciliates (Klobutcher et al., 1981). The telomeric protein in ciliate had been identified in 1986 as described above, which prompted biochemists to search a similar single-stranded telomeric DNA binding protein in higher eukaryotes. However, identification of such protein had been required to await availability of database mining. In 2001, Peter Baumann and Thomas R. Cech successfully identified fission yeast single-stranded telomere binding protein, Pot1, which has limited similarity to the portion of telomere protein TEBP from Oxytricha and other ciliates (Baumann and Cech, 2001). Following BLAST search with fission yeast Pot1 identified human POT1, which specifically binds to single-stranded TTAGGG repeat (Baumann and Cech, 2001). Identification of several telomeric proteins further accelerated the competition,

resulting in reports of the same protein from three independent studies in 2004. Titia

de

Lange

and

TRF2/TIN2-associated

co-workers proteins

performed

and

named

mass

spectrometry

novel

binding

of

partner

POT1-interacting protein 1, PIP1, based on its ability to interact with POT1 (Ye et al., 2004); Zhou Songyang group took a similar approach and identified PTOP, POT1- and TIN2-organizing protein (Liu et al., 2004b); while Susan Smith and colleagues utilized a yeast two-hybrid with TIN2 as a bait and reported TINT1, TIN2 interacting protein 1 (Houghtaling et al., 2004). A single name, TPP1, telomere protection protein 1, had been given later. Biochemical analysis of the identified six proteins (i.e. TRF1, TRF2, TIN2, RAP1, POT1 and TPP1) indicated those components make a complex on telomeric chromatin, which had been collectively named Telosome (Liu et al., 2004a) or Shelterin (de Lange, 2005), although the latter is more common in the current field. During the period of telomere protein searches, telomeric end structure had been visualized by electron microscopy, which demonstrated telomeres can form large duplex loop (called t-loop) in vitro in TRF2-dependent manner and t-loop can be isolated from cultured human cells (Griffith et al., 1999). The t-loop model is currently the most widely accepted model for end protection, proposing an integration of 3’-overhang into double-stranded repeats with the aid of shelterin function, especially TRF2. Telomeres connected to aging and cancer At the same period when replicative senescence was reported in early 60’s, another phenomenon that is highly relative to tumorigenesis was described: transformation of human culture cells had been characterized as the development of cytologically abnormal morphology following simian virus 40 (SV40) infection (Koprowski et al., 1962; Rabson and Kirschstein, 1962; Shein and Enders, 1962). Such transformed cells possess extended growth capacity compared to untransformed counterpart, which is followed by massive degeneration of the transformed cultures. This stage is termed “crisis”, leading to almost complete loss of cellular viability; while it is followed by “recovery” of the cultures, during which viable minor subpopulation rarely outgrow in the culture (Girardi et al., 1965). By the end of 70’s, p53 had been identified as a protein

exclusively expressed in transformed mouse cell lines (DeLeo et al., 1979) and co-immunoprecipitated with SV40 large-T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979). Ten years later, Bert Vogelstein and Arnold Levine groups had independently reported p53’s role as a tumor suppressor gene (Baker et al., 1989; Finlay et al., 1989). In the same period, the first tumor suppressor gene, Rb, which causes recessive mutations that predispose cells to retinoblastoma, had been identified (Friend et al., 1986), and shown to interact with the large-T antigen (DeCaprio et al., 1988). After the extensive researches on replicative senescence and crisis, those two phenomena were linked to the telomere biology as shortly described below (Fig. 2). Soon after the identification of telomerase in Tetrahymena (Greider and Blackburn, 1985), human telomerase activity in HeLa cell crude extracts were reported (Morin, 1989). As predicted by the end replication problem, telomeric DNA in human cell had been shown to shorten gradually during serial passage until replicative senescence in primary culture (Harley et al., 1990), as well as in vivo aging in blood samples (Hastie et al., 1990). Such gradual telomere shortening had also been reported in transformed human cells until crisis, after which post-recovery outgrown cell lines were shown to gain telomerase activity (Counter et al., 1992). Telomere shortening had been well explained by the fact that none of normal somatic human tissues, other than ovaries and testes, are positive for telomerase activity (Kim et al., 1994). In stark contrast, more than 90% of cultured immortal cells representing variety of human tissues and biopsies representing many tumor types were positive for telomerase activity (Kim et al., 1994). Following studies identified telomerase activity in human stem and progenitor cells, such as hematopoietic progenitor cells and T lymphocyte (Chiu et al., 1996; Weng et al., 1996), which is, however, not enough to maintain telomere length fully in hematopoietic stem cells (Vaziri et al., 1994). Although the underlying mechanism had been still unknown, those and other studies let Calvin Harley remodel the Olovnikov’s “Marginotomy theory” that predicted critical deletion of chromosome termini eventually causes cell death (Harley, 1991; Olovnikov, 1973). The same concept had been proposed by Woodring Wright and Jerry Shay as the two-stage mechanism hypothesis, in which gradual telomere shortening somehow activates checkpoint pathway (p53 and

Rb as key factors) to induce replicative senescence; while in the absence of functional p53 and Rb, cells keep dividing with progressive telomere shortening and enter crisis stage (Wright and Shay, 1992). Recovery from crisis by spontaneous activation of telomerase consequently gives rise to tumorigenic cells. Therefore, telomere shortening was proposed as a molecular basis of anti-tumorigenic cell cycle arrest and massive cell death in senescence and crisis, respectively. Identification of a gene encoding human telomerase catalytic subunit (hTERT a.k.a. hEST2) (Meyerson et al., 1997; Nakamura et al., 1997) enabled exogenous expression of hTERT in human normal fibroblast and epithelial cells, which resulted in extensive elongation of telomeres and immortalization of the normal cells (Bodnar et al., 1998; Nakayama et al., 1998). Ectopic expression of hTERT in SV40 large-T- and oncogenic H-ras-transformed human fibroblast and epithelial cells resulted in bypass of crisis and tumorigenic conversion of these cells (Hahn et al., 1999a). Conversely, inhibition of telomerase activity by a dominant negative DN-hTERT suppressed growth of human tumor-derived cell lines (Hahn et al., 1999b), demonstrating telomerase as a potential target of anti-cancer therapy. These experimental evidences established that telomere shortening underlies replicative senescence and crisis in human cultured somatic cells due to insufficient telomerase expression, and that maintenance of telomere length is essential for unlimited cellular growth. While telomerase activity had been strongly associated with tumor, there was a considerable exception. A number of immortalized in vitro human cell lines are capable of elongating telomeres without any detectable telomerase activity (Kim et al., 1994; Murnane et al., 1994; Bryan et al., 1995). These cells were characterized by very long and heterogeneous telomeres maintained by telomerase-independent

mechanisms,

referred

to

as

ALT

(alternative

lengthening of telomeres) (Bryan and Reddel, 1997), which had been also found in a subset of tumor-derived cell lines and tumors (Bryan et al., 1997). Roger Reddel and co-workers had elegantly shown that exogenous DNA sequences integrated into telomere of ALT cells are copied from the original telomere to another telomere, indicating HR-based mechanisms of ALT (Dunham et al., 2000). Although targeting telomerase by specific inhibitors had been a promising candidate for anti-cancer drug, the finding of ALT raised a significant implication

for therapeutic usages of telomerase inhibitors. The fact that ataxia telangiectasia patient, which is characterized by premature aging, predisposition to cancer, immunodeficiency, hypersensitivity to ionizing radiation, and neurological disorders, shows accelerated telomere shortening and increased telomere fusion phenotype suggested causal link between telomere maintenance and aging-related disorders (Metcalfe et al., 1996; Xia et al., 1996). In 1999, a direct evidence that causally links failure of telomere maintenance and diseases of aging human had come out. A patient of the X-linked form of human inherited disease, dyskeratosis congenita, suffers from premature aging disorders caused by defects in highly regenerative tissues, which is caused by mutations in the DKC1 gene that encodes the dyskerin protein. Kathleen Collins and colleagues had revealed that the dyskerin, which had been previously proposed to be involved in ribosomal function, is required for telomerase RNA generation and consequently telomerase activity and telomere maintenance (Mitchell et al., 1999). This finding was the first step to explore the causal relationship between telomere dysfunction and human degenerative disorders. Meanwhile, these findings and the fact that telomere gradually shorten in human stem cells (Vaziri et al., 1994; Lansdorp, 1998), together with mouse genetics studies described below, led to an idea that degeneration of self-renewal capacity of stem cells is partly due to a decline of telomere length or change in telomere structure, consequently underlying individual aging (Van Zant and Liang, 2003; Bell and Van Zant, 2004; Sharpless and DePinho, 2004). Thus, by early 2000’s, both diseases associated with aging and a physiological decline accompanied by aging had been connected to telomere biology. What mouse genetics taught us The telomere theory of aging and cancer was further strengthened by the development of mouse lacking telomerase RNA component (mTR), which was identified in 1995 by Carol Greider group (Blasco et al., 1995), together with human telomerase RNA by Bryant Villeponteau and co-workers (Feng et al., 1995). In contrast to human tissues, most mouse tissues possess active telomerase (Prowse and Greider, 1995), and telomeres of laboratory established

mouse strains are kept very long (up to several hundred kb) (Kipling and Cooke, 1990). First mTR-/- mice had been reported by Carol Greider and co-workers in 1997, which unexpectedly revealed that first few generations of KO animals are born successfully, suggesting that telomerase is not necessary for fertility and maintenance of renewing tissues during development (Blasco et al., 1997). However, careful analysis of the later generation animals had revealed that telomere repeats are lost gradually per generation of mice, resulting in chromosome instability, such as aneuploidy and chromosome end-to-end associations. Subsequent studies from Carol Greider and Ronad DePinho groups revealed that telomerase is indeed required for successive maintenance of highly proliferative organs, such as testis, bone marrow and spleen in later generation mTR-/- mice, which shows premature aging phenotypes at organismal level, including skin lesions, alopecia and hair graying (Lee et al., 1998; Rudolph et al., 1999), supporting the notion that telomere shortening underlies limited self-renewal capacity of stem cells in aged individuals. Severe telomere shortening in late generation mTR-/- mice activates p53-dependent cell cycle arrest pathway, and loss of both mTR and p53 synergistically enhances malignant transformation (Chin et al., 1999). Such transformed cells in p53-/- background often carry complex non-reciprocal translocations, a sign of BFB cycle during tumorigenesis (Artandi et al., 2000). These seminal works using telomerase deficient mice gave strong support to a “telomere crisis” model named by Fuyuki Ishikawa, in which the two-stage model was extended to incorporate BFB cycle as a driver of chromosome instability and consequently tumorigenesis during crisis stage (de Lange, 1995; Ishikawa, 1997; Artandi and DePinho, 2000). Current progress and future perspective Telomeric proteins, transcripts and structures As described, many important findings and concepts in telomere biology had been reported by early 2000’s. Although many other telomeric accessary factors have been identified by proteomic analysis (Déjardin and Kingston, 2009; Lee et al., 2011; Grolimund et al., 2013;

Bartocci et al., 2014) and the functions of

those accessary factors are quite important in telomere maintenance (Arnoult and Karlseder, 2015), shelterin is still recognized as a single constitutive protein component with a direct telomere-binding ability (Doksani and de Lange, 2014). Among shelterin components that directly bind to telomere, TRF2 has a central role in protecting telomeric ends from activating ATM-dependent DNA damage signaling pathway and from repairing through classical NHEJ, while ATR-dependent signaling is repressed by POT1 (Denchi and de Lange, 2007). Another telomere binding protein, TRF1, is also involved in suppression of ATR signaling through promoting telomeric semi-conservative replication (Sfeir et al., 2009). A recent identification of TZAP, telomeric zinc finger-associated protein, as a TTAGGG-binding protein, however, surprised the field since it was reported to bind directly and preferentially to long double stranded TTAGGG repeats (Li et al., 2017). Because TZAP competes with TRF2, negatively regulates telomere length and had been missed by conventional proteomics searches for shelterin-interacting proteins, it is an attractive possibility that TZAP composes an unknown telomeric complex. Chromosome ends had long been thought to possess a suppressive structure against transcription because of their enrichment of heterochromatic marks (Blasco, 2007). Artificial insertion of a reporter luciferase into subtelomere revealed 10 times less expression of the reporter compared to the one in non-telomeric chromatin, suggesting a transcriptional suppression near telomeres, which has been named as telomere position effect (TPE) (Baur et al., 2001). Despite this notion, a transcript that contains telomeric repeat had been reported in 2007 by Joachim Lingner’s group and called telomeric repeat-containing RNA (TERRA) (Azzalin et al., 2007), proving for the first time that some subtelomeric loci are actively transcribed. Ever since its finding, TERRA non-coding transcript has been associated with a proper telomere function through its interaction with various proteins, such as TRF1, TRF2, SUV39H1 and HP1 (Deng et al., 2009; López de Silanes et al., 2010; Scheibe et al., 2013; Porro et al., 2014). Interacting partners of TERRA includes telomerase RNA component as well, which suggests direct regulation of telomerase (Redon et al., 2010), although overexpression of TERRA does not affect telomerase activity in human cancer cells (Farnung et al., 2012). TERRA can also form

DNA:RNA hybrids with telomeric DNA, called R-loop, which has been implicated in telomere maintenance of ALT cells by regulating telomeric recombination (Arora et al., 2014). Because of its potential telomeric function in both telomerase-positive and telomerase-negative tumors, TERRA has been considered as an attractive candidate for therapeutic target (Cusanelli and Chartrand, 2015). Leading studies clearly demonstrated that telomeric end has at least two states: a closed- or capped-state that protects chromosome ends from activating DNA damage checkpoint including cell cycle arrest and damage repair pathways; and an open- or uncapped-state, in which both pathways are activated. Careful observation of telomere dysfunction induced foci (TIFs), a colocalization of DNA damage marker proteins with telomeres (Takai et al., 2003), in multiple tumor cell lines indicated the existence of a third state, an intermediate-state (Cesare et al., 2009) (Fig. 3). Many tumor cell lines as well as old normal human cells harbor unfused telomeres that are recognized as DNA damage, which indicates that these “intermediate-state” telomeres activate DNA damage checkpoint, yet still protected from being subjected to repair process (Cesare and Karlseder, 2012). The intermediate-state telomere is induced by extensive telomere shortening that prevents telomere from forming the protective closed-state structure (e.g. t-loop), and partial depletion of TRF2 (e.g. shRNA-based knockdown) that leads to a failure of the closed-state formation at an elongated telomere; while the open-state is a consequence of complete loss of TRF2-binding sites, or deletion of TRF2 (e.g. CRISPR/Cas9-based knockout) at an elongated telomere (Cesare et al., 2013). The intermediated-state telomere is proposed to induce replicative senescence without end-to-end fusion, while the open-state telomeres are fused and underlie deleterious chromosome instability and cell death during telomere crisis stage. The intermediate-state telomere can be also induced by a prolonged arrest in mitosis (Hayashi et al., 2012). The detailed mechanism is still unclear but such mitotic telomere deprotection requires Aurora B kinase activity, suggesting active pathway inducing the intermediate-state telomere during mitotic arrest. This partially explain a mechanism underlying cell death upon anti-tumor drug-induced mitotic arrest (Hayashi et al., 2015). The intermediate-state

telomere may thus function as a biological sensor for cellular stresses, such as prolonged mitosis and cellular chronological aging (Cesare and Karlseder, 2012). What remains to be studied is whether the three-state model is relevant to tumorigenesis in vivo, and whether natural telomeres can take any additional states other than described above. Telomere crisis The consequence of chromosome end-to-end fusion has been well studied in the last decade. Although recent massive genome sequencing of malignant tumor cells indicate frequent duplications in opposite orientations, which is indicative of BFB cycle (Campbell et al., 2010; Waddell et al., 2015), live cell analysis of epithelial cells that harbor chromosome fusions demonstrated that anaphase chromatin bridge formed between sister nuclei persists in the next cell cycle (Pampalona et al., 2012; Maciejowski et al., 2015). Persisted chromatin bridge results in either cytokinesis failure followed by tetraploidization (Pampalona et al., 2012), or resolution by enzymatic activities (Maciejowski et al., 2015). The latter was proposed to cause kataegis, a hypermutation pattern of clustered dC>dT and dC>dG changes at TpC site, and chromothripsis, a catastrophic rearrangement of one or a few chromosome regions in a single event (Maciejowski and de Lange, 2017). These models including BFB cycle hypothesis explain how chromosome accumulates deleterious rearrangement during telomere crisis. On the other hand, live cell observation of human fibroblast cells in telomere crisis indicated that cells often die during or after prolonged mitotic arrest. At least in fibroblast, chromosome end-to-end fusion was shown to trigger mitotic arrest by an unknown mechanism, which leads to mitotic telomere deprotection and cell death during the same mitosis or the following cell cycle (Hayashi et al., 2015). Cell death following mitotic arrest is dominant in telomere crisis of fibroblast, providing a novel insight about the mechanism of cell death during this catastrophic stage. It is worth noting that malignant tumors found in human patients are mostly of epithelial origin and fibroblast-derived sarcoma is very rare, suggesting distinct resistance to tumorigenesis in these cell types. This may be explained by strong preference of senescence in fibroblast cells compared to

apoptosis in epithelial cells upon exposure to stress (Georgakopoulou et al., 2016). Another possibility that needs to be addressed, however, is that end-to-end fusion may follow a different process in each cell type, consequently ending up with either tumorigenesis or cell death during telomere crisis. Telomere, aging, and disease It has been nearly 20 years since the first connection between telomere maintenance and dyskeratosis congenita was reported. Genetic diseases caused by malfunction of telomere maintenance pathway has been increasingly reported and collectively called the telomere syndromes (Armanios and Blackburn, 2012) or telomeropathies (Opresko and Shay, 2017). Mutations in genes known to function in telomere maintenance has now been identified in a variety of human genes involved in telomerase function (TR/TERC, TERT, DKC1, NOP10/NOLA3, NHP2/NOLA2, PARN, NAF1, and TCAB1/WRAP53) and telomere protection and replication (TIN2, RTEL1, POT1, CTC1, Apollo, and TPP1) (Blackburn et al., 2015; Martínez and Blasco, 2017; Opresko and Shay, 2017). The cause of the telomere syndromes has been linked to the failure of stem cell replenishment which highly demands functional telomere maintenance pathway (Blackburn et al., 2015). What human telomere syndromes and mouse genetics taught us is that telomere shortening caused by mutation in genes involved in telomere maintenance increases the risk for aging related diseases and mortality. Interestingly, mutations in the promoter of the TERT, which increases hTERT expression only a few-fold, have been reported to cause a familial and sporadic melanoma (Horn et al., 2013; Huang et al., 2013). This indicates that telomerase activity is highly regulated to be set in a certain range otherwise the risks of aging diseases, including cancer incidence, increase. This field of telomere biology attempts people’s mind since it may have direct implication on human aging and life span. Because of this, however, very careful studies will be required to dissect correlation and causality among telomere function and many aspects of human disease and health. Concluding remarks

As briefly discussed in the last section, the field of telomere biology will keep expanding to fulfil the demand of public interests about human health and disease without doubt. Comprehensive understanding of the role of telomere dysfunction in aging and tumorigenesis will thus drive applied research, potentially leading to the development of effective prognostic indicators, improved therapeutic treatment, and extension of a healthy life-span. As such, scientists are increasingly required to anticipate the impacts of their works on the public. In this respect, learning how the seminal works of the pioneers had influenced the current telomere biology would help to understand the dynamics of the field in near future. On the other hand, however, a number of leading studies were obviously the result of curiosity-driven basic research, which is an important lesson from the history. Some breakthroughs have even been the result of unexpected findings. It is therefore of great importance to maintain acknowledgement to the history of basic studies and maintain a harmonized cycle between basic and applied research for the sake of progress of science and human society. Acknowledgement I thank Jan Karlseder and Tomoichiro Miyoshi for critical reading of the manuscript. This work is supported by grants from the Senri Life Science Foundation, the Uehara Memorial Foundation, the Daiichi Sankyo Foundation of Life Science, the Nakajima Foundation, Grant-in-Aid for Young Scientists (A) (16H06176) and Grant-in-Aid for Scientific Research on Innovative Areas (16H01406).

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Figure Legends Fig. 1. The history of telomere biology, aging, and cancer field from early 1930’s to late 1980’s. The milestone studies related to telomeric DNA and proteins (top) and aging and cancer (bottom) are aligned in chronological order. Fig. 2. The history of telomere biology in aging and cancer field from late 1980’s to early 2000’s. The milestone studies related to telomeric DNA and proteins (top) and telomere biology in aging and cancer (bottom) are aligned in chronological order. Fig. 3. Three-state model of telomere structure. Closed state is achieved when enough telomeric repeats and TRF2 are available to form protective t-loop structure, in which single stranded 3’-overhang integrates into double stranded repeat locus. Closed state can suppress both ATM-mediated checkpoint signaling and classical NHEJ pathway. It is not well known how shelterin binds to t-loop structure. When telomeric repeats get shorter or TRF2 protein level decline to the level that is insufficient to support t-loop, telomere end becomes intermediate state. Intermediate telomere activates ATM checkpoint but still hold TRF2 enough to suppress NHEJ. When almost all telomeric repeats or TRF2 is removed, then chromosome end becomes open state, which resembles an internal DSB site and activates both checkpoint and NHEJ pathway. Intermediate state is relevant to telomere in replicative senescence, while open state is observed in crisis stage. Each component of shelterin is highlighted bellow. TRF1 and TRF2 can bind to TIN2 independently of each other, forming subcomplexes.

Telomeric DNA and proteins Telomere defined

McClintok 1931

1930

1940

Szostak & Blackburn 1982

Olovnikov 1971, 1973 Watson 1972

Muller 1938

Absence of fusion between natural end and broken end

Linear plasmid with Tetrahymena telomeres maintained in yeast

End replication problem proposed

BFB cycle hypothesis proposed

Telomere sequence of Tetrahymena Blackburn & Gall 1978

McClintok 1941

1950

Tetrahymena telomerase identified

1960

1970

Greider & Blackburn 1985

1980

p53 protein identified

Replicative senescence (Hayflick limit)

Deleo et al. 1979 Linzer & Levine 1979 Lane & Crawford 1979

Hayflick & Moorhead 1961

Cultured human and hamster cells transformed by SV40 Koprowski et al. 1962 Shein & Enders 1962 Rabson & Kirschstein 1962

Crisis defined Jensen et al. 1964

RB1 gene identified Friend et al. 1986

Aging and cancer

Fig. 1.

Telomeric proteins and structure hTRF1 identified

Nakamura et al. 1997 Meyerson et al. 1997

Zhong et al. 1992

Human telomeric repeat sequenced Moyzis et al. 1988

1985

Function of hTRF1/hTRF2 revealed

hTERT identified

hTRF2 identified Bilaud et al. 1996, 1997 Broccoli et al. 1997

1990

hPOT1 identified

3’-overhang identified

T-loop identified

Makarov et al. 1997

Griffith et al. 1999

1995

Checkpoint proposed

Telomerase activity associated with tumor cells

Hartwell & Weinert 1989

Kim et al. 1994

Harley et al. 1990 Hastie et al. 1990

Normal human cells immortalized by telomerase

Telomere biology in aging and cancer

Telomerase knockout mice Blasco et al. 1997 Lee et al. 1998

Shelterin/Telosome defined Liu et al. 2004 de Lange 2005

2000

Nakayama et al. 1998 Bodnar et al. 1998

Progressive telomere shortening in vitro and in vivo

Baumann & Cech 2001

van Steensel & de Lange 1997 van Steensel et al. 1998

2005

Tumor growth suppressed by DN-hTERT Hahn et al. 1999

DKC1 associated with telomerase ALT defined

Mitchell et al. 1999

Bryan & Reddel 1997

Fig. 2.

Subtelomere

TTAGGG repeats

3’-overhang

Open

Checkpoint

NHEJ

ON

ON

ON

OFF

OFF

OFF

3’

no TRF2 or no repeats

Intermediate lessTRF2 or less repeats

Closed enough repeats

T-loop

and enough TRF2

Shelterin components

TRF1

TRF2

RAP1

TIN2

TPP1

POT1

Fig. 3.