Telomeric Transcription and Telomere Rearrangements in Quiescent Cells

Review YJMBI-66431; No. of pages: 12; 4C:

Telomeric Transcription and Telomere Rearrangements in Quiescent Cells


phane Coulon 1 and Me
lina Vaurs 1 Ste
e Ligue contre le Cancer, Marseille, F-13009, 1 - CNRS, INSERM, Aix Marseille Univ, Institut Paoli-Calmettes, CRCM, Equipe labellise France


phane Coulon: [email protected] Correspondence to Ste https://doi.org/10.1016/j.jmb.2020.01.034 Edited by Brian Luke

Abstract Despite the condensed nature of terminal sequences, the telomeres are transcribed into a group of noncoding RNAs, including the TElomeric Repeat-containing RNA (TERRA). Since the discovery of TERRA, its evolutionary conserved function has been confirmed, and its involvement in telomere length regulation, heterochromatin establishment, and telomere recombination has been demonstrated. We previously reported that TERRA is upregulated in quiescent fission yeast cells, although the global transcription is highly reduced. Elevated telomeric transcription was also detected when telomeres detach from the nuclear periphery. These intriguing observations unveil unexpected facets of telomeric transcription in arrested cells. In this review, we present the different aspects of TERRA transcription during quiescence and discuss their implications for telomere maintenance and cell fate. © 2020 Elsevier Ltd. All rights reserved.

Quiescence is a common cell state The majority of the cells in adult human body tissues and organs are nondividing post-mitotic cells. In contrast to terminally differentiated cells, such as neurons, stem cells, or memory lymphocytes, can exit quiescence in response to defined extracellular signals. The ability of stem cells to enter and exit quiescence is required for tissue homeostasis and regeneration [1]. The molecular signature of quiescent cells includes low RNA content, a profound change in their transcriptional and epigenetic profiles, and the absence of proliferation markers (for review see Refs. [2e4]). Thus, quiescence is, by definition, a reversible state in which a basal metabolic and transcriptional activity is maintained to sustain cell survival. The budding yeast Saccharomyces cerevisiae enters into quiescence when essential nutrients, usually the carbon source, are exhausted. Similar to human cells, quiescent yeasts are characterized by profound changes in gene expression profile and by dramatically reduced protein synthesis [5]. These changes are accompanied by the reorganization of various cellular organelles and of the entire intracel0022-2836/© 2020 Elsevier Ltd. All rights reserved.

lular structure, reviewed by Sagot and Laporte 2019 [6]. Specifically, the remodeling of the microtubule network leads to extensive reorganization of the nucleus in quiescence that, in turn, modifies the genome organization [7]. Indeed, chromosomes themselves undergo modification in quiescence, including the strengthening of inter-telomere interactions and the dispersion of centromeres [8]. The compaction of chromosomes that represses transcription of certain domains of the chromatin is also a hallmark of arrested cells [8,9]. Thus, the survival of budding yeast during quiescence necessitates substantial changes in various cellular pathways, organelle structures, and topological reorganization of the genome. In the wild, quiescence is also a common cellular state present in all life forms, from unicellular to complex organisms that sustain life in the nondividing state. Furthermore, the control of the equilibrium between proliferation and quiescence clearly determines the fate of each organism in response to environmental changes. Disruption of this control can lead to cancer and other degenerative diseases in humans [10e13]. Investigating how cellular

Journal of Molecular Biology (xxxx) xx, xxx


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quiescence is achieved and maintained will contribute to the understanding of these diseases.

Fission yeast is a key model organism to study quiescence The fission yeast Schizosaccharomyces pombe is an excellent model to study the ability of cells to alternate between proliferation and quiescence, since it readily enters into a nondividing state (quiescence) upon nutritional stress, such as either nitrogen starvation or low glucose concentration [11,14]. Upon glucose deficiency, the S. pombe cells arrest rapidly without a single division. In contrast, upon nitrogen starvation, fission yeast cells divide twice without growth and then arrest synchronously with a haploid genome. This persistent arrest due to lack of nitrogen source, named G0, will be the focus of this review. Unlike rod-shaped vegetative cells, the G0 cells are small and round. In this state, the cells are still metabolically active and remain fully

viable for several weeks [15,16]. By facing these environmental changes, quiescent S. pombe cells modify their transcriptome, reducing the RNA mass by 85% compared to vegetative cells [17]. Although diversity in mRNA is still found in quiescence, mRNAs involved in cell maintenance, such as adaptation to stress and nutrient limitation, become relatively more prevalent, while those required for protein synthesis generally decrease. This highlights striking antagonistic changes in proliferative and quiescent cells, adapted to either cellular growth or maintenance, respectively. Genome reorganization is also a characteristic of quiescent fission yeast cells. Although several studies revealed a specific architecture of chromosomes that influences the transcription program of G1-arrested cells [18,19], it has still not been established as to whether these features are the same for nitrogen-starved cells. However, quiescence establishment is also accompanied by a massive reorganization of cytoskeleton in the absence of nitrogen [20], and in contrast to budding

Fig. 1. Telomere hypercluster localizes to the nuclear envelope in the nitrogen-starved fission yeast cells. (A) The Rabl configuration: the nucleolus is located opposite to the centromeres that are held by the spindle pole body (SPB) and telomeres are maintained at the nuclear periphery. (B) Schematic representation of the nucleus of an S. pombe cell arrested after nitrogen starvation. Rabl configuration is preserved in G0-arrested cells. Telomeres form a single focus that localizes to the nuclear periphery and faces the SPB that gathers centromeres. Telomere hypercluster is positioned in close proximity to the nucleolus. (C) Anchoring of the telomeres to nuclear periphery relies on (i) the interaction between Bqt4, a component of the inner membrane protein complex Bqt3-Bqt4, and the telomeric protein Rap1 and (ii) the interaction between the chromatin remodeler Fft3 that binds to long-terminal repeat (LTR) elements and the inner nuclear membrane (INM) protein Man1. The nuclear envelope is a safe zone that anchors and stabilizes heterochromatin regions and inhibits improper recombination.


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yeast, S. pombe G0-arrested cells retain the Rabl configuration of their chromosomes [21]. In this configuration, initially observed by Carl Rabl in rapidly dividing nuclei of salamanders [22], the nucleolus is located opposite to the centromeres that are held by the spindle pole body (SPB), while most telomeres are maintained at the nuclear periphery (Fig. 1A) [23,24]. More specifically, in quiescent fission yeast cells, the telomeres form a unique cluster that attaches to the nuclear envelope, thus tethering the nucleolus at the nuclear periphery as well (Fig. 1B). Although G0 cells do not divide, the chromosomes of S. pombe cells are still subjected to mutations that threaten genome integrity [25,26]. Mostly, the production of endogenous reactive oxygen species (ROS) by respiration is a source of DNA lesions [27]. For limiting the accumulation of mutations, G0arrested cells use efficient DNA repair mechanisms. In the repair of double-strand DNA breaks (DSBs), the nonhomologous end-joining (NHEJ) pathway prevails over homologous recombination (HR) in quiescence since haploid cells lack the sister chromatids, and hence, homologous sequences for repair by HR [28,29]. This bias toward NHEJ in quiescent cells is also a feature of mammalian cells that spend most of their time in the G1 phase [30e32].

Telomeres in S. pombe Telomeres are nucleoprotein structures that protect chromosome ends from degradation. Telomeric DNA consists of G-rich repetitive sequences, ending in a 30 single-stranded overhang (G-tail). These sequences are bound by telomeric proteins that form a complex named Shelterin. The function of Shelterin is to protect the natural chromosome ends by repressing DNA repair pathways and controlling the telomerase-based telomere maintenance [33,34]. Telomeric proteins and their related functions are evolutionarily conserved among eukaryotes, including S. pombe, which telomere-bound proteins exhibit as a shelterin-like structure [35]. Terminal sequences in fission yeast are 300 bp long and encompass a degenerated telomere repeat (G26TTAC[A]). The Taz1 protein specifically binds to duplex DNA, Pot1 recognizes the G-tail, and the four telomeric proteins Tpz1, Rap1, Poz1, and Ccq1 bridge Taz1 to Pot1 through a network of proteinprotein interactions [36,37] (Fig. 1C). The telomerase holoenzyme includes the catalytic subunit Trt1, the regulatory subunit Est1, and the TER1 RNA [38,39]. In yeast and other eukaryotes, telomerase activity maintains telomere homeostasis to counteract the natural telomere attrition that occurs at each cycle of DNA replication [40]. In the absence of

telomerase activity, telomeres progressively shorten until they reach a critical size when the cells either permanently arrest or die [41e44]. This arrest, caused by activation of DNA damage checkpoint resulting from deprotection of short telomeres during replicative senescence, is called a crisis [45,46]. In rare cases, cells eventually recover from growth by elongating telomeres through either reactivation of telomerase or alternative recombination-based mechanisms [47,48]. In fission yeast, survivors emerge after approximately 100 generations, either by circularizing their chromosomes or by activating alternative telomerase-independent pathways of telomere maintenance [44,49]. The shelterin complex also ensures the localization of telomeres to the nuclear periphery in cycling cells. Indeed, the six telomeres of S. pombe form 1 to 3 foci within the nucleus that are tethered to the nuclear membrane through the interaction between Rap1 and Bqt4, a component of the inner membrane protein complex Bqt3-Bqt4 [50] (Fig. 1C). Although the function of telomere positioning is not well defined in fission yeast, tethering of telomeres to the perinuclear region stabilizes the subtelomeric heterochromatin and is thought to create a specialized environment that prevents collisions between transcription and replication machinery [51,52]. Studies in budding yeast also revealed that tethering of telomeres at the nuclear periphery suppresses recombination at chromosome ends [53,54]. Thus, in a general manner, attachment of chromosomes ends to the nuclear membrane is likely to contribute to telomere stability. Subtelomeric regions contain a mosaic of multiple segments of homologous sequences that span ~50 kb of the telomere-proximal DNA region. The subtelomeric elements 1 (STE1), 2 (STE2), and 3 (STE3) contain centromere-homologous sequences (cenH) that are located several kilobases away from TERRA TSS (Fig. 2) [55e57]. The cenH sequences are embedded in tlh genes (tlh1 and tlh2). These sequences are part of the open reading frames of putative telomere-linked helicases (Tlh) of the RecQ family. Expression of the tlh genes is induced in cells undergoing telomere crisis caused by the loss of telomerase [58] but seems to be repressed under nitrogen starvation [17,59]. Subtelomeres are heterochromatinized regions, where histone 3 methylation at lysine 9 (H3K9me) is highly enriched, and heterochromatin protein 1 (HP1) homologs are associated. Two mechanisms are responsible for establishing heterochromatin at subtelomeres, the RNA interference (RNAi) machinery that uses cenH sequence as a template, and the Shelterin (Fig. 3). The RNAi machinery recruits the methyl-transferase Clr4-complex (CLRC), which methylates the H3K9 for heterochromatin establishment [56]. H3K9me serves as a binding site for the


Coulon and M.e
Vaurs, Telomeric Transcription and Telomere Rearrangements in Quiescent Cells, Please cite this article as: S.e Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2020.01.034

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Fig. 2. Organization of the subtelomeric regions in S. pombe. Schematic illustration of the chromosomes I, II, and III. The number of subtelomeres varies from four to six per haploid genome, depending on the presence or absence of subtelomeric sequences between the telomeres and the rDNA repeats at the ends of chromosome III. Zoom into the Subtelomeric region of chromosomes I and II. Subtelomeric regions contain a mosaic of common segments. The telomereproximal sequences contain subtelomeric-elements 1 (STE1), 2 (STE2), and 3 (STE3) and the centromere-homologous sequences (cenH) that are embedded within tlh1/2 genes encoding the putative telomere-linked helicases. The homologous repeated sequences (HRS) contain the TERRA transcription start site.

heterochromatin proteins (Swi6, Chp1, and Chp2; HP1 family). Chp2 plays an important role in the recruitment of SHREC, a histone deacetylase complex (HDAC), that participates in the heterochromatin establishment by ensuring a low level of histones 3 and 4 acetylations (a transcriptional activation histone mark) [60e63]. Thus, hypermethylation and hypoacetylation of histone tails at subtelomeres contribute to telomere silencing. The shelterin complex also promotes heterochromatin assembly through its association with Clr4-complex. This is mediated by the Ccq1 protein [64], which also cooperates with Taz1 to recruit SHREC [63]. The anchoring of subtelomeric regions to the nuclear membrane is another important condition for transcriptional silencing. The interaction between Man1, an inner nuclear membrane protein, and the chromatin remodeler Fft3 that binds to long terminal

repeats within subtelomeric regions, creates chromatin domain boundaries and anchors subtelomeric chromatin to the nuclear envelope (Fig. 1C) [65]. Thus, heterochromatin assembly at the chromosome ends relies on several protein complexes to ensure the establishment of the silent histone marks. Natural attrition of telomeres is a progressive process that is linked to cell division. However, telomere shortening has also been observed in nondividing differentiated somatic cells, e.g., in certain brain regions and in skeletal muscle [66,67]. One possible explanation is that G-rich telomeric DNA repeats can be damaged by ROS in these postmitotic cells. This could, in turn, trigger the DNA repair mechanisms and provoke telomere attrition [68e70]. Whether it happens in post-mitotic or quiescent stem cells, these observations raise the question of how telomeres are preserved in


Coulon and M.e
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Telomeric Transciption in Quiescent Cells

Fig. 3. Heterochromatin establishment at telomeres in S. Pombe. The establishment of heterochromatin at telomeres relies on the RNA interference (RNAi) machinery and the Shelterin. The cenH transcription by the RNA Polymerase II leads to the production of double-stranded RNA (dsRNA) that is processed into small interfering RNA (siRNA) by Dicer (Dcr1). siRNAs are loaded into RITS complex (Ago1), guiding it back to the complementary nascent RNA (transcribed by RNA Polymerase II), where it directs the deposition of H3K9me2 through the histone methyl-transferase Clr4 (CLRC). H3K9me serves as a binding site for the heterochromatin proteins (Swi6 and Chp2). SHREC, a histone deacetylase complex (HDAC), participates in the heterochromatin establishment by keeping the acetylation at a low level. The Shelterin promotes heterochromatin assembly through its association with CLRC. This is mediated by the Ccq1 protein, which also recruits SHREC.

quiescence. We previously showed that intact telomeres are maintained in quiescence and that the viability of wild-type S. pombe cells is not affected over time [71]. More recently, we assessed the localization of telomeres in G0-arrested cells [21]. We found that these cells mainly display a single telomeric cluster anchored to the nuclear envelope. As in vegetative cells, the tethering to the nuclear periphery depends on Bqt4 protein. Furthermore, this unique cluster is localized next to the nucleolus facing the spindle pole body (SPB), where centromeres are attached to the nuclear envelope through kinetochores. These observations show that G0-arrested fission yeast cells maintain a Rabl organization in which telomeres form a single cluster (Fig. 1A). This particular organization likely participates in the protection of telomeres and the maintenance of their stability in quiescence.

Telomeric transcription in S. pombe In humans and budding yeast, telomeres are transcribed into a long noncoding RNA named TERRA [72e74]. Transcription of TERRA starts within the adjacent subtelomeric sequences and terminates randomly within telomeric repeats so that TERRA transcripts contain a variable number of

telomeric G-rich sequences. In fission yeast, chromosome ends produce a variety of telomeric transcripts in addition to TERRA (Fig. 4A) [75]. This includes ARIA, which is made exclusively of C-rich telomeric repeats, and two antiparallel RNA species (ARRET and aARRET), which are transcribed from subtelomeres and are devoid of telomeric sequences. Rap1 and Poz1 telomeric proteins are known to negatively control the TERRA level [75,76], whereas telomere shortening induces the expression of TERRA, likely due to the decreased occupancy of Rap1 and Taz1 proteins at short telomeres [77,78]. Experimental evidence confirms the model in which only limited amounts of TERRA are produced at long telomeres due to low H3K9ac at TERRA promoters, limiting the RNA polymerase II-mediated transcription, while the level of TERRA increases at critically short telomeres, characterized by higher H3K9ac, [76,77]. TERRA transcripts from humans and mice are partially polyadenylated [72,74]. In budding yeast, TERRA is also polyadenylated, although conflicting observations were reported regarding its level [73,79]. In fission yeast, only a subset of these TERRA molecules is polyadenylated, which may protect them from degradation. Strikingly, these polyadenylated TERRA molecules that are characterized by the presence of very short telomeric tracts


Coulon and M.e
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Telomeric Transciption in Quiescent Cells

Fig. 4. Telomeric transcriptome and subtelomeric rearrangements in fission yeast. (A) Schematic representation of the S. pombe telomeric transcripts. The subtelomeric region is shown as a light blue line, and telomeric repeats are represented by a dark blue line. TERRA transcription start site (TSS) and homologous repeated sequences (HRS) are located within the subtelomeric elements 1 (STE1). The RNA Polymerase II associates with chromosome ends to produce G-rich TERRA molecules and subtelomeric RNA species transcribed in the direction opposite to TERRA (ARRET). Moreover, C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (aARRET) are also transcribed. Rap1 and Poz1 negatively control transcription at chromosome ends. Upon telomere shortening, a fraction of TERRA with short telomeric repeats is polyadenylated. This polyadenylated TERRA can physically interact with telomerase. (B) Possible mechanism for STEEx formation in quiescent S.pombe cells. Resection at unprotected telomeres might occur in quiescence generating a recombinogenic 30 -overhang within the telomere proximal HRS. Homology search and strand invasion by HR machinery might be facilitated by the presence of TERRA R-loop within the distal HRS. Break induced replication (BIR)-related process might allow D-loop migration, and the lagging-strand synthesis might then ensure duplication of the subtelomeric block. The reiteration of this process might lead to STEEx formation.


Coulon and M.e
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Telomeric Transciption in Quiescent Cells

at their 3 ’ end can associate with telomerase in a way that is independent of telomerase RNA (Fig. 4A) [77,80]. According to the model proposed by the group of P. Chartrand in budding yeast [81,82], the interaction of telomerase with polyadenylated TERRA would promote telomerase recruitment and elongation of those telomeres from which the TERRA molecules originated. TERRA molecules can also be engaged in the formation of RNA-DNA hybrids at telomeres, and those structures called telomeric R-loops that may inhibit telomerase action [73]. In yeast and human telomerase-negative cells, TERRA may target these telomeric R-loops in homology-directed repair [83e86]. Since the discovery of TERRA [72,74], its evolutionary conserved function has been confirmed, and its involvement in telomere length regulation, heterochromatin establishment, and telomeric recombinational repair has been demonstrated [87e89].

Telomeric transcription in G0 As mentioned above, when fission yeast cells enter into quiescence, transcription is highly reduced. Nevertheless, we observed that in the wild-type quiescent cells, the level of telomeric transcription, and in particular, the level of TERRA, is higher than that in cycling cells. This intriguing observation raises the question as to why and how the cells maintain transcription at telomeres in quiescence. This is an unresolved issue. A part of the answer may come from our findings that TERRA transcription promotes the rearrangements of eroded telomeres in quiescent cells [71]. Indeed, when the cells arrest in G0 with short telomeres due to the lack of telomerase activity, the STE1 subtelomeric region is duplicated at high frequency in the total absence of cell division. We named these events, STEEx for STE1-Expansion (Fig. 4B). STEEx increases with telomere shortening and time in quiescence. They are promoted by HR in a Rad51-dependent manner and are likely controlled by checkpoint pathways [71,90]. Duplication of subtelomeric sequences primarily occurs internally in cis and then propagates to other chromosome extremities in trans. Importantly, STEEx is initiated at homologous repeated sequences (HRS) flanking the STE1 telomeric region. In the absence of DNA replication, the initiation of subtelomeric rearrangements was surprising. The observation that TERRA is enhanced when telomere length decreases and the fact that HRS contains the transcription start site of TERRA (TSS) clearly linked TERRA to STEEx. Indeed, entry into quiescence exacerbates the fact that telomere attrition induces telomeric transcription. Furthermore, TERRA accumulates over time in quiescence and correlates with subtelomeric ampli-

fication. Strikingly, the absence of RNAseH1 exacerbates STEEx formation. Although we do not have a formal proof that stable RNA-DNA hybrids are formed in quiescence, we hypothesize that TERRA molecules engaged in telomeric R-loops promote telomere reorganization, as it stimulates recombination in proliferating cells lacking telomerase activity [85]. We present a possible mechanism for STEEx formation in which R-loop formed by TERRA invasion promotes duplication of a subtelomeric block through a process that is related to breakinduced replication (BIR) (Fig. 4B). Interestingly, we uncovered a mode of telomere repair mechanism specific to post-mitotic cells. However, it is not clear as to why the cells recombine their eroded telomeres in quiescence. Most importantly, rearranged telomeres prevent cells from properly exiting quiescence. Indeed, STEEx cells either die in G0 or fail to re-enter the cell cycle [71]. Thus, STEEx may appear as a proliferative barrier for quiescent cells, and then, a parallel can be drawn between the crisis in replicative senescence and STEEx [90]. In this context, we can speculate that cells maintain a certain level of transcription at telomeres in quiescence to keep them on alert. Indeed, if terminal sequences are either damaged or eroded, this basal transcription would trigger extensive erosion and lead to STEEx formation. Thus, telomeric transcription may serve as a standby mode in quiescence to keep cells, with intact telomeres, in a poised state in response to environmental signals. An intermediary state has been proposed for stem cells and T-lymphocytes and is referred to as G-alert and G0(A), respectively [91,92], and it might be possible that telomere stability is involved in this case as well. Another interesting observation we made is that in the absence of Bqt4, transcription of TERRA is highly upregulated in quiescent cells [21]. As mentioned above, the Rap1-Bqt4 interaction tethers telomeres to the nuclear envelope, and this anchoring mode is conserved in quiescent cells. This clearly reveals the importance of telomere position in post-mitotic cells and defines the nuclear periphery as a transcription-repressive area. When we provoked the detachment of telomeres from the nuclear envelope in cells harboring short telomeres (in bqt4D cells deprived of their telomerase activity), the transcription reaches an extremely high level. This, in turn, promotes a massive accumulation of STEEx and then further prevents cells from properly exiting the quiescence. In this context, chromosome end positioning seems to exacerbate transcription at eroded telomeres and that way reveals telomeric defects. Therefore, we can propose that in cells harboring intact ends, telomere positioning to the nuclear envelope (i) reduces TERRA transcription, (ii) prevents rearrangements of subtelomeres, and


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then (iii) preserves telomere integrity. This defines the nuclear periphery as a safe zone where recombination-dependent transcription is restricted in post-mitotic cells. These observations reveal the complexity of telomere maintenance mechanism in quiescent cells in which telomere transcription and telomere localization seem to control the ability of cells to exit from quiescence.

Transcriptional regulation of TERRA in quiescence One of our most intriguing observations is likely that, in G0-arrested cells, the telomeric transcription is kept at a higher level than in vegetative cells, while the RNA mass of S. pombe is highly reduced. This raises the question as to how TERRA is regulated in G0. Although this question remains unsolved, some of our data may allow us to understand its function. In proliferative mammalian cells, telomere length, epigenetic state, RNAi, and nonsense-mediated RNA decay seem to regulate the levels of TERRA [93,94]. In proliferating budding and fission yeasts, rDNA decay also regulates the TERRA level [73,87], and telomere length, as well exerts control over the telomere transcript level, as pointed out by a correlation between the TERRA levels and the length of the telomeric tracts [77,86]. More specifically, in S. pombe, the H3K9ac mark is found at critically short telomeres and may then play a role in the upregulation of TERRA [76,77]. Clearly, in quiescent fission yeast cells, as well as in vegetative cells, the shortening of telomeres promotes TERRA transcription [71]. Likewise, it appears that time in quiescence correlates with telomeric rearrangements and also induces telomeric transcripts [71]. Although we have no direct evidence that heterochromatin marks may influence TERRA levels in quiescence, a recent study investigated the genome-wide distribution and the level of H3K9 methylation in fission yeast quiescent cells [95]. It appeared that constitutive heterochromatic regions in vegetative cells become subject to dynamic regulation during cellular quiescence. In particular, within subtelomeric regions, H3K9me2 levels seem to decrease rapidly upon nitrogen removal and to persist over time in quiescence. This is reminiscent of recent observations showing that constitutive heterochromatin, including telomeres, is used to establish the specific G0-transcription program [96]. Thus, it might indicate that the reduction of H3K9me2 contributes to the positive regulation of telomere transcription in quiescence. However, only a subset of transcripts (subtelomeric transcripts but not TERRA) was found to be upregulated in proliferating cells lacking Swi6 and Clr4 proteins [78]. Further investigations will be required to assess heterochromatin status in quies-

cent cells, such as the determination of methylation and acetylation levels, in order to understand its connection with telomeric transcription. We will also need to establish how these histone marks are deposited, particularly if they differ from those in vegetative cells. By studying telomere organization in quiescence, we also found that fission yeast retains a Rabl conformation in which the telomeric cluster is tethered to the nuclear periphery next to the nucleolus where the ribosomal DNA is gathered. This surprising observation led us to think that the proximity of telomeres to the nucleolus influences telomeric transcription [18]. Indeed, the RNA polymerase I-dependent rDNA transcription is significantly reduced in G0 and correlates with an increase of H3K9 methylation [96,97]. This regulation is necessary to adjust the active/silent rDNA repeat ratio, and this is controlled by RNA interference (RNAi) in which Dicer prevents extensive heterochromatin formation [13,97]. As mentioned above, the RNAi machinery is also involved in telomeric heterochromatin formation [56]. Thus, in quiescence RNAi controls H3K9me level, increasing it at rDNA and possibly reducing it at telomeres [95,97], two genomic regions that are found in close proximity in quiescence [21]. In this context, we may hypothesize that the methyl-transferase Clr4, the histone deacetylase complex, and the RNAi components are titrated away from the telomeric regions to the nucleolus in G0-arrested fission yeast cells. This hypothesis is confirmed by the observation that Clr4dependent H3K9me and RNAi proteins play an important role in the regulation of rDNA in G0 by using the constitutive heterochromatin to repress transcription [96]. Thus, we may speculate that the displacement of Clr4-dependent RNAi factors responsible for H3K9 methylation, from telomeres to nucleolus, could establish rDNA silencing and consequently promote TERRA transcription at chromosome termini in G0-arrested cells. Although the regulation of TERRA transcription in quiescence remains largely uncharacterized, these observations indicate that the length and the localization of telomeres may both have an impact on heterochromatin status of subtelomeric regions. Therefore, the establishment and the dynamics of histone marks may certainly control the global transcription at telomeres in quiescent cells.

Concluding remarks In this review, we focused on the transcription at telomeres related to noncoding RNA, namely TERRA. Because subtelomeric regions also contain tlh genes that are repressed under nitrogen starvation [17,59], this raises the question as to how the cell differently controls the transcription of


Coulon and M.e
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Telomeric Transciption in Quiescent Cells

subtelomeres (tlh genes) and telomeres (TERRA). Further investigations in this area will be required to determine the subtleties of the control of transcription at chromosomes ends in vegetative and quiescent cells. Work in budding yeast clearly established that entry into quiescence is associated with the substantial reorganization of various components of cellular machinery and organelles. The genome also undergoes massive topological rearrangements, including chromosome condensation and telomere clustering. This is also a characteristic of the fission yeast Schizosaccharomyces pombe, in which the telomeres form a unique focus at the nuclear periphery, nearby the nucleolus. This specific position correlates with an elevated transcription, which itself may impact the telomere maintenance and cell fate if the terminal sequences are eroded. We may then wonder if transcription of telomeres is a consequence of the profound reorganization of the genome or is an integral part of the G0-transcription program. This pending question deserves close attention. In mammals, including humans, quiescent cells include adult stem cells. Features of quiescent adult stem cells that comprise changes in their metabolism, gene expression, epigenetic, and cell cycle regulation, contribute to the quiescence maintenance, self-renewal, and proliferation [98]. In addition, these cells share some architectural characteristics of quiescent yeast cells, such as condensed chromosomes and cell size reduction. So far, telomere localization in quiescent cells has not been investigated in mammals. Interestingly, it has been reported that quiescent hematopoietic stem cells with short telomeres have an elevated tolerance to accumulating genomic alterations, which transmission to progenitor cells is prevented by senescence and apoptosis [99]. In the light of these findings, telomere stability and telomere localization, as well as TERRA transcription in quiescent stem cells, need to be addressed in the future.

Acknowledgment We are very grateful to Benjamin Roche and Dmitri Churikov for reading the manuscript and critical
li for continuous comments. We thank Vincent Ge and generous support. SC is supported by “Agence Nationale de la Recherche” ANR-16-CE12-0015 TeloMito, l’“Association de la Recherche sur le Cancer (ARC) Projet Fondation ARC” and
ropo ^le-PACA Emergence”. “Cance Received 18 October 2019;

Received in revised form 29 January 2020; Accepted 30 January 2020 Available online xxxx Keywords: telomere; quiescence; TERRA; fission yeast

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