Stem cells at odds with telomere maintenance and protection

Abstract

Telomeres are distinctive structures that protect the ends of linear chromosomes and ensure genome stability. They are composed of long tracks of repetitive and G-rich DNA that is bound by shelterin, a dedicated six-subunit protein complex. In somatic cells, shelterin protects telomeres from the DNA damage response and regulates telomere length. Telomere repeats are replenished by telomerase, a specialized ribonucleoprotein composed of telomerase reverse transcriptase and an integral RNA component. Telomere protection and telomerase regulation have been primarily studied in somatic cells. However, recent evidence points out to striking differences in the context of embryonic stem cells. In this review, we discuss insights into telomere protection in ESCs vs. somatic cells and summarize findings on telomerase regulation as a function of pluripotency.

Mechanism of Telomere protection: the shelterin complex

Linear chromosomes pose two challenges for eukaryotic cells, known as the “end-replication” and “end-protection” problems. Semi-conservative DNA replication fails to copy chromosomes to the very end, leading to loss of terminal DNA sequence. In addition, chromosome ends resemble broken DNA that can potentially activate a DNA damage response (DDR) and trigger aberrant DNA repair. The solution to both problems is inherent to telomeres; nucleoprotein structures consisting of double-stranded (ds) TTAGGG repeats that terminate in a single-stranded (ss) G-rich 3′ overhang. Telomeres are bound by a six-subunit protein complex, termed shelterin, that silences various DNA damage signaling and repair reactions [1]. To solve the end-replication problem, cells employ telomerase, a specialized reverse transcriptase that adds telomere repeats during rounds of cellular division [2]. Seminal discoveries, largely in somatic cells, shaped our understanding of how shelterin silences the DDR at chromosome ends and shed light on telomere maintenance by telomerase. Recent and unexpected findings identified major differences in telomere protection and telomerase reactivation in embryonic stem cells (ESCs). In this review we discuss insights and implications of telomere maintenance and protection as a function of pluripotency.

Telomere protection in somatic cells – the shelterin complex

The six-subunit shelterin complex composed of TRF1, TRF2, POT1, RAP1, TIN2, and TPP1, prevents activation of the DNA damage response pathway at chromosome ends and inhibits unwanted DNA repair activities [3]. TRF1 and TRF2 bind double-stranded telomere DNA and act as anchors for the remaining subunits of the complex. TIN2 serves as a protein scaffold that bridges TRF1 and TRF2 with TPP1, which in turn recruits POT1 to telomeric repeats [1]. POT1 binds the single-stranded portion of telomeres and blocks the recruitment of RPA, thereby preventing the activation of ATR-dependent DNA damage response pathway at chromosome ends [4,5]. TRF1 facilitates replication fork progression at TTAGGG repeats that would otherwise trigger ATR signaling [6].

TRF2 plays a major role in telomere protection in somatic cells by suppressing the activation of ATM-dependent DDR pathway. Furthermore, telomeres depleted of TRF2 are substrates for non-homologous end-joining (NHEJ) that lead to end-to-end chromosome fusions [7,8] (Figure 1A). TRF2 functions by promoting a secondary protective structure termed t-loop as well as actively repressing ATM signaling and NHEJ. (reviewed in [3]). In addition, TRF2 suppresses homologous recombination [9] and is a co-repressor of microhomology-mediated end-joining by inhibiting PARP1-mediated signaling [10]. TRF2 also regulates the length of the single-stranded telomere overhang by preventing excessive 5’-exonuclease activity by CtIP and Exo1 [11]. Loss of function analysis indicated that RAP1, which is recruited to telomere through TRF2, is largely dispensable for chromosome-end protection [12]. However, in certain experimental settings, RAP1 promotes TRF2-mediated suppression of NHEJ [13,14]. In addition to fulfilling these non-overlapping functions in telomere protection, the shelterin complex modulates telomerase recruitment and activity [15,16].

Figure 1. Consequences of telomere deprotection in somatic cells and pluripotent stem cells.

Telomeres lose their protective cap (purple spheres) and become deprotected as a consequence of telomere erosion or upon depletion of shelterin components. A) In somatic cells, deprotected telomeres are bound by DNA damage response factors (orange spheres). This triggers the activation of the DNA Damage Response (DDR) pathway that causes end-end-chromosome fusions and irreversible exit from the cell cycle through apoptosis and/or senescence. B) In contrast, in pluripotent stem cells, telomere deprotection triggers gene deregulation, activation of alternative pathways of telomere protection, sustained proliferation, and inhibition of differentiation potentials.

Alternative mechanisms of telomere protection

Recent evidence suggests that some aspects of the key mechanisms required for chromosome end-protection in somatic cells are dispensable in specific developmental stages. For example, during meiosis, shelterin is replaced by a specialized complex that facilitates telomere tethering to the nuclear envelope to enable homologous chromosome pairing. This phenomenon termed “telomere cap exchange” is established when the TERB1/2-MAJIN complex binds to telomere DNA, displaces shelterin, and forms a link between the inner nuclear membrane and chromosome ends [17]. This process is vital since it aids chromosome movements and facilitates pairing of homologous chromosomes. In effect, during this stage, chromosome ends are protected independent of shelterin.

In addition, analysis of embryonic stem cells (ESCs) derived from the inner mass of the blastocyst indicates that the response to telomere deprotection in the early stages of development is different from what we learned in somatic cells. Specifically, in somatic cells, when telomere becomes critically short, they fail to recruit sufficient shelterin and – depending on the cell type – trigger cellular senescence, apoptosis, or autophagy. In contrast, mouse ESCs with critically short telomeres show an impaired differentiation potential [18,19] without losing proliferative capacity. In a similar manner, human pluripotent cells with critically short telomeres undergo cellular proliferation and DNA synthesis for extended periods without undergoing cellular senescence [20]. The molecular mechanism underlying these differences remains to be fully elucidated but two recent publications analyzing the consequences of TRF2 inhibition provide further evidence in support of the unique nature of telomere protection in mouse pluripotent stem cells [21,22]. The results show that wheras TRF2 is essential in all somatic cells analyzed to date, its function appears to be dispensable in mESCs (Figure 1B). Intriguingly, TRF2 depleted cells can grow indefinitely when undifferentiated but rapidly lose viability upon differentiation. This data clearly shows the mechanism of telomere protection – at least the TRF2-dependent ones – are tightly associated with cellular differentiation and raise a set of fundamental questions regarding the mechanism of telomere protection in pluripotent stem.

TRF2-independent telomere protection in mESCs

TRF2 deletion in normal somatic cells triggers remarkable phenotypes of rampant telomere fusions, robust activation of ATM-dependent DNA damage signaling, and irreversible cell cycle arrest or cell death [7]. So how can ESCs proliferate in the absence of TRF2? Given that ESCs are fully proficient in detecting and responding to DNA damage, the difference between pluripotent and somatic cells is inherent to suppression of DNA damage at telomeres. At least two models for TRF2-mediated end protection have been proposed in somatic cells. The first includes the formation of a secondary protective structure termed t-loop and direct suppression of the DNA damage response machinery. The t-loop model postulates that TRF2 stabilizes the invasion of the terminal G-rich telomeric overhang into a portion of the double-stranded telomeric DNA hiding the chromosome termini from the DNA damage response machinery [23,24]. Surprisingly, TRF2 null ESCs do not show any reduction in t-loop frequency [22], suggesting the existence of a TRF2-independent mechanism to establish these secondary structures. Future work will most likely establish how t-loops are established in ESCs and whether they play a protective role in these cells.

A second mechanism for TRF2-mediated telomere protection in somatic cells is linked to its ability to suppress the DNA damage response at the level of the ATM kinase and its downstream signaling pathway [8,25]. Interestingly, in TRF2-depleted ESCs triggering DNA damage by depletion of other telomeric factors (e.g. POT1B) or the chromatin remodeling factor BRD2, unleashes the telomere dysfunction phenotype observed in TRF2 deficient somatic cells [21]. Critically, in the presence of TRF2, depletion of POT1b or BRD2 does not activate a telomere dysfunction phenotype. These data indicate that while TRF2 depletion is not sufficient to fully engage the DDR pathway in ESCs, it is necessary for full telomere protection. Furthermore, these data suggest that ESC-specific factors are likely to be involved in establishing a novel mechanism of telomere protection in the early stages of development.

Telomere dysfunction triggers a 2C-like state in pluripotent stem cells

Telomere dysfunction in pluripotent stem cells triggers major changes in gene expression that have a profound effect on cell fate and telomere function. Cells with critically short telomeres fail to silence the pluripotency genes Nanog and Oct4 resulting in impaired differentiation. These defects are likely caused by genome-wide alterations in chromatin accessibility mediated by the Polycomb Repressive Complex 2 (PRC2) activity [18]. Similarly, depletion of TRF1 in mESCs leads to significant changes in gene expression mediated by PRC2 [26]. The Telomeric repeat-containing RNA (TERRA) has been proposed to control this process through the recruitment of the PRC2 complex to gene promoters. In contrast to TRF2 depletion in somatic cells that shows no significant changes in gene expression, deletion of TRF2 in ESCs results in significant transcriptional changes [21,27].

Notably, TRF2 null ESCs upregulate genes typically expressed at the 2-cell (2C) stage embryo, including the MERVL family of endogenous retroviruses and the zinc-finger family of transcription factors, ZSCAN4. The latter appears to play a role in the protection of telomeres lacking TRF2 [21]. Activation of ZSCAN4 and other 2C genes in ESCs has been also noted in response to telomere shortening, in cells treated with genotoxic stress, and following perturbation to chromatin landscape [28–30]. ZSCAN4 activation has been proposed to play an important role in maintaining genome stability and telomere length in ESCs, possibly through the downregulation of DNA methyltransferases [31]. Disruption of either DNMT1 or DNMT3a/3b in ESCs leads to telomere elongation in a homologous recombination (HR)-dependent manner [32]. ZSCAN4 promotes degradation of DNMT1, thus inducing DNA hypomethylation, which could then promote telomere recombination and telomere elongation [31]. ZSCAN4 was also shown to be ensure genome stability during reprograming of iPSCs [33], and protects fragile microsatellite regions in 2C-like cells [34].

ZSCAN4 function may not be restricted to ESCs since the transcription factor is expressed in a small fraction of human cancer cell lines. In these cells, ZSCAN4 expression may promote DNA hypomethylation and facilitate the activation of the alternative lengthening of telomeres (ALT) mechanism of telomere elongation [35,36]. However, the precise mechanism of action of ZSCAN4 remains poorly characterized and further work is required to determine the significance of ZSCAN4 and DNA methylation for telomere maintenance during embryonic development and in cancer cells.

Shelterin and the regulation of gene expression

In summary, emerging data reveal that loss of telomere protection can have a major impact on the global transcriptional program in pluripotent cells. These observations raise the interesting question of how events that take place at chromosomal termini affect gene expression throughout the genome. One hypothesis is that loss or delocalization of telomere-associated protein might have a direct effect on gene expression. In this regard, it is interesting to note that the binding partner of TRF2, RAP1, has an established extra-telomeric function as a transcriptional regulator from yeast to mammals [37–39]. Furthermore, in yeast, telomere shortening triggers the re-localization of RAP1 to gene promoters [40]. Depletion of RAP1 in mouse ESCs induces a 2-C-like stage, marked by upregulation of ZSCAN4 and MERVL (Barry et al, biorxiv, https://doi.org/10.1101/2021.11.02.467017). It is therefore conceivable that telomere erosion, as well as depletion of TRF2, could impact transcriptional function of RAP1. An alternative hypothesis is that telomere dysfunction triggers changes in the levels of the long-noncoding telomeric transcript TERRA [41,42] that would in turn induce genome-wide epigenetic deregulation [26]. Consistent with these findings, the interaction between TRF2 and TERRA facilitates heterochromatin formation at telomere repeats as well as pericentromeric repeats [43,44], and further implicate TRF2 in controlling gene expression in pluripotent stem cells. TRF2 has also been linked to gene expression though association with G-quadruplexes containing promoters [45], suggesting that its depletion could affect gene expression directly. Future work will likely pinpoint to the mechanism(s) that link TRF2 deletion and gene deregulation and elucidate the cell specificity of these processes.

Telomere maintenance by telomerase

To counteract telomere shortening, cells rely on telomerase, a ribonucleoprotein complex composed of a reverse transcriptase (TERT) that catalyzes the addition of telomere sequence and a non-coding RNA (TERC) that provides the template to guide repeat addition [46]. Telomerase is associated with additional factors that mediate processing and maturation of TERC and those that critical for complex assembly and trafficking through the nucleoplasm [47]. Cryo-electron microscopy analysis revealed that the telomerase holoenzyme adopts a flexible two-lobed structure with TERC acting as a scaffold. The catalytic core makes up the first lobe comprising TERT and the template region of TERC that is embedded within the reverse transcriptase domain. The second lobe contains a heterotetramer of Dyskerin, NOP10, NHP2 and GAR [46] (Figure 2). Of note, active telomerase also contained an H2A-H2B dimer that interfaces with an essential RNA motif. While the functional significance of the latter interaction remains to be uncovered, it is tempting to speculate that the H2A-H2B component might link telomerase activity to histone deposition during replication.

Figure 2. Telomerase assembly and recruitment to telomeres.

Following TERC synthesis, dyskerin, Nop1, Gar1 and Nhp2 bind to the 3’ H/ACA stem loop. Binding of TCAB1 facilitates recruitment to Cajal bodies, where hTR remains until recruited by hTERT to form the active telomerase holoenzyme complex. hTERT expression in cells is regulated by multiple mechanisms depending on cell type. In cancer cells, hTERT promoter mutations increase expression of hTERT mRNA, whereas pluripotent cells use alternative splicing to ensure high levels of hTERT expression. Telomerase is recruited to telomeres via an interaction between with TPP1 while telomerase retention is driven by base pairing of hTR with the 3’-end of the telomere DNA and mediates telomere elongation.

Telomerase expression in human cells is restricted to specific cell types and developmental stages. Telomerase activity is high in the germline and pluripotent cells and diminished in differentiated cell. In the developing human embryo, telomerase is first expressed in the blastocyst stage, primarily in the inner cell mass to help establish full-length telomeres [48]. As development progresses, the reverse transcriptase is repressed as a function of cellular differentiation [49], with the exception of resident and progenitor stem cell compartments in proliferative tissues including intestinal crypts and hair follicle cells. TERT silencing in somatic cells is thought to exert a tumor-suppressive effect by limiting cellular lifespan of differentiated cells [48,50]. Instead, minimal telomerase activity in stem cells is necessary to counteract telomere erosion and maintain proper tissue function (reviewed in [51]. Mutations in telomerase pathways genes, including TERT and TERC are associated with several short telomere syndromes, including dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson syndrome (HHS), as well as lung fibrosis. Such mutations lead to telomerase insufficiency, telomere loss, and ultimately result in premature exhaustion of stem cell pools [52].

hTERT promoter regulation as a function of pluripotency

Cells of the inner cell mass and the derived human ESCs are unique cellular contexts for telomere dynamics as they possess high levels of telomerase able to significantly extend telomeres. Robust telomerase reactivation was also reported during nuclear reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) [53] and high telomerase activity is essential to sustain telomeres in both murine and human pluripotent cells [54–56]. Tight regulation of telomerase is primarily dictated by the levels of human TERT (hTERT), which is limiting for telomerase assembly. Accumulation of hTERT mRNA is regulated transcriptionally and post-transcriptionally. Several studies identified hTERT cis-regulatory elements and transcription factors associated with hTERT promoter in different cell types. The core hTERT promoter contains binding sites for pluripotency and growth-related transcription factors, including Myc, Klf4, and Sp1 [57]. The effect of transcriptional activation on telomerase expression is most evident in cancer cells, where activating hTERT promoter mutations were first identified in familial and sporadic melanoma and later found to be widespread in many tumors [58]. Cancer-associated hTERT promoter mutations (−124C>T and −146C>T) generate a novel binding motif for ETS/Tcf transcription factors that increases hTERT promoter activity [59] (Figure 2). The activity of the hTERT promoter is further regulated by deposition of both regulatory histone marks and DNA methylation. Paradoxically, while promoter methylation typically leads to gene silencing, hypermethylation of the hTERT promoter is associated with increased hTERT levels [60,61]. The impact of histone marks on telomerase expression was explored in a series of experiments where murine and human TERT regulatory elements were swapped. Interestingly, replacing intragenic regions from mouse introns 2 and 6 with the corresponding human sequences, recapitulated the tight hTERT expression pattern characteristic of human cells, including strict repression upon differentiation [62]. These studies indicate that histone marks at multiple sites within the hTERT locus, including intragenic elements contribute to hTERT regulation.

A critical role for alternative splicing during hTERT accumulation

It has become evident that transcriptional regulation does not fully account for the strict control of telomerase levels in pluripotent vs. differentiated cells. Specifically, deletion of hTERT cis-acting enhancers had little impact on telomerase activity in embryonic stem cells. Engineering cancer-associated hTERT mutations in hESCs did not enhance telomerase activity [16], nor did it prevent telomerase silencing upon differentiation to fibroblasts [63]. Lastly, when positioned upstream of a luciferase reporter gene in differentiated cells that lack detectable telomerase activity, a minimal hTERT core promoter was reported to drive the expression of luciferase reporter gene [64–68]. Collectively, these studies implicated post-transcriptional processes in the regulation of telomerase as a function of pluripotency. Alternative splicing emerged as a key step during the regulation of telomerase. hTERT mRNA is subject to extensive splice with ~16 splice variants reported in multiple cell types including ESCs [69]. Common and well-studied mRNA variants include hTERT α and β splice isoforms formed by exclusion of exon 6 and exons 7–8, respectively. Partial elimination of the reverse transcriptase domain in α/β variants reduced overall telomerase activity. Genetic screening of splicing factors revealed that formation of α/β hTERT splicing isoforms is regulated by binding of regulatory factors Nova1 and Ptbp1 to hTERT intronic sequences [70,71].

Recent work identified a splicing event centered around exon-2 that is critical for the robust accumulation of hTERT mRNA in human pluripotent cells (Figure 2). Notably, hTERT transcripts lacking exon-2 are abundant in somatic cells when telomerase activity is diminished. In contrast, inclusion of exon-2 is predominant in pluripotent cells and is both necessary and sufficient for the accumulation of hTERT transcript. Upon performing a small-scale RNAi screen to uncover regulators of the hTERT splicing, we uncovered a role for the nuclear speckle protein and splicing regulator, SON, in promoting hTERT exon-2 inclusion in pluripotent cells [64]. Of note, both introns 2 and 6/8 are predicted to form stem-loop structures through RNA:RNA pairing that influence hTERT alternative splicing [72]. Structured RNAs in the nucleus are commonly bound by binding proteins with a variety of functions affecting splicing, RNA processing and nuclear export [73]. Despite the accumulation of hTERT mRNA upon inclusion of hTERT exon-2 is promoted, hTERT activity was lower than would be predicted based on mRNA expression alone [64]. These observations potentially implicate other regulatory processes such as nuclear sequestration and translational regulation in determining telomerase activity.

Regulation of human telomerase RNA – hTERC

In contrast to hTERT, which is tightly regulated and low in abundance, hTERC is ubiquitously expressed and highly abundant. Nevertheless, defects in hTERC maturation and modification have severe consequences on the rate of holoenzyme formation and impair telomerase activity. Precursor hTERC molecules contain a 5’ methylguanosine cap (m7G) and a short-encoded tail (< 10 nucleotides) at the 3’-end. hTERC precursors can go through a direct maturation pathway that is poorly understood. Alternatively, hTERC maturation proceeds via an indirect pathway that involves reversible adenylation and acquisition of a tri-methyl cap. hTERC precursors are also oligo-adenylated by PAPD5 (polyA RNA polymerase), a modification that is subsequently reversed by the disease-associated poly(A) ribonuclease (PARN), which promotes hTERC degradation. Mutations in the PARN are associated with DC and compromise telomerase activity in iPSCs derived from DC patients [65]. Reversible oligo-adenylation of hTERC by PAPD5 slows the rate of RNA maturation and allows the accumulation of inactive hTERC that can serve as a buffer against fluctuations in transcription rates in cells [74]. Inhibition of PAPD5 by small-molecule inhibitor in DC patient iPSCs and HSCs was recently shown to increase active hTERC and consequently telomerase activity, leading to stabilization of telomere length [75], thus demonstrating the impact of seemingly small perturbations in telomerase RNA steady-state levels to telomere homeostasis.

Telomerase holoenzyme assembly and recruitment to telomeres

Following TERC maturation and TERT translation, processing and assembly of telomerase holoenzyme is a multistep process that involves many factors and trafficking of telomerase through Cajal bodies prior to reaching its substrate. Following hTERC maturation, a pair of identical complexes of the RNA-binding proteins GAR1-NOP10-NHP2-Dyskerin coat the RNA and stabilizes its secondary structure [46]. Subsequent assembly into a telomerase particle occurs via interactions between hTERT and the template pseudoknot and CR4/5 stem- loop regions of hTERC. This interaction is facilitated by TCAB1 binding to hTERC to induce specific conformational change bringing the CR4/5 and pseudoknot regions into alignment with hTERT [47]. Biochemical experiments and cryo-electron microscopy revealed that the dyskerin complex and TCAB1 are components of the mature telomerase holoenzyme. In addition to its role in promoting catalytic activity, TCAB1 is required for trafficking of telomerase to Cajal bodies. Single particle tracking of hTERC and hTERT in cancer cells enabled the visualization of telomerase as it trafficked through subnuclear sites and found that following its association with RNA-binding proteins, hTERC accumulates in Cajal bodies in a TCAB1 dependent manner. On the other hand, hTERC exit from Cajal bodies is dependent on hTERT [76]. Intriguingly, hTERT itself does not enter Cajal bodies, so it is still unclear where does full assembly of the telomerase holoenzyme occur (Figure 2).

Following its exit from Cajal bodies, telomerase is recruited to the telomere through a two-step recruitment mechanism. First, telomerase accumulation is driven by scanning interactions between the shelterin component TPP1 and hTERT [77,78]. Next, stable association of telomerase with telomere ends is facilitated by binding between the ssDNA telomeric end and the template region of hTERC template [76,78] to facilitate telomere elongation. These observations reinforce previous observations in ESCs that the TEL-patch TERT-interacting domain on TPP1 is required for both steps of telomerase recruitment and activating telomere repeat synthesis, showing modulation of the interaction between TPP1 and TERT alters the telomere-length setpoint in TPP1-mutant ESCs [79]. Interestingly, multiple isoforms of human TPP1 regulate telomere length in ESCs and germ cells [80]. While these isoforms do not seem to alter the processivity of repeat addition, it is possible that they alter telomere length setpoint in ESCs, by modulating the TPP1 - hTERT interaction.

Notably, depletion of TCAB1 and Coilin in cancer cells lead to gradual shortening of telomere length but had limited effects on telomere length in hESCs [81], likely due to high expression of hTERT. Consistent with this idea, TCAB1-depleted HCT116 cancer cells could only maintain their telomere length when hTERT was overexpressed exogenously [81]. These studies highlight significant differences in telomerase dynamics between human ESCs and cancer cells and underscore the need to further explore live-cell imaging of telomerase particles in pluripotent cells.

Concluding Remarks

Telomere length regulation and telomere protection are actively regulated mechanisms that play pivotal role during embryogenesis, tissue homeostasis, and cancer development. The discovery that a novel mechanism of telomere protection is at play during early embryogenesis raise the question of whether similar protective modes exist during tissue homeostasis and cancer development (see outstanding questions box). Furthermore, these findings highlight the need to better understand the mechanistic basis of TRF2-independent mode of telomere protection in ESCs. One can envision that cancer cells hijack these processes to mitigate telomere dysfunction and allow cancer cell proliferation. With regards to telomerase regulation, a previously unappreciated role for alternative splicing is now linked to developmental regulation of hTERT expression. These novel findings underscore a potential therapeutic benefit for targeting splicing to boost hTERT activity in premature aging and other telomere biology disorders.

Outstanding question box.

• Depleting TRF2 and RAP1 in ESCs increases the expression of 2C genes, including endogenous retrovirus and ZSCAN4. What is the underlying mechanism by which telomere binding proteins regulate gene transcription?

• How does ZSCAN4 induction protect chromosome ends from DDR activation and end-to-end chromosome fusions?

• Formation and stabilization of t-loops in pluripotent cells is TRF2-independent. The underlying mechanism of t-loop formation/stabilization remains outstanding.

• Are there adult stem cells or even cancer cells that employ similar telomere protection mechanism as ESCs?

• In addition to splicing, what other post-transcriptional processes regulate telomerase reactivation in pluripotent cells?

• Is hTERT exon2 splicing de-regulated in cancer cells?

Highlights:

• TRF2 is dispensable for telomere protection in pluripotent cells

• Loss of TRF2 in embryonic stem cells triggers a 2C-like stage

• Alternative splicing promotes hTERT mRNA accumulation in embryonic stem cells

• Minor impact of hTERT transcriptional regulation as a function of pluripotency