Review

Telomere Length Maintenance, Shortening, and Lengthening†

ZHENRONG ZHAO 1, XINGHUA PAN 2, LIN LIU 1**, NA LIU 1,2*

1, State Key Laboratory of Medicinal Chemical Biology; Department of Genetics and Cell Biology, College of Life Science, Nankai University, Tianjin 300071, China 2, Department of Genetics, Yale School of Medicine, Yale University, New Haven, CT 06520, USA

* Correspondence to Na Liu, State Key Laboratory of Medicinal Chemical Biology; Department of Genetics and Cell Biology, College of Life Science, Nankai University, Tianjin 300071, China E-mail: [email protected], or [email protected]. ** Correspondence to Lin Liu, Nankai University, E-mail: [email protected].

†

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.24537]

Received 11 December 2013; Accepted 13 December 2013 Journal of Cellular Physiology © 2013 Wiley Periodicals, Inc. DOI 10.1002/jcp.24537

Abstract Telomere plays key roles in maintaining chromosome stability and cell replicative capacity. Telomere shortening occur concomitant with aging. Abnormal short telomere associated with some diseases, such as dyskeratosis congenita, idiopathic pulmonary fibrosis and aplastic anemia. Telomere is longer in pluripotent stem cells than in somatic cells. During preimplantation development, the telomeres lengthen significantly. Furthermore, during somatic cell reprogramming, telomere elongation is of great importance in the acquisition of authentic pluripotency. This review focuses primarily on regulatory mechanisms of telomere length, telomere length maintenance in pluripotent cells, telomere length extension in early embryo development, and also on telomere rejuvenation in somatic cell reprogramming. Telomere related diseases were also touched in this review. Key words: telomere, telomerase, reprogramming, pluripotent stem cells

Contents: Introduction 1. Telomere and telomere related proteins Telomere structure Shelterin 2. Telomere shorten 3. Telomere lengthen regulation mechanisms Telomerase depended elongation Telomerase independed elongation (ALT mechanism) 4. Telomere elongation Telomere extended in early development Telomere in pluripotent stem cells Telomere lengthening in cells reprogramming SCNT iPS cells 5. Telomere related disease

Introduction The ends of linear chromosomes are formed by a special heterochromatic structure, known as telomeres. Telomeres are GC rich repeated DNA sequences enclosed by associated proteins at the chromosome ends that are essential for maintenance of chromosomal genomic stability. Telomere shortening and loss can result in chromosome fusions, which then lead to chromosomal breakage-fusion-breakage cycles during cell division, eventually causing genomic instability shown by genomic copy addition, deletion, mutation, and translocation (Blackburn, 2001; McClintock, 1941; Palm and de Lange, 2008). Telomere length can be maintained at a stable state in the present of telomerase. Now, some evidences showed that pluripotent stem cells and cancer cell possess longer telomere than in the somatic cells, in these cells telomerase activity are also higher (Huang et al., 2011; Stadtfeld et al., 2008). Telomere play important role in ES cells self-renewal and proliferation (Flores et al., 2005; Hiyama and Hiyama, 2007; Morrison et al., 1996; Niida et al., 2000; Wang et al., 2005). Beseide of telomerase, telomere can be also extanded by homorecombination. Following the development and differentiation, the telomere will be shorten (Forsyth et al., 2002; Wright et al., 1996). During the progress of somatic cell reprogramming, the telomere extandence is an indispensable step. Telomere abnormal will induce some diseases, such as cancer, aging, DC, and so on. We intend to systematically review the telomere and telomere length regulation mechanisms, especially in pluripotent stem cells and somatic cell reprogramming. In the following parts, we will describe it in detail of the telomere structure and telomere length regulation. In addition we will discuss several telomere-related diseases.

1. Telomere and telomere related proteins Telomere structure Telomeres are specialized terminal capping structures at the end of eukaryotic chromosomes, composed of TTAGGG repeats and a variety of proteins such as shelterin and telomerase. Telomeres ensure chromosome stability and protect the ends from degradation and from fusing with other chromosomes. They act as safeguards to prevent the loss of important genetic information during DNA replication due to the inability of DNA polymerase to replicated DNA to the ends of linear chromosomes (Blackburn, 2001). Telomere length varies greatly between species, from approximately 300 base pairs in yeast (Shampay et al., 1984) to many kilo-bases in humans (about 5~15kb) and in mouse (20~30kb). Chromosomes are progressively loss with each round of cell division. The loss of telomeric repeats triggers senescence in cells and prevents the cancer (Watson, 1972). Eukaryotic telomeres normally terminate with 3′ single-stranded-DNA overhang, which is essential for

telomere maintenance and capping. This 3’ single stranded repeat DNA looped back and annealed to double strand to form a large physical loop structure, named telomeric loop (also named T loop). T-loops were first identified by electron microscopy of purified telomeric restriction fragments from human and mouse cells (Griffith et al., 1999). T loops may provide a general mechanism to mask telomere termini from cellular activities that can act on DNA ends and regulate the elongation and shortening of telomeres (Griffith et al., 1999). In the T-loop structure, the 3’ overhang has been proposed to invade the double-stranded telomeric DNA, on the basis of pairing with the C-strand and displacing the G-strand, forming a D-loop (Displacement Loop). The strand invasion takes place at a distance from the physical end of the telomeres and therefore results in a large T-loop structure. They are a conserved aspect of telomere structure and have been speculated to protect telomeres and regulate telomerase (de Lange, 2005). In normal somatic cells telomeres shorten after each cell division because of the end replication problem and the absence of telomerase activity (Allsopp et al., 1992). However, telomeres can be lengthened by telomerase when it is activated in germ line cells, stem cells and tumor cells (Gomez et al., 2012). Besides this way there is an alternative mechanism (ALT) (Bryan et al., 1997) can also maintain telomere length. Apart from the role to protect chromosome ends, T-loops structure may also result in telomere elongation or shortening. Because the 3' overhang strand invasion in T-loops is similar to the structure in recombination dependent replication. And small linear or circular telomeric DNA may form through improper resolution of T-loops (Nittis et al., 2008). Telomere shelterin As mentioned above, telomere is special DNA sequences. In mammalian cells, telomere consists of a double-stranded region and a single stranded 3’ overhang. Telomeres can bind to several protein, which is called shelterin complex. The telomere shelterin is composed of six telomere-specific proteins (Trf1, Trf2, Pot1, Tin2, Tpp1, and Rap1). Three shelterin proteins, Trf1, Trf2, and Pot1 directly bind to telomere sequences. The other three shelterin components, Tin2, Tpp1, and Rap1, cannot directly recognize the telomeric DNA, but together with Trf1, Trf2 and Pot1 they form a complex that allows cells to distinguish telomeres from sites of DNA damage and regulate the elongation of the telomere (de Lange, 2005). The shelterin can affect the telomere length by blocking the action of telomerase. When the telomere is too long, more shelterin are attached to the TTAGGG repeat arrays to inhibit the telomerase. At a telomere which is too short the shelterin becomes relaxed, so that the telomerase can reconstruct its length (de Lange, 2005). The double-strand telomeric DNA at mammalian telomeres is bound by two shelterin related proteins, the TTAGGG repeat-binding factors (Trf1 and Trf2) (Broccoli et al., 1997; Chong et al., 1995; Griffith et al., 1999). Among them Trf1 was the first protein isolated based on its specificity for double-stranded TTAGGG repeats (Zhong et al., 1992). Trf1 can induce a shallow bend in duplex TTAGGG repeats and might stimulate the

folding of telomeres. Trf1 binds DNA as a dimer using a large conserved domain near the N-terminus of the protein for Trf1-Trf1 interactions (Bianchi et al., 1997). Trf1 is a suppressor of telomere elongation. Studies have shown that long term overexpression of Trf1 in the telomerase-positive tumor cell line results in a gradual and progressive telomere shortening. Conversely, telomere elongation was induced by expression of a dominant-negative Trf1 mutant that inhibited binding of Trf1 to telomeres. Trf1 does not detectably affect the expression of telomerase activity, maybe the binding of Trf1 controls telomere length by inhibiting the action of telomerase at the ends of individual telomeres (van Steensel and de Lange, 1997). Tin2 can enhance some of Trf1’s architectural effects (Kim et al., 2003). As we described above, in vitro telomeric DNA can be isolated as large duplex loops, called T-loops. The shelterin subunit Trf2 is a main player in the generation of T-loops. Trf2 also plays a key role in the repression of the telomeric DNA damage response. Pot1 (protection of telomeres), was identified as the most conserved component of shelterin complex (Baumann and Cech, 2001). Unlike Trf1 and Trf2 binding to double strand telomere, Pot1 specifically binds the single-stranded DNA (ssDNA) overhangs at the ends of telomere and suppresses unwanted DNA repair activities (Loayza and De Lange, 2003). Tpp1 can’t bind to telomere sequence directly, but binds to the Pot1-ssDNA complex and enhances the Pot1-ssDNA interaction. It is identified as a binding partner of Pot1. Tpp1-Pot1 association enhanced Pot1 affinity for telomeric single strand DNA (Baumann and Cech, 2001; Wang et al., 2007; Xin et al., 2007). The binding of the Pot1-Tpp1 complex to telomeric ssDNA serves to cap telomere ends and prevent telomerase access to the ssDNA template. Tpp1 and Tin2 are also required to bridge the Trf1 and Trf2 complexes. Overexpression of Tpp1 enhanced Tin2-Trf2 association. Conversely, knock down Tpp1 reduced the ability of endogenous Trf1 to associate with the Trf2 complex. Coordinated interactions among Tpp1, Tin2, Trf1, and Trf2 may ensure robust assembly of the telosome, telomere targeting of its subunits, and, ultimately, regulated telomere maintenance (O'Connor et al., 2006). Rap1 is closely linked to Trf2. Although Rap1 can bind to DNA templates in the absence of Trf2 with a preference for double strand-single strand junction, when Trf2 and Rap1 are in a complex, its affinity for double-strand telomeric sequences is 2-fold higher than Trf2 alone and more than 10-fold higher for telomeric 3’ends (Arat and Griffith, 2012). Accordingly, Rap1 was dispensable for the essential functions of Trf2. It is involved in the repression of ATM signaling, NHEJ (non-homologous end-joining), and HDR (homology-directed repair) (Li et al., 2000). Especially Rap1 was critical for the repression of homology-directed repair (HDR), which can alter telomere length. Human Rap1 affects telomere length homeostasis and an experiment has suggested that Rap1 can repress telomere fusions (Li and de Lange, 2003).

2. Telomere shorten Telomeres cannot be fully duplicated during cell division because of the end-replication problem, and this insufficiency causes telomere shortening at a rate of 30±50 bp per cell division (Martens et al., 2000). This problem is caused by the DNA polymerase’s characteristic of requiring an RNA primer to initiate duplication. When the RNA primer binds to the very last nucleotides (ending in 3’-OH) of the leading strand, polymerase binds to the heteroduplex DNA-RNA double strand and starts polymerization, moving along the DNA strand; as DNA polymerase moves away, the RNA primer dissociates, leaving a gap (a non-duplicated DNA strand) behind; the newly synthesized strand is therefore shorter than the template strand correspondent to the size of the RNA primer (Calado, 2009). This is the main reason for telomere shortening. Besides of this, cellular environment also plays an important role in regulating telomere length and telomerase activity. Oxidative stress can shorten telomeres. Suppression of oxidative stress by antioxidative agents, such as vitamin C, extends the replicative life span by reducing the rate of telomere shortening (von Zglinicki, 2002). Chronic psychological stress may also lead to telomere shortening and lowered telomerase function (Epel et al., 2004). When telomeres shorten sufficiently, the cell is arrested into senescence. Thus, telomere length can serve as a molecular clock of the aging process or potential for further cell division. On the one hand it can increase the initiation of tumour by inducing chromosomal instability and genetic alterations that lead to cellular transformation. On the other hand, tumour cells need to stabilize telomere shortening to avoid an accumulation of too high levels of instability that would ultimately kill the cancer cell. This hypothesis appears to be in line with the observation that most human cancers have critically short telomeres but at the same time show a reactivation of telomerase (Jiang et al., 2007).

3. Telomere lengthening regulation mechanisms Telomere length is influenced by changes in the activity of telomerase, the reverse transcriptase that elongates telomeres (Greider and Blackburn, 1985), as well as by the so called alternative lengthening of telomeres (ALT pathway), which relays in homologous recombination between telomeric sequences, known as telomeric sister-chromatid exchange (T-SCE) (Dunham et al., 2000). Telomerase depended elongation: The telomeres are progressively lost with each round of cell division, which can be extended by telomerase. Telomerase is a reverse transcriptase (a RNA dependent polymerase). Telomerase is made of protein and RNA subunit that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes. Telomerase is also called telomere terminal transferase. Telomerase is a ribonucleoprotein

composed of dyskerin, catalytic subunit Tert (telomerase reverse transcriptase), and Terc (telomerase RNA component) which serve as the template to synthesize telomere DNA. Dyskerin protein stabilizes the telomerase complex (Greider and Blackburn, 1985). The telomerase adds telomere repeats to chromosome ends to offset the loss of telomere sequences that occurs due to the end-replication problem, the inability of DNA polymerase to replicate fully the lagging DNA strand (Martinez and Blasco, 2011). In the absence of sufficient levels of telomerase, telomeres shorten progressively with cell division, ultimately leading to loss of telomere protection and a DNA damage response that induces senescence or cell death. Terc knock-out (-/-) mouse strain lacks functional telomerase and is characterized by continuous telomere shortening from one generation to the next, eventually leading to telomere dysfunction, premature aging, and a reduced lifespan (Blasco et al., 1997). To avoid the erosion of telomeres, some of the germ line cells, stem cells and cancer cells activate the activity of telomerase (Greider and Blackburn, 1985). Telomerase expression is restricted to embryonic development as well as to adult stem cell compartments. In somatic cells, telomerase activity is very low, almost undetectable. So telomere shortens with each cell division. When the telomere length reaches a point, it will result the cell senescence and dies. Telomerase independed elongation (ALT mechanism): In some mammalian cancer cells and immortalized cell lines, telomeres are extended in a telomerase-independent manner called ALT (Alternative lengthening of telomeres). It has been suggested that the ALT mechanism rely on the homologous DNA recombination (Dunham et al., 2000; Reddel et al., 2001). Telomere homologous recombination is usually carried on with the telomere replication. Telomeres are repetitive DNA sequences and thus, have multiple sites of homology necessary for homologous recombination. ALT cells contain high levels of ECTRs (extra-chromosomal telomeric repeats), which might be linear as well as circular (T-looped) (Cesare and Griffith, 2004; Tokutake et al., 1998). According to different templates, ALT mechanism can be classified into 4 types, namely the inter-telomeric recombination (Henson et al., 2002), T-loops dependent recombination, rolling circle dependent recombination and linear ECTRs DNA-dependent recombination. In the inter-telomeric recombination, one telomere invades double-stranded DNA of another telomere and uses it as a copy template resulting in a net increase in telomeric DNA within the cell (Henson et al., 2002). Telomere can be lengthened by copying its own sequences, without the need for using another telomere as a copy template. Telomere tag can be amplified without the involvement of other telomeres, indicating that telomere elongation can also occur by intra-telomeric DNA copying. This is the first direct evidence that the ALT mechanism involves more than one method of telomere elongation (Muntoni et al., 2009). In the rolling circle-dependent recombination, a dependent T-circle (a circle of ECTR DNA) and a chromatin are involved. The chromatin’s 3' single-stranded

overhang will invade into the duplex T-circle and use it as a rolling template to elongate telomeres without limitations (Regev et al., 1998). In one aspect, the linear extra-chromosomal telomere repeats (linear ECTR) are the products of the ALT mechanism. In another aspect, linear ECTRs could be used to elongate telomeres by end-joining reactions or by homologous recombination and copy template (Henson et al., 2002). ALT cells are usually characterized by a remarkable heterogeneous telomere length within a given cell and the presence of promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA and telomere-binding proteins. These ALT-associated PML bodies (APBs) may be used to identify cells which use the ALT pathway, their function is still unknown (Nittis et al., 2008). In turn, these telomere-elongation mechanisms are regulated by the epigenetic status of telomeric chromatin (Blasco, 2007a). In particular, both telomeric and subtelomeric repeats are enriched in histone methylation marks characteristic of repressed heterochromatic domains such as trimethylation of H3K9 and H4K20, and they show binding of the heterochromatin protein 1 (HP1).

4. Telomere elongation Telomere extended in early embryo development As we known, the telomere in oocyte is shorter than in somatic cells. Studies have shown that compared to oocytes the telomere length in zygotes is extended, and telomere continued to elongate from two-cell to four-cell embryos until the blastocyst stage (Liu et al., 2007). So something must have happened during the early embryo development. Is the activation of telomerase, the ALT mechanism or other special mechanism related to the matters? As the telomerase activity is crucial for the maintenance of telomere length, it was examined during the embryos’ development. Telomerase activity was not detected in mature spermatozoa, oocytes, and morula (Wright et al., 1996). Telomerase activity was detected until the embryo develops into the blastocyst stage. That so say telomere length extended in this period was not dependent on telomerase. But it has been demonstrated that the telomere in oocytes can be lengthened after fertilization or parthenogenetic activation. At fertilization, the paternal genome exchanges protamines for histones, undergoes DNA demethylation, and the demethylation continues during the early cleavage embryos (Morgan et al., 2005). The extensive genome-wide demethylation provides a favorable environment for the telomere lengthening via a recombination-based mechanism (Liu et al., 2007). In contrast, a strong up-regulation of telomerase was observed at the blastocyst stage. So at the transition from morula to blastocyst telomeres are significantly elongated (Schaetzlein, 2004). Interestingly, just as

telomerase activity increases in blastocyst, the proteins for recombination and DNA-damage repair, as well as T-SCE, decrease markedly, suggesting that cells may use different mechanisms to elongate telomeres at different stages of development (Liu et al., 2007). Telomeres do not elongate appreciably after the blastocyst stage (Schaetzlein, 2004). Thus, telomere length is established during early cleavage stages by ALT mechanism, and activation of telomerase in blastocyst only maintains telomere length during subsequent development (Liu et al., 2007). As previously mentioned, ALT mechanism may be another method to maintain telomere length. It can be classified into telomere sister chromatin exchange (T-SCE) and telomere inter-chromosomal exchange (T-ICE). In general, homology recombination is expected to replace the same amounts of DNA. However, within the telomere repetitive sequences, the exchanging strand can invade at multiple points, resulting in unequal T-SCEs and elongation of telomeres (Bailey et al., 2004). T-ICE is an exchange resembling T-SCE except that it occurs between the telomeres of different chromosomes. It is puzzling that in somatic cells there is no net gain of telomere after the recombination (Bailey et al., 2004), while in early cleavage embryos telomeres are lengthened. So it is possible that T-SCE and recombination do not directly lengthen telomeres in early embryos, but, instead, that the telomeres are lengthened by a DNA polymerase-mediated extension after recombination (Liu et al., 2007).

Telomere in pluripotent stem cells ES cells (Embryonic stem cell) were derived from ICM (inner cell mass，ICM) at the blastocyst stage, which can differentiate into all kinds of cells including germ cells (Evans and Kaufman, 1981; Thomson et al., 1998). A significant signature for ES cells is that which can proliferate indefinitely. Compared with somatic cells, telomere length is longer in pluripotent stem cells, which means there might be some relationship between telomere length and pluripotency. ES cells and other pluripotent embryonal carcinoma cells display high levels of telomerase activity and Tert expression, both of which are rapidly downregulated during differentiation (Armstrong et al., 2005), which means high level of telomerase activity contributes to the longer telomere in pluripotent cells. Very interesting, ALT mechanism also take part in telomere regulation in ES cells (Huang et al., 2011; Varela et al., 2011). During this process, Zscan4 play an important role (Zalzman et al., 2010). Zscan4, with higher expression in 2-cell stage of the early embryo development, is necessary for the implantation of blastocyst and self-renew of ES cells (Carter et al., 2008; Falco et al., 2007). In ES cells, following Zscan4 knockdown, telomere shorten quickly. Zscan4 might involve in telomere ALT mechanism in ES cells (Zalzman et al., 2010).

Telomere reprogramming The 2012 Nobel Prize in Physiology or Medicine was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent". Differentiated cell can turn back to the pluripotent state by somatic nuclear transfer or iPS cells method. During this process, the short telomeres in differentiated cells were also reprogrammed. Telomere elongation during somatic cell reprogramming is of great importance in the acquisition of authentic pluripotency. In the following parts, we will illustrate the telomere reprogramming during the SCNT and iPS cells formation separately. SCNT: Previous studies with cloned animals by means of somatic cell nuclear transfer (SCNT) into enucleated oocytes have rendered contradictory results regarding whether telomeres are elongated or not during nuclear reprogramming (Lanza et al., 2000; Shiels et al., 1999). Dolly, the first sheep cloned from a cultured mammary cell from a 6-year-old animal by using SCNT. In order to figure out the actual physiological age of its cells experiments were taken to analyze Dolly’s telomeres, and the results showed that they were shorter compared with age-matched controls (Shiels et al., 1999). The above indicated that the cells didn’t get ‘rejuvenated’ during reprogramming. However, follow-up analyses across a variety of SCNT-derived animals give rather different results, with the majority of cloned animals presenting a normal telomere length (Marion and Blasco, 2010a). The experiments covered different species like mice and cattle as well as different cell types like fibroblasts cells, cumulus cells and granulosa cells. These results demonstrated that the telomeres were able to be restored. But why Dolly’s telomeres were so unusually short? It has been suggested that they may be the consequence of differences in the species, in donor cell type, as well as in nuclear transfer procedures (Marion and Blasco, 2010a; Schaetzlein, 2004). Since cloned sheep derived from fibroblast cells successfully restored telomere length (Clark et al., 2003), it cannot be the reason that account for Dolly’s short telomere. And then attention was focused on differences in telomere lengths depending on different cell types. Scientists produced cloned cattle by using nuclei of donor cells derived from muscle, oviduct, mammary, and ear skin. Among them telomere length varied remarkably. Telomere lengths in cloned cattle derived from muscle cells of an old bull were longer than those of a donor animal. Consistent with Dolly’s telomeres, those derived from oviductal and mammary epithelial cells of an equally old cow were shorter than any found in control cattle (Miyashita et al., 2002). Among different cell types, there may be many differences in telomere binding proteins, the ability of telomerase to extend telomeres or signaling pathways (Lanza et al., 2000), so that SCNT can not successfully trigger all the elongation of telomeres. But when and how did the telomeres elongate during SCNT. Different with the telomere extension in early embryo development stage, during SCNT before the morula stage there is no recombination-based elongation or the activation of telomerase. When come to the blastocyst stage the telomeres are restored to normal length

(Schaetzlein, 2004) and the telomerase activity is also detected. These results indicate that the

telomere

elongation during SCNT happens at the morula to blastocyst transition by activating telomerase (Marion and Blasco, 2010a). Recently a group of research investigated the telomere lengthening mechanism during the SCNT. They found that telomere elongate markedly in cloned embryos reconstructed with telomerase-deficient somatic cells, leading to improved telomere capping function, which indicated that telomerase-independent mechanism is also have function in SCNT program (Le et al., 2013). That might be due to factors involved in telomere lengthening via the ALT mechanism in SCNT or early embryos, such as Zscan4 (Falco et al., 2007; Zalzman et al., 2010). iPS cells: Beside of SCNT, some transcription factors can directly reprogram the somatic cell back to pluripotent state, these cells also called induced pluripotent stem cell (iPS cells) (Takahashi and Yamanaka, 2006). iPS cells hold great promise in future clinical applications, especially in the treatment of degeneration disorders caused by aging. However, not all of the iPS cells have higher quality of pluripotency, which partially due to the insufficient telomere lengthening. In some iPS cells with higher quality, telomere length was significantly increased in iPS cells compared to parental differentiated cells, reaching intermediate levels to those of control ES cells in early passages, and then reaching telomere length comparable to control ES cells at later passages (Esteban et al., 2009; Liao et al., 2009; Liu et al., 2008; Wu et al., 2009; Yu et al., 2007). Telomerase activity has been found upregulated in iPS cells compared with their parental somatic cells (Stadtfeld et al., 2008; Takahashi et al., 2007). During reprogramming, telomere elongation is usually mediated by telomerase and that iPS cell telomeres acquire the epigenetic marks of ES cells, including a low density of trimethylated histones H3K9 and H4K20 and increased abundance of telomere transcripts. Both telomere length and telomere heterochromatic marks acquire ES cell features during iPS cell generation. It is interpreted that reprogramming changes the accessibility of telomerase to telomeres, allowing their progressive elongation until telomeres reach the length characteristic of ES cells (Marion et al., 2009). Telomeres are efficiently elongated in iPS cells derived from old animals, demonstrating that telomeres can be efficiently rejuvenated during nuclear reprogramming. And telomerase activity is the primary mechanism to mediate telomere re-elongation during reprogramming (Marion et al., 2009). However telomere length and the level of telomerase exhibit heterogeneity in different iPS cell lines (Wang et al., 2012). In some fully reprogramming iPS cells, the telomere length is more similar to ES cells (Agarwal et al., 2010; Marion and Blasco, 2010b; Marion et al., 2009; Mathew et al., 2010; Suhr et al., 2009; Vaziri et al., 2010). The heterogeneity reflects in the following aspects, in some iPS cell lines telomeres lengthened at early passages, and continued elongation by passages. While in other iPS cells, telomere length maintained or slightly elongated at early passages and became shorter with the passages. As previously mentioned telomeres are

elongated mainly by telomerase, in iPS cells the expression level of Tert and Terc is upregulated relatively early during the reprogramming process, preceding the activation of the endogenous Oct4, Nanog and Sox2 pluripotency genes. Telomerase needs a number of cell divisions to restore telomere length in iPS cells (Siegl-Cachedenier et al., 2007), which suggests that most telomere elongation occur post-reprogramming (Marion et al., 2009). A less efficient reprogramming of iPS cells derived from patients with germline telomerase mutations, such as dyskeratosis congenita, some cases of aplastic anemia, and idiopathic pulmonary fibrosis, as telomere elongation during iPS cell generation requires an active telomerase complex (Marion et al., 2009). Recently, several groups obtained iPS cells with telomerase lesion cells (Agarwal et al., 2010; Marion et al., 2009; Wang et al., 2012), which means telomerase independent mechanisms also take part in the telomere reprogramming in iPS cells. iPS cells also show higher telomere recombination frequencies than parental cells, reaching similar values to those of ES cells (Marion et al., 2009). It is because the density of H3K9m3 and H4K20m3 histone heterochromatic marks at the telomeres of iPS cells are decreased. The decreased heterochromatic marks at telomeres promote the homologous recombination events between telomeric repeats and thus extend the telomere. So during the reprogramming to iPS cells these epigenetic alterations are accompanied by telomere elongation. In dyskeratosis congenita (DC) patient specific iPS cells, TERC (the RNA component of the telomerase) is upregulated as a feature of the pluripotent state (Agarwal et al., 2010). Chromatin immunoprecipitation (ChIP) in human iPS cells was performed, and enhanced binding of OCT4 and NANOG in the TERC locus was detected (Agarwal et al., 2010). Telomere sister chromatin exchange events are indicative of an active recombination-based mechanism for telomere length maintenance, and were observed at an elevated frequency in Terc–/– iPS cells. Moreover, Zscan4, recently shown to have an important role in recombination-based telomere length maintenance in ES cells, was observed to be highly expressed in Terc–/– iPS cells (Wang et al., 2012). The transient expression of Zscan4 in ES cells is associated with rapid telomere elongation (Zalzman et al., 2010). Addition of Zscan4 in the portion of iPS cells with the ability to generate full-term mice (Jiang et al., 2013). Together, these results indicate that both telomerase-dependent and telomerase-independent ALT mechanisms play critical roles in telomere length maintenance in iPS cells. But compared with SCNT, ALT mechanism is substantially more effective in SCNT than in iPS cells (Le et al., 2013).

5. Telomere related disease It is widely known that telomere shortens with each cell division, and finally leading the cell senescence. A number of age-related pathologies and premature aging syndromes are also characterized by telomere

shortening, and this indicates that short telomere can provoke aging. Telomere shortening promotes aging may by inducing apoptosis and cell cycle arrest, and then leading to cell loss and tissue dysfunction (Blasco, 2005; Blasco, 2007b). Previous studies showed that the mTerc-/- mice exhibit progressive telomere shortening and loss of proliferative cell types with each progressive generation (Blasco et al., 1997; Lee et al., 1998). Short telomeres link more directly to a fundamental feature of aging: a reduced capacity to respond to acute and chronic illness (Flores and Blasco, 2009). Several human premature ageing syndromes are characterized by a faster rate of telomere attrition with age, and these have provided important insights into the consequences of telomere loss. One of these syndromes is dyskeratosis congenita (DC). Dyskeratosis congenita is a rare disorder that is characterized by a triad of clinical symptoms: dystrophic nails, skin hyperpigmentation and oral leukoplakla (Carroll and Ly, 2009). All forms of dyskeratosis congenita are associated with very short telomeres in peripheral blood lymphocytes (Alter et al., 2007). Mutations in the three main components of telomerase holoenzyme, TERC and TERT, as well as dyskerin (DKC) have been linked to the disease (Carroll and Ly, 2009). Defects in telomere length have been implicated in the pathology of several age-related diseases and premature ageing syndromes. Some mouse models which are deficient for telomerase activity also demonstrate that short telomeres in the mouse can lead to similar disease states to those associated with both normal ageing and premature ageing syndromes in human (Blasco, 2005). Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease, characterized of cough, dyspnea, impaired gas exchange, and reduced lung volume and finally leads to respiratory failure and death within 3-5 years (Kropski et al., 2013). It is also an age-related disease. Detailed experiments have revealed that two components of the telomerase complex, TERT and TERC are the most commonly identified genetic risk factor in IPF (Kropski et al., 2013). TERT and TERC complex act as components of telomerase, they have certain impact on the length of telomere and mutations in them make up one-sixth of pulmonary fibrosis families (Armanios, 2012). It is proposed that telomere dysfunction may cause an irreversible stem cell failure in alveolar epithelial cells and then result in the IPF (Armanios et al., 2007). However IPF patients have short telomeres even when no mutation in telomerase is detected (Armanios, 2012). Sporadic IPF is the very case (those who report no family history) that have significantly shorter telomeres than age-matched controls with intact telomerase (Alder et al., 2008; Cronkhite et al., 2008). So the telomerase cannot be the direct reason of this progressive worsening disease. As previously mentioned IPF is a degenerative disease, it has a significant requirement for intact regenerative potential to sustain the high turnover demands, and finally resulting substantial erosion of telomeres and end to end fusion (Lee et al., 1998; Rudolph et al., 1999). So it is the short telomeres, not telomerase level, cause idiopathic pulmonary fibrosis. The absence of telomerase alone is

therefore not sufficient to mediate degenerative disease (Hao et al., 2005). Another telomere related disease is tumor. As we described above, in somatic cells, there is no telomerase activity. Their telomeres progressively shorten with each cell division. When one or more telomeres reach a critically short length, the de-protected chromosome ends are recognized by DNA damage responses factors, such as p53, and p16. When the function of p53 is normal, the result is typically senescence, in which the cells continue to live but are irreversibly blocked from further cell division. These cells enter into senescence. The impact of telomere is dramatically altered when checkpoint pathways are disrupted, and which has important implication for cancer biology. In particular, the cells with p53 mutation, bypass of replicative senescence and further telomere attrition. Despite the presence of critically short telomeres, removal of p53 function rescued many of the cellular defects, including growth arrest in cell culture, testicular atrophy and intestinal and germ-cell apoptosis ??. In the absence of p53, telomeres shorten and the resultant genomic instability promotes tumorigenesis. Telomere shorten promotes the development of cancer in the setting of p53 deficiency. Intact p53 pathway inhibits the tumor development in the setting with short telomere (Deng et al., 2008).

Acknowledgements This work was supported by China MOST National Major Basic Research Program (973 Program) (2012CB911202), National Natural Science Foundation of China (31000651), and China Scholarship Council (201208120050).

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