# TELS1 stabilizes t-loops independently of TRF2 and controls telomere length in pluripotent cells

Joey Dufourd, Thi Hai Yen Huynh, Sandrine Sauzet, Quynh Huong Lai, Francesco Abagnale, Sihem Zitouni, Marie-Josèphe Giraud-Panis, Serge Urbach, Eric Gilson, Mathieu Tardat, et al.

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## To cite this version:

Joey Dufourd, Thi Hai Yen Huynh, Sandrine Sauzet, Quynh Huong Lai, Francesco Abagnale, et al. TELS1 stabilizes t-loops independently of TRF2 and controls telomere length in pluripotent cells. Cell Reports, 2025, 44 (9), pp.116260. 10.1016/j.celrep.2025.116260. hal-05360233

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**Cell Reports**

# TELS1 stabilizes t-loops independently of TRF2 and controls telomere length in pluripotent cells

## Graphical abstract

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## Authors

Joey Dufourd, Thi Hai Yen Huynh, Sandrine Sauzet, ..., Eric Gilson, Mathieu Tardat, Jérôme Déjardin

## Correspondence

mathieu.tardat@igh.cnrs.fr (M.T.),
jerome.dejardin@igh.cnrs.fr (J.D.)

## In brief

Dufourd et al. describe a pluripotent cell-specific mechanism for telomere protection involving t-loop regulation and ubiquitin signaling. Their work reveals how telomeres can remain protected despite the loss of canonical loop structures, highlighting alternative strategies for chromosome end protection in mouse stem cells.

## Highlights

* TELS1 stabilizes telomere loops in mouse embryonic stem cells

* Linear telomeres remain protected from DNA damage signaling in mESC

* Telomere loops restrict telomerase access to chromosome ends

* UBR5 prevents DNA damage signaling at telomeres lacking t-loops in mESC

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## Article

# TELS1 stabilizes t-loops independently of TRF2 and controls telomere length in pluripotent cells

Joey Dufourd,<fnref n="1" />,<fnref n="5" /> Thi Hai Yen Huynh,<fnref n="1" />,<fnref n="5" /> Sandrine Sauzet,<fnref n="1" />,<fnref n="5" /> Quynh Huong Lai,<fnref n="2" /> Francesco Abagnale,<fnref n="2" /> Sihem Zitouni,<fnref n="1" /> Marie-Josèphe Giraud-Panis,<fnref n="2" /> Serge Urbach,<fnref n="3" /> Eric Gilson,<fnref n="2" />,<fnref n="4" /> Mathieu Tardat,<fnref n="1" />,<fnref n="5" />,* and Jérôme Déjardin<fnref n="1" />,<fnref n="6" />,*

## SUMMARY

Telomeric loops (t-loops) are thought to protect chromosome ends, with their stabilization generally requiring the shelterin protein TRF2. However, the mechanisms operating in pluripotent cells remain unknown. Here, we identify TELS1 as a TRF2-independent t-loop stabilizer in pluripotent cells. TELS1 binds single-stranded, G-rich telomeric DNA tracts likely present within duplex telomeric regions and promotes strand invasion in vitro, consistent with a direct role in t-loop formation. When targeted to telomeres in differentiated cells, TELS1 is able to substitute for TRF2 in making the t-loop, which partially protects from ATM activation. In TELS1-deficient pluripotent cells, telomeres lack t-loops but remain protected and become more accessible to telomerase, resulting in elongation. A genome-wide CRISPR screen identifies *Ubr5* as essential for this tolerance. These findings validate the t-loop as an essential structure for end protection and uncover a telomere protection pathway unique to pluripotent cells that appears to function independently of shelterin.

## INTRODUCTION

Mammalian telomeres terminate with a 3' single-stranded DNA (ssDNA) overhang, serving two main functions: it acts as a substrate for telomerase-mediated elongation and can fold into duplex DNA to form the telomere-loop (t-loop). In most somatic and cancer cells, the shelterin complex is associated specifically with telomeres, safeguarding chromosome ends from unwanted DNA damage responses (DDRs) and repair mechanisms.<fnref n="1" /> Telomere repeat binding factor 2 (TRF2, encoded by the *Terf2* or *Trf2* gene) is a critical component of the shelterin complex. TRF2 stabilizes other shelterin proteins on telomeres (through interaction with the TIN2 and RAP1 shelterins), inhibits DDR at telomeres,<fnref n="2" />,<fnref n="3" /> facilitates telomeric DNA replication,<fnref n="4" /> and is both necessary and sufficient for t-loop stabilization.<fnref n="5" />⁻⁹ Moreover, TRF2 interacts with the MRN complex,⁹⁻¹¹ a DNA damage sensor involved in ATM activation.¹² This interaction inhibits MRN's endonuclease-associated activity and likely also ATM activation.⁹,¹³ Consequently, TRF2 withdrawal leads to multiple outcomes: partial dissociation of other shelterins from telomeres, t-loop disruption, ATM and DDR activation, and ultimately telomere fusions, senescence, or cell death.<fnref n="2" />,¹⁴,¹⁵

By concealing the 3' overhang, the t-loop is a structure proposed to play a central role in end protection by preventing detection by DNA damage sensors and ATM activation.⁸ In particular, the concomitant loss of the t-loop and ATM activation in settings involving TRF2 perturbations represents a key observation supporting the importance of t-loops for protection.¹⁶ However, despite substantial evidence supporting the “t-loop model of end protection,” direct causal proof remains to be fully established. The challenge lies in isolating the role of the t-loop structure from the broader functions of TRF2 and other shelterin in DDR suppression.<fnref n="5" />,¹⁷,¹⁸ A recent preprint¹⁹ describes that TRF2-dependent t-loops could be rescued by artificially inducing the tetramerization of the TRF1 shelterin, a TRF2 paralog that shares the same domain organization but differs in partners and function. In this context, TRF1-induced t-loops effectively suppressed ATM activation and telomere fusions, providing the first evidence that t-loops are structures that play a crucial role in end protection.

T-loops can be directly observed from genomic DNA spreads by electron microscopy after telomeric DNA enrichment⁸ or by super-resolution microscopy using telomere-specific fluorescence *in situ* hybridization.<fnref n="5" /> Both methods require stabilization

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<footnote n="1">¹Institut de Génétique Humaine, CNRS UMR9002, INSERM, Université de Montpellier, Equipe Labellisée la Ligue contre le cancer, 141 rue de la Cardonille, 34000 Montpellier, France</footnote>

<footnote n="2">²University Côte d'Azur, CNRS UMR7284/INSERM U1081, Institute for Research on Cancer and Aging, Nice (IRCAN), Faculty of Medicine, Nice, France</footnote>

<footnote n="3">³Institut de Génétique Fonctionnelle, Université de Montpellier, CNRS, INSERM, Montpellier, France</footnote>

<footnote n="4">⁴Department of Medical Genetics, IHU RespirERA, CHU, Nice, France</footnote>

<footnote n="5">⁵These authors contributed equally</footnote>

<footnote n="6">⁶Lead contact</footnote>

<footnote>*Correspondence: mathieu.tardat@igh.cnrs.fr (M.T.), jerome.dejardin@igh.cnrs.fr (J.D.)
https://doi.org/10.1016/j.celrep.2025.116260</footnote>
of the displacement-loop (i.e., covalent crosslinking of the invading 3' overhang) with psoralen in living cells. These approaches have generally shown that only a minority of telomeric single molecules have resolvable loops, typically ranging from 20% to 50%.⁶,⁸,¹⁶,¹⁷ Those figures do not align well with the absolute requirement of the t-loop in end protection, which would imply that most telomeric DNA molecules conceal their 3' end in a loop at all times. However, they are considered a technical consequence of the experimental conditions, which may break these structures upon fiber spreading or be caused by incomplete psoralen crosslinking of the invading overhang. Alternatively, it may indicate that t-loops are dynamic structures that cannot be captured on all telomeres.

In contrast to our understanding of telomere regulation in fibroblasts or cancer cells, telomere regulation and protection during early pre-implantation development is poorly understood. Intriguingly, this brief developmental window is the only period in the life of somatic cells when telomeres significantly lengthen to reach a maximal size.²⁰ Mouse pluripotent embryonic stem cells (mESCs) employ poorly understood mechanisms for telomere protection and t-loop formation, where TRF2 appears dispensable.²¹,²² This observation further suggests that the t-loop, rather than TRF2, may provide the essential protective function. These findings also imply the existence of an alternative pathway or protein involved in t-loop stabilization during pre-implantation development. Upon losing stemness, TRF2 becomes rapidly crucial for telomere protection, indicating a developmental switch where TRF2 assumes an essential role for protection and t-loop formation, while the mechanism used by pluripotent cells to stabilize t-loops likely phases out.

Here, we identify telomere-loop stabilizing protein 1 (TELS1) as a telomere-specific protein that stabilizes t-loops in pluripotent cells independently of TRF2. Manipulating TELS1 expression allows us to show that even with significantly reduced t-loops, shelterin-proficient telomeres remain protected in these cells. In the absence of t-loops and DDR, the telomeric overhang becomes accessible and bound by telomerase, leading to telomere lengthening. This tolerance pathway, which we propose is integral to the extensive elongation telomeres experience at this developmental stage, is supported by the ubiquitin ligase UBR5, which prevents ATM activation on shelterin-proficient linear telomeres. Our data suggest that t-loops protect chromosome ends from detection by DNA damage sensors, regardless of how they form.

## RESULTS

### TELS1 is a pre-implantation-specific telomere-binding protein

As telomeres still form t-loops in the absence of TRF2 in ESCs,²¹ these cells provide an ideal system to explore t-loop formation and functions independently of TRF2. To investigate how t-loops form in pluripotent cells and to identify potential contributors, we employed a hybridization capture method known as proteomics of isolated chromatin segments (PICh) to isolate telomere chromatin from mouse ESCs and analyze its composition.²³ PICh identified 204 proteins enriched 3-fold over input chromatin across three independent experiments.

Enriched factors included most known telomere interactors, such as shelterin proteins (the most abundant telomere components in PICh), and telomerase (Figures 1A and S1A). Among the uncharacterized factors identified, we focused on zinc fingers and scan domain protein 20 (ZSCAN20) because this protein harbors two Myb domains (Figure 1A). Myb domains are relatively rare in mammalian proteomes, present in only 44 proteins within the annotated mouse proteome, yet they are featured in several known mammalian telomere-binding proteins. These include TRF1,²⁴ TRF2,²⁵,²⁶ repressor activator protein 1,²⁷ and telomere repeat binding bouquet formation protein 1.²⁸ Hence, ZSCAN20 was prioritized. Given that this protein has no currently known molecular function,²⁹,³⁰ we propose renaming it "telomere-loop stabilizing protein 1" (TELS1) for reasons detailed below.

TELS1 expression was observed throughout pre-implantation development and continued upon ESC differentiation, with its mRNA detected in the oocyte, during pre-implantation stages, and in neurons differentiated from ESCs (Figures S1B and S1C). Mass-spectrometric analysis of telomeric protein preparations suggested that TELS1 is present in minute amounts on ESC telomeres (Figure 1A), approximately two to three orders of magnitude less abundant than shelterin subunits but comparable to telomerase levels. Expression of TELS1 fused to green fluorescent protein (GFP) in mouse zygotes (Figure S1D) demonstrated telomere colocalization by TRF1 co-staining. Colocalization by telomere immuno-FISH was almost exclusive in interphase ESCs (Figures 1B and 1C) and comparable to that observed with TRF1-GFP and TRF2-GFP, which were used as references. TELS1-GFP also colocalized with endogenous TRF1, TRF2, and RAP1 (Figure S1E) and was colocalized with TRF1 at most chromosome extremities in metaphases (Figure S1F). We then derived ESC and mouse embryonic fibroblast (MEF) cell lines with a doxycycline-inducible TELS1-GFP-V5 expression transgene, which allowed us to perform chromatin immunoprecipitation using the V5 epitope. This construct reproduced the specific telomeric localization of TELS1 in ESCs, while intriguingly, most of the TELS1 signal was non-telomeric in MEFs (Figure S1G). Chromatin immunoprecipitation confirmed the specificity for telomeric DNA in ESCs vs. MEFs (Figure S1H). Due to the very low abundance of endogenous TELS1 on ESC telomeres (Figure 1A), its detection by immunofluorescence and conventional microscopy was challenging. We, therefore, relied on super-resolution microscopy and 3D reconstructions to monitor the subnuclear localization of TELS1. Signals could be detected as foci on the majority of ESC telomeres, but the majority was at non-telomeric localizations. As a control for antibody specificity, no telomere-specific signal was detected in Tels1 CRISPR KO ESC clones, while most non-telomeric foci remained detectable, suggesting that the telomeric foci observed in WT ESCs were coming from endogenous TELS1 and non-telomeric foci corresponded to background staining (Figures 1D and S1L-S1L for details on the Tels1 KO and antibody characterization). Consequently, we concluded that TELS1 is a bona fide telomere component in pluripotent stem cells, likely associated also at the zygotic stage, but non-telomeric in differentiated cells.
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<figure><img src="image_7.png" /><figcaption>**Figure 1. TELS1 is a telomere-binding protein**</figcaption></figure>

(A) Average mass spectrometry signal intensity for each protein found in PICh and enriched 3 times over input chromatin (average of n = 3 replicates). Proteins are ranked according to their abundance in PICh.

(B) Immuno-FISH experiment using a telomere-specific probe (in red) in ESCs with TRF1-GFP, TRF2-GFP, and TELS1-GFP (in green) and visualized with an apotome microscope. Scale bars are 5 µm. Full z-stack projections are shown.

(C) Percentage of co-localization between GFP constructs and the telomere FISH signal. Average of n = 3 replicates. *p* value and two-way ANOVA, ns: non-significant.

(D) Left: scheme representing z-stack, partial z-stack, and y-stack representations. Right: an immuno-FISH experiment showing the localization of endogenous TELS1 (green) and telomeres (red) in WT and *Tels1* KO ESCs. Partial z-stack projections were obtained by OMX super-resolution microscopy. Percentage of colocalization are given in the merged panels. Average of n = 2 replicates. Scale bars are 5 µm. Quantification of non-telomeric TELS1 foci in partial z-stacks in each cell, from m = 21 and 12 nuclei, respectively. Error bars are standard deviations.

## TELS1 binds to G-rich telomeric ssDNA

Given that TELS1 appeared to be specifically associated with telomeres during pre-implantation stages but not in differentiated cells such as MEFs, it is likely that the protein is not recruited through mere sequence-specific dsDNA binding. We thus investigated the potential mechanisms behind the protein's recruitment. We first characterized the TELS1 interacting proteome to determine if TELS1 interacted with any known telomere-binding proteins, such as members of the shelterin complex, which could explain its localization to telomeres. For this, we used the doxycycline-inducible TELS1-GFP-V5 cell line, which recapitulates exclusive TELS1 localization to telomeres in ESC. While we mostly identified other SCAN domain-containing proteins (Figure 2A), we did not detect the enrichment of shelterins in the TELS1-GFP-V5 interactome.

Considering that TELS1 (ZSCAN20) contains a SCAN domain, this result aligns with previous findings that SCAN domains can homo- or hetero-dimerize³¹ and suggests the specificity of our procedure. To further explore this, we reduced the amounts of the entire shelterin complex at telomeres by partially knocking down both TRF1 and TRF2 using siRNA (Figure S2A). These knock-down conditions were sufficient to trigger significant telomere deprotection (Figure S2B). Despite significantly reduced shelterin proteins (by 70% for TRF1 and TRF2 in the *Tels1* KO background, Figure S2A), endogenous TELS1 was still bound to telomeres. We then hypothesized that telomere repeat-containing RNA (TERRA), produced from telomeres,³² might be involved in recruiting TELS1. To test if TELS1 localization was RNA dependent, we treated cells with RNase A prior to fixation. RNase A treatment was effective
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<figure><img src="image_9.png" /><figcaption>Figure 2. TELS1 binds the G-rich ssDNA</figcaption></figure>

(A) Left: silver stained gel showing proteins recovered from immunoprecipitation of TELS1-V5-GFP in mouse ES cells. Expression of TELS1-V5-GFP was induced by addition of doxycycline in the medium. Three independent replicates are shown. MW kDa: molecular weight in kiloDalton. Right: most enriched proteins recovered from immunoprecipitation with TELS1-V5-GFP. "+" indicates proteins harboring an SCAN domain; TELS1 is also highlighted in red.

(B) Left: immunofluorescence experiments in mouse ES cells expressing TELS1-V5-GFP treated or not with RNAse A (0.1 mg/mL for 30 min) before fixation. Scale bars are 5 µm. Right: quantification of colocalization between TRF1 (red) and TELS1-V5-GFP (green) staining. Average of n = 3 replicates.

(C) Coomassie-stained gel showing *in vitro*-produced GFP-TELS1 after purification from insect cells. MW kDa: molecular weight in kiloDalton.

(D) Left: electrophoretic mobility shift assay (EMSA) of 6xHis-TELS1-GFP with telomeric ³²P-labeled G-strand radioactive probe or ³²P-labeled scramble radioactive probe. Right: quantification of the intensity of the bound and the free fraction. Average of n = 3 replicates.

(E) Left: EMSA of 6xHis-TELS1-GFP with a ³²P-labeled telomeric G-strand radioactive probe without competition or with specific competition (cold telomeric G-strand probe) or non-specific competition (cold scramble probe). Right: quantification of the intensity of the bound fraction normalized by the intensity of bound fraction without competition. Average of n = 3 replicates. Error bars are standard deviations.

since it eliminated the TERRA signal and disrupted the localization of the RNA-binding protein NONO to splicing speckles (Figures S2B and S2C). However, it did not impact TELS1-GFP association with telomeres (Figure 2B), indicating that TERRA was not crucial. In contrast to the MEF telomere proteome,³³ the mESC telomere proteome is enriched in proteins typically found at stressed replication forks.³⁴ This led us to assess TELS1's *in vitro* specificity for non-dsDNA species, including telomeric ssDNA, as these are potentially generated during replication stress. Purified from insect cells (Figure 2C), TELS1 specifically bound to G-rich telomeric ssDNA, with an estimated apparent dissociation constant (Kd) of 120 nM, but did not bind to a probe of the same nucleotide composition but with a different sequence (Figures 2D and 2E). Additionally, TELS1 was inefficient at binding C-rich telomeric DNA and showed inefficient, non-specific binding to double-stranded DNA (dsDNA) *in vitro* (Figure S2D). While the mechanism of TELS1 binding to ES telomeres remains unclear, its specificity for ssG-rich telomeric DNA suggests a specific contribution of this binding to the recruitment mechanism (more details on this below).

## TELS1 stabilizes t-loops independently of TRF2 in pluripotent cells

Super-resolution imaging of ESCs transiently expressing TELS1-GFP, together with TRF1 immuno-labeling to mark telomeres, revealed a profound reorganization of a majority of TRF1 foci into toroids (donut shapes), with TELS1-GFP occupying the center (Figures 3A and 3B). Absence of real co-localization at this resolution is consistent with the lack of interaction between TELS1 and shelterin. TRF1 organization into toroids is rarely observed in non-transfected cells or upon transient expression of TRF2-GFP. These alterations hint at a potential role for TELS1 in the three-dimensional organization of telomeric chromatin. Recent proposals suggest that an unknown protein, distinct from TRF2 but expressed during early developmental stages and bound to telomeres, may be involved in t-loop formation and telomere protection.³⁵ Given that TELS1 overexpression could transform the 3D shape of TRF1 foci into looped structures, we investigated its potential involvement in telomere looping. We used psoralen/UV crosslinking to prepare t-loop spreads⁶,⁸ from wild-type (WT), *Trf2* CRISPR knock-out (KO) ESCs, *Tels1* CRISPR KO ESCs, and double *Trf2*; *Tels1* CRISPR KO ESC
A

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B

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C

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D

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(legend on next page)
clones (see Figures S1I, S1J, and 3A for a detailed description of these KOs). T-loops were visualized by fluorescence *in situ* hybridization using super-resolution microscopy and quantified as described.⁶,¹⁶ T-loop frequencies in WT ESCs (25% on average) were comparable to those obtained by others in fibroblasts⁶ (Figure 3C). In agreement with Ruis et al.,²¹ t-loop frequencies and sizes were unaffected in *Trf2* KO ESCs (Figures 3C and S3B), confirming that t-loops form independently of TRF2 in these cells. However, t-loop frequencies dropped by three to four-fold in *Tels1* KO cells (5 and 8%; two independent KO clones were analyzed), levels comparable to the frequencies seen in *Trf2* KO embryonic fibroblasts, where TRF2 is crucial for t-loop stabilization.⁶,¹⁶ Hence, the loss of TELS1 drastically affected t-loops in ESC, comparable to the significant impact on t-loops observed with TRF2 loss in fibroblasts. *Trf2*; *Tels1* double KO cells also had reduced t-loops. We excluded the possibility that reduced t-loops were due to inefficient psoralen crosslinking of telomere dsDNA in the *Tels1* KO context (Figure S3C). Since the 3' ssDNA overhang is necessary to form the classical t-loop structure, we checked whether *Tels1* KO telomeres had lost their 3' overhang. Using native in-gel hybridization assays, we quantified the amounts of 3' telomeric ssDNA in *Tels1* KO vs. WT cells. We found no significant loss (Figure S3D), excluding this possibility. We then monitored the subnuclear localization of TELS1 deletion constructs fused to GFP in ESCs (Figures 3D and 3E) to identify which region of TELS1 is important for telomere targeting. In addition to the *Myb* and SCAN domains, TELS1 harbors 10 C-terminal zinc fingers, known to bind DNA, RNA, or proteins potentially. The truncated constructs are expressed at comparable levels in ESCs (Figure S3E). Analyzing their relative localization by co-staining with TRF1 revealed that neither the *Myb* domain (TELS1ΔMyb), nor the SCAN domain (TELS1ΔSCAN), nor the first four zinc fingers (TELS1ΔZ1-4) were crucial for telomere targeting. In addition to telomere localization, TELS1ΔSCAN showed a diffuse nuclear distribution, suggesting that the SCAN domain might contribute to exclusive telomere localization. Deletion of the last six zinc fingers (TELS1ΔZ5-10) completely abolished telomere accumulation, implicating this region in telomere targeting. T-loop frequencies were restored to WT levels upon stable re-expression of the full-length TELS1 protein in *Tels1* KO cells. We verified that t-loop rescue was not due to changes in telomere composition due to exogenous TELS1 expression. The

rescue cell line had a normal growth and did not show detectable signs of telomere dysfunction (Figures S3H-S3J). Moreover, we performed PICh to evaluate whether telomere composition was affected in this cell line. While TELS1 was about 20 times more abundant on telomeres in the rescue cell line compared to WT ESCs, these levels had no significant impact on shelterin recruitment and telomere steady state composition (Figure S3G), consistent with normal growth of this cell line. In contrast, t-loops were not restored upon stable expression of TELS1ΔZ5-10, the version of TELS1 that failed to target telomeres and expressed at similar levels as full-length TELS1 (Figures 3F and S3F). This indicated that t-loop stabilization required TELS1 binding to telomeres in pluripotent cells.

## TELS1 favors dsDNA invasion by G- and C-rich ssDNA *in vitro*

Inspired by the large body of work on TRF2, we next tested whether TELS1 was able to stimulate dsDNA invasion by an ssDNA oligonucleotide *in vitro*³⁶,³⁷ (Figure 4A). Since TELS1 can bind to telomeric G-rich strands, we reasoned that this could be mimicked *in vitro* by using a negatively supercoiled plasmid containing a small (103 bp) telomeric sequence insert. Negative supercoiling leads to weakening of the double helix, which transiently creates small regions of melted DNA within the plasmid, momentarily exposing the G-rich strand to which TELS1 could bind. Purified TELS1 fused with a C-terminal FLAG tag and produced in 293T cells (Figure S4A) was first incubated with a negatively supercoiled plasmid (pBB) containing the telomeric sequence, and a 90 nt ssDNA oligonucleotide was subsequently added to the mix (T15G containing 15 G-rich telomeric repeats, T15C containing 15 C-rich telomeric repeats and one sequence, BP19, complementary to another part of the plasmid backbone). TELS1 stimulated stable-strand invasion of both the G- and C-rich strands (Figures 4B and 4D) but not of the oligonucleotide complementary to the backbone (Figure S4B). Telomeric oligonucleotides did not co-migrate with a plasmid that did not contain a telomeric target, suggesting that they bind through sequence specific hybridization. Also, strand invasion was relatively stable after TELS1 acted on the plasmid, since the DNA species are separated and visualized after protein degradation.

Considering that POT1a and POT1b are more abundant than TELS1 at telomeres and exhibit approximately 10 to 20-fold higher affinity for G-rich telomeric ssDNA *in vitro*,³⁸,³⁹ we sought

### Figure 3. TELS1 stabilizes t-loops in pluripotent cells

(A) Co-immunostaining of TELS1-GFP or TRF2-GFP (green) and TRF1 (red) of ESC transfected with a TELS1-GFP or a TRF2-GFP expression construct. Left: full projection image of two neighboring cells in the same field (one non-transfected on top, and one transfected at the bottom) obtained by super-resolution microscopy. Right and top: insets showing a 4× magnification of indicated parts of the left images. Right bottom: 3D reconstitution using the Imaris software of TELS1-GFP or TRF2-GFP distribution (green) relative to TRF1 (red). Scale bars are 5 µm.

(B) Quantification of TRF1 signals having a toroid shape in normal and TELS1-GFP- and TRF2-GFP-expressing cells. m = 11, 10, and 8 nuclei counted for each condition.

(C) Top: cropped examples of single telomere molecules, linear or forming a terminal loop, representative of molecules found in the indicated genetic backgrounds (WT: wild-type). Bottom: quantification of telomere loops. Average of *n* = 3, *n* = 3, *n* = 6, *n* = 3, and *n* = 3 experiments in WT, *Trf2* and *Tels1* KO#1, and *Tels1* KO#2 and *Trf2*; *Tels1* double knockout (D.KO), respectively.

(D) Left: scheme of the tested TELS1 deletions fused in frame with GFP. Right: their subnuclear localization (green) relative to TRF1 (red) used to label telomeres by immunofluorescence. Scale bars are 5 µm.

(E) Graph showing the extent of colocalization of each construct with TRF1. Average of *n* > 7 replicates.

(F) T-loops quantification in *Tels1* KO#1 cell lines stably expressing full-length TELS1 or TELS1Δ5-10. Average of *n* = 3 replicates. *p* value and two-way ANOVA, \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001, and \*\*\*\* *p* ≤ 0.0001. ns: non-significant. Error bars are standard deviations.
<figure><img src="image_17.png" /><figcaption>B</figcaption></figure>

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<figure><img src="image_20.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Figure 4.** TELS1 favors dsDNA strand invasion *in vitro*</figcaption></figure>

(A) Outline of the strand invasion experiment. Red highlights the target of the single-strand oligonucleotides T15G and T15C (in the telomere sequence of 103 bp), and blue, the target of the single-strand oligonucleotide BP19 (in the ampicillin resistance gene). The figure was generated with BioRender.

(B) Representative invasion assay performed with TELS1-FLAG using the T15G and T15C oligonucleotides.

(C) Representative invasion assay performed with hPOT1 using the T15G and T15C oligonucleotides.

(D) Quantification of invasion assays showing the relative ratio of invaded plasmid as a function of the concentration of protein. Error bars are standard deviations from triplicate experiments.

(E) Proposed TELS1 t-looping mechanism scheme.

to verify if POT1 could perform like TELS1 in the strand invasion assay. Hypothesizing that replication stress or transcription can generate transiently open regions within dsDNA *in vivo*, we thought that TELS1 could bind such regions in ESC telomeres, while POT1 could not if too small. Indeed, while POT1 strongly binds the 3' overhang, it cannot stably associate with internal

ssDNA telomeric sequences if too short (2 repeats or less), while it can associate with longer internal telomeric sequences.39

We used human POT1 that recapitulates the function of both mouse paralogs and verified its high affinity for G-rich ssDNA *in vitro* (Figure S4C). We then used hPOT1 in the strand invasion assay. Stable-strand invasion was not increased with hPOT1,
suggesting that, unlike TELS1, hPOT1 is unable to stabilize dsDNA opening within the plasmid to allow strand invasion (Figures 4C and 4D). Hence, like TRF2 but unlike hPOT1, TELS1 alone directly stimulated strand invasion at telomeric DNA, likely by favoring further dsDNA melting upon binding. Based on these data, the results of EMSA, and the tendency for TELS1 to dimerize, we propose a mechanism of t-loop formation in ES cells in Figure 4E. TELS1 binding requires the accessibility of short G-rich ssDNA at discrete regions within the duplex telomeric DNA in ES cells (presumably due to replication stress or transcription of these telomeres) to which TELS1 can bind (short “bubbles” or short-gapped regions). In support of this, super-resolution imaging of telomeric DNA and TELS1 shows that endogenous TELS1 makes discrete contacts with the telomeric DNA signal (see arrowheads in Figure S2A). TELS1 binding may lead to further opening and stabilizing of these opened regions. TELS1 oligomerization might be important to bring the 3' overhang in spatial proximity to the opened dsDNA region, thereby favoring strand invasion and the formation of a D-loop.

## ESC telomeres without t-loops remain protected

Tels1 KO ESCs displayed a mostly unaffected proliferation rate and could be continuously grown in culture for extended times (up to 100 days). These cells also maintained high levels of the pluripotency markers OCT4 and NANOG, showed an unperturbed cell cycle distribution (Figure S5A), and could be differentiated into embryoid bodies. Tels1 KO cells maintained telomere protection, as indicated by negligible increases in γH2A.X accumulations, a DDR marker, on telomeres, also known as telomere dysfunction-induced foci (TIF; Figure 5A).40 A small number of cells in the analyzed population showed increased TIF, but this was also observed in the WT condition. In contrast, Trf2 CRISPR KO ES clones displayed a subtle yet significant activation of the DDR at telomeres, as previously described,22 which did not significantly increase in Tels1 KO cells (Figure 5A). Consistently, Tels1 KO cells did not show an increase in telomere fusions (Figure 5B). The lack of telomere DDR triggered by Tels1 loss might be explained if TELS1 was important for DDR activation. However, this was unlikely since telomere-specific damage was triggered by expressing the TRF1-FokI nuclease41 both in WT and Tels1 KO cells (Figure S5B). Trf2 KO ES clones exhibited a few telomere fusions (around 3%), always involving short chromosomal arms (Figure 5B). Chains of chromosomes fused by their telomeres, like those observed in Trf2 KO embryonic fibroblasts,14 were never seen. Furthermore, double Tels1; Trf2 KO cells did not show increased fusions compared to Trf2 KO cells alone. Thus, in pluripotent cells, deficiencies in TRF2 alone, t-loops alone (i.e., TELS1 depletion), or both combined did not appear to greatly impair chromosome-end protection.

## T-loops control telomerase recruitment

To understand the biological significance of TELS1's association with telomeres, and thus the role of t-loops in mouse ESCs, we used PICH to compare telomere composition in WT versus Tels1 KO ESCs. Although TELS1 loss did not affect the association of shelterin proteins with telomeres (Figures 6A and S6A), in line with the generally unaffected telomere protection in these cells, we observed an increase in telomerase reverse transcriptase

(TERT) association. Given that Tels1 KO telomeres maintained their 3' ssDNA overhangs, which did not form stable t-loops, this increased telomerase recruitment might be due to more accessible 3' overhangs, the natural binding sites for telomerase (Figure 6B). This increased association was confirmed by two orthogonal methods: a TERT-specific chromatin immunoprecipitation (ChIP) showed an increase of telomeric DNA in Tels1 KO cells (Figure S6B), and TERT-TRF1 proximity ligation assays indicated a significantly higher number of telomeres recruiting telomerase in KO cells (Figures 6C and S6C).

In non-pluripotent cellular contexts, telomerase recruitment is affected by several telomeric factors,42,43 but the roles of t-loops have never been tested. We analyzed telomerase recruitment to telomeres upon TRF2 removal in immortalized MEFs.14 In this differentiated cell type, stable t-loops strictly depend on TRF2. TRF2 withdrawal triggers a stereotypical cascade of events16: t-loops are first disrupted concomitantly with ATM activation, activation of DDR, overhang resection, and eventual telomere fusions by non-homologous end-joining (NHEJ). We observed transient telomerase recruitment to an increased number of telomeres 24 h after Trf2 KO induction, which completely disappeared by 48 h, as the DDR was activated at telomeres (Figures 6D and S6D). T-loops were disrupted 24 h after Trf2 KO induction (Figure S6E), consistent with increased telomerase recruitment at this time point. Telomere overhangs started disappearing only after 48 h, likely due to DDR activation and overhang resection (Figure S6F). Altogether, these findings suggested that t-loops restricted telomerase access to telomeres, independently of how t-loops are stabilized (by TELS1 or by TRF2), and that DDR activation by ATM might inhibit telomerase recruitment to linear telomeres.

Considering telomerase's known protective role in Saccharomyces cerevisiae, where telomeres were not shown to form t-loops,44,45 and its proposed protective functions in human cancer cell lines,46,47 we investigated whether telomerase played a protective role in Tels1 KO cells where telomeres tend to adopt a linear conformation that favors telomerase binding. Knocking out telomerase in both WT and Tels1 KO ESCs using CRISPR (Figures S6G and S6H for KO description) did not significantly affect TIF levels (Figure S6I), indicating that neither telomerase nor t-loops contribute significantly to telomere protection in pluripotent cells. Finally, we tested whether telomerase recruitment was productive in ESCs and could lead to telomere lengthening. In vitro established ESC populations have extremely long telomeres, measuring changes in telomere length is challenging. Since telomeres that long cannot be measured using a PCR approach or long read sequencing, we used a telomere restriction fragment assay and pulse-field gel electrophoresis. Using this method, a sufficient amount of time in culture is required to register resolvable shifts in telomere size due to telomerase elongation. We selected a WT and a Tels1 KO ESC clone with TRF2 of comparable sizes (about 75 and 85 kb, respectively) and monitored size changes over ~50 days. While there was no measurable telomere lengthening in WT cells within that period, Tels1 KO cells showed progressive telomere elongation (500–700 bp/day, Figure 6E), and this elongation was telomerase dependent (Figure S6J). This underscores the role of TELS1 in stabilizing mESC t-loops, which control telomerase recruitment and lengthening activity.
<figure><img src="image_22.png" /><figcaption>**Figure 5. T-loop loss does not trigger the DNA damage response in mouse ES cells**</figcaption></figure>

(A) Left: images of telomere DNA damage in the indicated cell lines. Full z-stack projections are shown. Scale bar represents 2 µm. “TIFs” outset shows an example of a TIF in WT siPOT1a cell. Right: quantification of telomere dysfunction-induced foci (TIFs) represented as the percentage of TRF1 foci colocalizing with γH2A.X in the indicated cells. Average of *n* = 3 replicates; a minimum of 50 nuclei counted per replicate. Graph representing the percentage of TRF1 foci colocalizing with γH2A.X in each nucleus in WT, *Tels1 KO#1* cells, *Trf2 KO* cells, and *Trf2 KO; Tels1 KO* cells. Average of *n* = 3 replicates.

(B) Typical telomere FISH performed on metaphase spreads obtained from the indicated genotypes. DAPI is pseudo-colored in red; telomere FISH in green. White numbers are the average percentage values of telomere fusion/metaphase. From 2 replicates; *m* > 12 metaphases per replicates. *p* value and two-way ANOVA, \* *p* ≤ 0.05, \*\* *p* ≤ 0.001. ns: non-significant. Error bars are standard deviations.

**UBR5 supports a robust tolerance mechanism for shelterin-proficient linear telomeres in ESCs**

We used well-characterized MEF cells to test whether TELS1 could substitute for TRF2 in promoting t-loops and protecting chromosome ends. We engineered MEF lines with a hydroxyta-

moxifen-inducible *Trf2 KO*¹⁴ to express doxycyclin-inducible TELS1. Since TELS1 does not bind MEF telomeres effectively on its own, we enhanced TELS1's telomere binding by fusing the C-terminal end of TELS1 to the zinc fingers 9-11 from ZBTB48/TZAP, a domain known for its specific binding to
A

<figure><img src="image_23.png" /></figure>

B

<figure><img src="image_24.png" /></figure>

C

<figure><img src="image_25.png" /><figcaption>TRF1-TERT</figcaption></figure>

ESC

D

<figure><img src="image_26.png" /></figure>

MEF Trf2<sup>F</sup>- CreErt2

<figure><img src="image_27.png" /></figure>

Trf2 KO
induction

E

<figure><img src="image_28.png" /></figure>

(legend on next page)
telomeric dsDNA.48,49 This fusion significantly improved TELS1 recruitment to MEF telomeres, whereas TELS1 alone exhibited very little telomere binding, consistent with our earlier findings (Figure S7A). However, exploring for optimal experimental conditions, we noticed that inducing moderate or high expression of the TELS1<sup>29-11</sup> fusion led to weak CHK1 activation and telomere entanglements (Figures S7B and S7C), a feature we hypothesized was likely linked to the ability of TELS1 to favor strand invasion. TELS1 overexpression did not affect telomeres in MEF, while it induced their entanglements in ESCs (Figure S7C). Hence, large amounts of TELS1 on telomeres were toxic regardless of the cell type or the telomere-targeting mechanism. Consequently, in t-loop rescue experiments in *Trf2* KO MEFs, TELS1<sup>29-11</sup> fusion expression had to be maintained at very low levels (using 5 ng/mL of doxycyclin, instead of the usual 1 µg/mL concentration) to prevent toxicity and likely ATR activation (Figure S7C). As shown earlier, t-loops were disrupted 24 h after TRF2 KO induction in MEF. However, even 72 h after *Trf2* removal, t-loops were maintained at normal levels upon minute expression of the TELS1<sup>29-11</sup> fusion (Figure S7D), suggesting that, as seen in ESCs, low amounts of telomeric TELS1 are sufficient to promote t-loop stabilization also in MEFs. Interestingly, t-loop levels were also slightly restored to significant levels in non-induced TELS1<sup>29-11</sup> fusion cells, likely due to the leakiness of the doxycycline-inducible promoter used (Figure S7B). In fact, t-loop levels increased with higher doxycycline induction and increased TELS1<sup>29-11</sup> expression (Figure S7D), suggesting a TELS1-dependent dose effect on t-loop stabilization. We concluded that minimal amounts of telomeric TELS1 can stabilize t-loops in MEFs without TRF2. TELS1<sup>29-11</sup>-dependent t-loops were indistinguishable in size and appearance from TRF2-dependent t-loops in MEFs (Figure S7E), suggesting rescue of this structure. We then analyzed the timing and extent of ATM activation and the DDR response following TRF2 withdrawal to measure the protective contribution of TELS1<sup>29-11</sup>-dependent t-loops. ATM activation and the telomeric DDR levels were unchanged at 72 h of *Trf2* KO induction upon t-loop rescue (Figure S7F). We reasoned that ATR activation by TELS1<sup>29-11</sup> might obscure our interpretation of telomeric DDR in the context of *Trf2* KO. Therefore, we performed the same rescue experiments in the presence of ATM or ATR inhibitors. As expected, ATM inhibition drastically suppressed TIFs in both parental and TELS1<sup>29-11</sup> rescued MEFs. In contrast, while ATR inhibition had no significant effects on TIFs in the *Trf2* KO context, TIFs were significantly reduced in the *Trf2* KO MEFs rescued by TELS1<sup>29-11</sup>. This suggested that, provided TELS1<sup>29-11</sup> toxicity

is kept in check by ATR inhibition, the t-loops it forms has a significant contribution to end protection. Moreover, TELS1<sup>29-11</sup> partly reduced telomere fusions associated with TRF2 loss in MEFs (Figure S7H). Hence, the absence of a DDR at telomeres in *Tels1* KO mESCs suggested the presence of a stem cell-specific tolerance mechanism for shelterin-proficient linear telomeres. This mechanism appears robust enough to suppress the ATM activation typically observed in differentiated cells when shelterin-proficient telomeres lose their t-loops. To investigate this, we conducted a genome-wide CRISPR dropout screen using the “Brie” library.⁵⁰ Our goal was to identify genes whose inactivation specifically impairs *Tels1* KO ESCs with linear telomeres, without affecting WT ESCs that maintain looped telomeres (Figure 7A). Both telomere configurations maintain a comparable level of shelterin (Figure 6A). Dropout genes were analyzed with MAGeCK-MLE.⁵¹ The screen proved robust, as known essential genes in ESCs were depleted at similar rates in both WT and *Tels1* KO cells (Figure S7I). Importantly, none of the genes encoding for shelterin were synthetic lethal or even significantly more toxic to *Tels1* KO vs. WT ESCs (Figure S7I), suggesting that individual shelterin are not involved in TELS1 function. We considered genes as synthetically lethal in the *Tels1* KO background if they exhibited an essentiality score (β-score) above −0.4 in WT cells and below −0.8 in *Tels1* KO cells. Only two genes met these criteria: *Exoc1*, involved in vesicular trafficking, and *Ubr5*, encoding an E3 ubiquitin ligase implicated in various cellular processes, including DNA damage signaling. Given *Ubr5*'s known association with the DDR, we focused our subsequent analyses on this gene. Validation using siRNA-mediated knockdown of *UBR5* confirmed its important role: *Tels1* KO ESCs exhibited growth arrest upon *UBR5* depletion by siRNA, whereas WT mESC growth remained largely unaffected (Figures S7J and S7K). Notably, *UBR5* knockdown in *Tels1* KO cells led to significant telomere deprotection, as evidenced by increased γH2A.X telomere dysfunction-induced foci, a phenomenon not observed in the WT background (Figure 7B).

Interestingly, the deprotection phenotype in *Tels1* KO cells upon *UBR5* knockdown was not accompanied by 53BP1 recruitment or telomere fusions. Telomere DDR signaling appeared predominantly ATM dependent (Figure 7C), indicating that while *UBR5* depletion in *Tels1* KO cells activates ATM signaling presumably due to telomere linearity, it does not trigger downstream DDR signaling or repair mechanisms. This likely reflects the continued presence of shelterin components at these linear telomeres.

Figure 6. TELS1 and TRF2-dependent t-loops control telomerase recruitment

(A) Representative replicate of a PICh performed in WT vs. *Tels1* KO#1 cells. Proteins are ranked according to their relative abundance in PICh.

(B) Model scheme of WT vs. *Tels1* KO telomeres.

(C) Proximity ligation assay (PLA) measuring spatial proximity (in red) between telomerase (TERT) and TRF1, used as a telomere marker. Left: typical nuclei from WT, *Tels1* KO, *Tert* KO, and *Tels1*; *Tert* DKO are shown. Right: quantification of the number of specific PLA signals in WT vs. *Tels1* KO cells. Average of *n* = 3 replicates.

(D) Proximity ligation assay measuring spatial proximity (in red) between telomerase (TERT) and TRF1, upon deletion of *Trf2* in mouse embryonic fibroblasts (MEFs) at the indicated time points. Left: typical nuclei and PLA signal from MEFs WT and after *Trf2* KO are shown. Right: quantification of the number of specific PLA signals at the indicated time points. Average of *n* = 3 replicates.

(E) Telomere length analysis by terminal restriction fragment (TRF) analysis in WT and *Tels1* KO#1 mouse ESCs at the indicated time points. Top: telomeric DNA is probed with a ³²P-labeled [CCCTAA]₆ probe. Bottom: ethidium bromide staining of DNA after TRF. *p* value and two-way ANOVA, *p* ≤ 0.05. ns: non-significant. Error bars are standard deviations.
<figure><img src="image_29.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

(legend on next page)
# DISCUSSION

## A TRF2-independent pathway for t-loop stabilization

Our study uncovers a developmentally regulated, TRF2-independent mechanism for t-loop stabilization mediated by TELS1. This discovery challenges the prevailing model where TRF2 is central to t-loop formation and suggests that alternative mechanisms exist, particularly during key developmental windows. Notably, pre-implantation development, a brief period of significant telomere elongation in somatic cells, may depend on this pathway. This positions TELS1 and t-loops as central regulators of early telomere dynamics.

## T-loops in telomere length regulation during pre-implantation development

T-loops were historically hypothesized to serve three roles: end protection, telomerase-independent elongation, and regulation of telomerase access.<sup>8</sup> While their protective function has been studied in detail, their role in telomere length control has never been directly demonstrated in cells. Our findings provide the first functional evidence that t-loops modulate telomerase recruitment, regardless of whether they are stabilized by TRF2 or TELS1. This control is likely exerted through sequestration of the 3' overhang, aligning with the original hypothesis of their involvement in length regulation. Intriguingly, TELS1 localizes to telomeres as early as fertilization, a stage where telomerase-independent elongation has been proposed.<sup>52</sup> As embryos reach the morula stage, telomeres lengthen rapidly via telomerase. This suggests a model where t-loops may be absent or transiently destabilized, enabling elongation until blastocyst-stage TELS1 binding reinstates t-loop formation, restricting further telomerase access. These insights open avenues for revisiting early embryonic telomere biology.

## Linear telomere damage tolerance in mESCs requires UBR5

In mouse ESCs, shelterin proficient telomeres can remain linear yet avoid triggering full DDRs, despite being ATM competent.<sup>21,22</sup> Our study identifies the E3 ligase UBR5 as a key factor in this tolerance mechanism. This is particularly striking given that UBR5 promotes telomere fusions in differentiated cells lacking TRF2.<sup>11,53</sup> Hence, UBR5 plays fundamentally different roles depending on cell identity, underscoring the distinct nature of telomere protection in pluripotent cells. The fact that linear telomeres in ESCs signal damage through ATM but evade fusion suggests that TELS1-mediated t-loops are the primary protective strategy in UBR5-deficient mESCs. The interplay between TELS1 and UBR5 thus reveals a unique, stem cell-specific end-protection mechanism.

## Shelterin-independent evidence for the t-loop model of end protection

Contributions of t-loops to end protection have mainly been inferred from TRF2 depletion or mutations in its TRF homology domain (TRFH), which might affect its dimerization or tetramerization, recruitment of telomere-associated factors, and telomere-binding stability. Hence, fully demonstrating that t-loops are protective structures has remained challenging using TRF2 alone. Our data confirm the t-loop model established for TRF2: in mESCs, TELS1 forms t-loops that efficiently protect from ATM activation, provided the tolerance pathway supported by UBR5 is suppressed. In MEF, TELS1 can make t-loops in the absence of TRF2 and does also seem to confer partial protection from ATM activation. In *Tels1* KO mESCs deficient for UBR5, linear telomeres, despite likely being TRF2 proficient, elicit a DDR, mirroring the “intermediate state” of telomeres seen in cancer and senescent cells.<sup>54</sup> Thus, our findings establish t-loops as bona fide protective structures, independently of shelterin.

## Comparative analysis of TRF2 and TELS1 t-loops

Although TELS1 (ZSCAN20) and TRF2 are structurally unrelated and recruited to telomeres through distinct pathways, the t-loops stabilized by each exhibit strikingly similar features: comparable frequency, morphology, and functional outcomes, including telomerase exclusion and suppression of ATM activation. This strongly suggests that both proteins converge on a common structural mechanism: effective masking of the 3' G-rich overhang.

TRF2-dependent t-loops require high local concentrations of TRF2 and rely on TRF2's ability to modify DNA topology, specifically by inducing negative supercoiling, to facilitate strand invasion and loop formation. However, despite being abundantly bound to telomeres in mESCs, TRF2 fails to generate t-loops in this context. We propose that this failure might stem from the presence of small single-stranded gaps within the telomeric duplex. These interruptions likely dissipate topological stress, thereby preventing the formation of stable TRF2-induced t-loops, even when TRF2 is present at high levels.

Paradoxically, these gaps might enable TELS1 recruitment. TELS1 likely stabilizes t-loops by binding to pre-existing and accessible small G-rich single-stranded regions within telomeric chromatin. Although the precise determinants of TELS1 binding await further elucidation, our data suggest that even minimal amounts of TELS1 are sufficient to stabilize strand invasion and loop formation, provided these permissive DNA structures are present. Moreover, TELS1 appears capable of multimerization, which may promote t-loop stabilization, analogous to the role of TRF2 oligomerization. This alternative mode of action, i.e., binding accessible substrates rather than altering topology,

**Figure 7. UBR5 supports tolerance to shelterin-proficient linear telomeres**

(A) Rationale for the synthetic lethal screen performed to identify *Tels1* KO cells vulnerabilities.

(B) Left: microscopy immunofluorescence image of the indicated cell lines for TRF1 (red) and γH2A.X (green) at day 4 after siRNA knockdown. DNA was counterstained with DAPI. Full z-stack projections are shown. Scale bar represents 5 µm. Right: quantification of telomere dysfunction-induced foci (TIFs) represented as number of TRF1 foci colocalizing with γH2A.X per nucleus in the indicated cells. *n* = 2 replicates.

(C) Immunofluorescence experiment showing the accumulation of γH2A.X (green) at telomeres, indicated by TRF1 (red). The indicated cells were treated with ATM inhibitor (250 nM AZD0156, overnight incubation), ATR inhibitor (1 µM VE822, for 1 h), and mock (DMSO, overnight) as control. Error bars are standard deviations.
may explain why TELS1, but not TRF2, can promote t-loop formation in mESCs, offering a powerful new model to investigate t-loop dynamics independently of shelterin.

## Limitations of the study

Some mechanistic insights in this study were obtained using tagged TELS1 constructs that we could not achieve to express at similarly low levels as the endogenous protein. Although we confirmed that, when weakly induced, these constructs localize similarly to the endogenous protein and do not overtly perturb telomeric chromatin composition and protection, we acknowledge that overexpression may introduce non-physiological effects or promote interactions not occurring under native conditions. Additionally, while we establish that TELS1 is required for t-loop formation in mouse embryonic stem cells, the precise molecular mechanism by which TELS1 recognizes telomeric DNA and promotes loop formation remains to be elucidated. These aspects will require future dedicated biochemical and structural studies to refine the model proposed here.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jérôme Déjardin (jerome.dejardin@igh.cnr.sfr).

### Materials availability

Unique reagents generated in this paper are indicated in the key resources table and will be available upon request.

### Data and code availability

* The raw proteomics data of this paper have been deposited in the Proteomics IDEntifications (PRIDE) database (https://www.ebi.ac.uk/pride/) under the accession numbers PXD066469, PXD066470, and PXD066260.

* This paper analyzes existing, publicly available data, accessible at Wang et al.⁵⁵ and Tippmann et al.⁵⁶

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

The authors would like to acknowledge Bernard de Massy for critically reading the manuscript and Evgeniy A. Ozonov and Antoine H.F.M Peters for providing resources for the RNA-seq analyses of pre-implantation embryos. We would like to thank Francois Dossin and Ludovic Deriano for the kind gift of the CAS9 vectors used in the synthetic lethal screen. We acknowledge the imaging facility MRI of the France-Bioimaging national infrastructure supported by the French National Research Agency (ANR-10-INSB-04, "Investments for the Future"), and the facilities of the Montpellier Proteomics Platform (PPM, BioCampus Montpellier). Work in the J. Déjardin lab is supported by the ERC (CoG grant METACHROM), the ANR (Earlytel), the Labex EpiGenMed, and by La Ligue contre le cancer, "program equipe labelisee." J. Déjardin is also a recipient of a Chaire d'Excellence (ENDLOOP) from the France 2030 initiative. The E.G. lab is supported by the ANR (Earlytel) and the Inserm cross-cutting program AgeMed (from Aged Cells to Medical Applications).

## AUTHOR CONTRIBUTIONS

M.-J.G.-P. Q.H.L., F.A., and E.G. performed initial strand invasion experiments. J. Dufourd, M.T., S.Z., S.S., T.H.Y.H., and J. Déjardin performed the other experiments and analyzed the results. Bioinformatic analyses were performed by J. Dufourd. S.U. performed MS analyses. J. Dufourd, M.T., M.-J.G.-P., S.S., and Y.H. prepared the figures. J. Déjardin wrote the manuscript with input from the others.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

During the preparation of this work, the author(s) used ChatGPT in order to improve language. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Animals and mouse embryo culture

* Ethical statement

* Cell culture

* METHOD DETAILS

* Plasmids and mRNA generation

* mRNA microinjection

* Immunofluorescence and microscopy

* FISH on metaphase spreads

* Fluorescence-activated cell sorting

* Immunoblotting

* TELS1 pull-down assay

* *In vitro* production of proteins

* Band shift and strand invasion assays

* Antibodies

* Chromatin immunoprecipitation and slot blotting

* siRNA RNA knockdown

* RNA extraction and RT-qPCR

* PICh

* Psoralen crosslinking and chromatin spreading for t-loop assays

* Probes radiolabeling

* Analysis of ss telomeric 3' overhang and total telomeric DNA

* CRISPR-Cas9 screen

* Clonogenic survival assay

* QUANTIFICATIONS AND STATISTICAL ANALYSES

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.116260.

Received: November 21, 2024

Revised: July 3, 2025

Accepted: August 19, 2025

Published: September 10, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Rabbit monoclonal anti-ATM</td><td>CST</td><td>Cat# 2873; RRID: AB_2062659</td></tr><tr><td>Rabbit polyclonal anti-ATM (phospho S1981)</td><td>R&amp;D System</td><td>Cat# AF1655; RRID: AB_2062935</td></tr><tr><td>Rabbit polyclonal anti-TRF1</td><td>This paper</td><td>Biologics Corp (Indianapolis, USA)</td></tr><tr><td>Rat monoclonal anti-TRF1</td><td>Abcam</td><td>Cat# ab192629; RRID: AB_3675954</td></tr><tr><td>Rabbit polyclonal anti-TRF2</td><td>T. De Lange</td><td>N/A</td></tr><tr><td>Rabbit monoclonal anti-TRF2</td><td>CST</td><td>Cat# 13136; RRID:AB_2722641</td></tr><tr><td>Rabbit polyclonal anti-ZSCAN20 (TELS1)</td><td>Sigma</td><td>Cat# AV33522; RRID: AB_1859027</td></tr><tr><td>Mouse monoclonal anti-FLAG M2</td><td>Sigma</td><td>Cat# F1804; RRID: AB_262044</td></tr><tr><td>Mouse monoclonal anti-GFP</td><td>Roche</td><td>Cat# 1181440001; RRID: AB_390913</td></tr><tr><td>Mouse monoclonal anti-gamma H2A.X</td><td>Millipore</td><td>Cat# 05-636; RRID: AB_309864</td></tr><tr><td>Rabbit monoclonal anti-gamma H2A.X</td><td>CST</td><td>Cat# 9718; RRID: AB_2118009</td></tr><tr><td>Rabbit monoclonal anti-CHK1 (phospho S345)</td><td>CST</td><td>Cat# 2348; RRID:AB_331212</td></tr><tr><td>Mouse monoclonal anti-CHK2</td><td>Millipore</td><td>Cat# 05-649; RRID: AB_2244941</td></tr><tr><td>Rabbit polyclonal anti-CHK2 (phospho T68)</td><td>CST</td><td>Cat# 2661; RRID:AB_331479</td></tr><tr><td>Rabbit polyclonal anti-KAP1 (phospho S824)</td><td>Bethyl</td><td>Cat# A300-767A; RRID: AB_669740</td></tr><tr><td>Mouse monoclonal anti-PCNA</td><td>Millipore</td><td>Cat# MABE288; RRID: AB_11203836</td></tr><tr><td>Rabbit monoclonal anti-p54nrb</td><td>Abcam</td><td>Cat# ab133574; RRID:AB_3675483</td></tr><tr><td>Mouse monoclonal anti-V5</td><td>Thermo Fisher</td><td>Cat# R960-25; RRID: AB_2556564</td></tr><tr><td>Mouse monoclonal anti-a-TUBULIN</td><td>Sigma</td><td>Cat# T9026; RRID:AB_477593</td></tr><tr><td>Goat anti-Rabbit Alexa Fluor™ 568</td><td>Thermo Fisher</td><td>Cat# A-11011; RRID: AB_143157</td></tr><tr><td>Goat anti-Rabbit Alexa Fluor™ 488</td><td>Thermo Fisher</td><td>Cat# A-11008, RRID:AB_143165</td></tr><tr><td>Goat anti-Mouse Alexa Fluor™ 488</td><td>Thermo Fisher</td><td>Cat# A-11001; RRID: AB_2534069</td></tr><tr><td>Goat anti-Rat Alexa Fluor™ 568</td><td>Thermo Fisher</td><td>Cat# A-11077; RRID: AB_2534121</td></tr><tr><td>anti-Phospho P53 (Ser15)</td><td>Cell Signaling</td><td>Cat# 12571; RRID:AB_2714036</td></tr><tr><td>anti-UBR5</td><td>Cell signaling</td><td>Cat# 65344; RRID:AB_2799679</td></tr><tr><td>anti-OCT4</td><td>Santa Cruz</td><td>Cat# sc-5279; RRID:AB_628051</td></tr><tr><td>anti-NANOG</td><td>Abcam</td><td>Cat# ab80892; RRID:AB_2150114</td></tr><tr><td>anti-SOX2</td><td>Abcam</td><td>Cat# ab15830; RRID:AB_443255</td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody</td><td>Cell signaling</td><td>Cat# 7076; RRID:AB_330924</td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell signaling</td><td>Cat# 7074; RRID:AB_2099233</td></tr><tr><td>anti-TERT IgM antibody</td><td>Novus Biologicals</td><td>Cat# NB100-317; RRID:AB_2201588</td></tr><tr><td>Goat anti-mouse IgM</td><td>Abcam</td><td>Cat# ab102445; RRID:AB_10710440</td></tr><tr><td>Control IgG rabbit</td><td>Abcam</td><td>Cat# ab171870; RRID:AB_2687657</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>MAX Efficiency™ DH10Bac™ Competent Cells</td><td>Gibco</td><td>Cat# 10361012</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Colcemide™ KaryoMAX™</td><td>Gibco</td><td>Cat# 15212012</td></tr><tr><td>ATM inhibitor (AZD0156)</td><td>CliniSciences</td><td>Cat# NB-64-35525; CAS:1821428-35-6</td></tr><tr><td>ATR inhibitor (VE822)</td><td>gift from Jihane Basbous</td><td>CAS: 1232416-25-9</td></tr><tr><td>Lipofectamine 3000</td><td>Invitrogen</td><td>Cat# L3000015</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>Cat# 13778150</td></tr><tr><td>Hygromycin</td><td>Sigma-Aldrich</td><td>Cat# H3274</td></tr><tr><td>Puromycin</td><td>Gibco</td><td>Cat# A1113803</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Blasticidin</td><td>Invivogen</td><td>Cat# Anti-bl-1</td></tr><tr><td>Leukemia inhibitory factor</td><td>This paper</td><td>N/A</td></tr><tr><td>DMEM, high glucose, GlutaMAX™<br>Supplement, pyruvate</td><td>Gibco</td><td>Cat# 31966047</td></tr><tr><td>Fetal calf serum (MEF)</td><td>Eurobio</td><td>Cat# CVFSVF00-01</td></tr><tr><td>Fetal bovine serum (ES)</td><td>Sigma</td><td>Cat# F7524</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>Cat# 15140122</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>Cat# 25030081</td></tr><tr><td>β-mercaptoethanol</td><td>Gibco</td><td>Cat# 11528926</td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Gibco</td><td>Cat# 11140035</td></tr><tr><td>Gelatin from cold water fish skin</td><td>Sigma</td><td>Cat# G7041</td></tr><tr><td>Opti-MEM I Reduced Serum Medium GlutaMAX</td><td>Life technologies</td><td>Cat# 51985026</td></tr><tr><td>Psoralen</td><td>Sigma</td><td>Cat# P8399</td></tr><tr><td>Vectashield without DAPI</td><td>Eurobio</td><td>Cat# H-1000</td></tr><tr><td>DAPI Solution</td><td>Thermo Scientific</td><td>Cat# 62248</td></tr><tr><td>Dynabeads protein G</td><td>Life Technologies</td><td>Cat# 10004D</td></tr><tr><td>ChromoTek GFP-Trap® Magnetic Agarose</td><td>Proteintech</td><td>Cat# gtma-20</td></tr><tr><td>LightCycler® 480 SYBR Green I Master</td><td>Roche</td><td>Cat# 4887352001</td></tr><tr><td>TALON® Metal Affinity Resin</td><td>Clontech</td><td>Cat# 635502</td></tr><tr><td>Sephacryl® S-400 HR</td><td>Cytiva</td><td>Cat# GE17-0609-01</td></tr><tr><td>Protein L Magnetic Beads</td><td>Thermo Scientific</td><td>Cat# 88849</td></tr><tr><td>EX-CELL 420 media</td><td>Sigma</td><td>Cat# 14420C</td></tr><tr><td>PerfectHyb™ Plus</td><td>Sigma</td><td>Cat# H7033</td></tr><tr><td>S-Trap micro columns</td><td>Protifi</td><td>N/A</td></tr><tr><td>D-Biotin</td><td>Invitrogen</td><td>Cat# B20656</td></tr><tr><td>Anti-flag M2 affinity gel</td><td>Sigma</td><td>Cat# A2220</td></tr><tr><td>3X FLAG Peptide</td><td>Sigma</td><td>Cat# F4799</td></tr><tr><td>High-Capacity streptavidin agarose</td><td>Thermo Scientific</td><td>Cat# 20361</td></tr><tr><td>Dynabeads™ My One Streptavidin C1</td><td>Invitrogen</td><td>Cat# 65002</td></tr><tr><td>MinElute PCR purification kit</td><td>Qiagen</td><td>Cat# 28006</td></tr><tr><td>Tetracyclin free serum</td><td>SIGMA</td><td>Cat# F2442</td></tr><tr><td>TELS1-FLAG</td><td>This paper</td><td>N/A</td></tr><tr><td>TelC-Alexa488</td><td>PNABIO</td><td>Cat# F1004</td></tr><tr><td>TelC-Cy3</td><td>PNABIO</td><td>Cat# F1002</td></tr><tr><td>Doxycyclin</td><td>Sigma</td><td>Cat# D9891; CAS: 24390-14-5</td></tr><tr><td>Human POT1</td><td>Origene</td><td>Cat# TP316275M</td></tr><tr><td>SeaKem Gold Agarose</td><td>LONZA</td><td>LON50152</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>PLA Duolink® In Situ Red Starter Kit Goat/Rabbit</td><td>Thermo Fisher</td><td>Cat# DUO92105</td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</td><td>Life Technologies</td><td>Cat# C10425</td></tr><tr><td>Human and Mouse Pluripotent Stem Cell Analysis Kit</td><td>BD Biosciences</td><td>Cat# 560477</td></tr><tr><td>SilverQuest™ Silver Staining Kit</td><td>Life technologies</td><td>Cat# LC6070</td></tr><tr><td>SuperScript™ III First-Strand Synthesis System</td><td>Life technologies</td><td>Cat# 18080051</td></tr><tr><td>Nuclear Extract Kit</td><td>Active Motif</td><td>Cat# 40010</td></tr></table>

Deposited data

<table><tr><td>PICh Rescue</td><td>This paper</td><td>PRIDE: PXD066469</td></tr><tr><td>PICH Tels1 KO</td><td>This paper</td><td>PRIDE: PXD066470</td></tr><tr><td>IP-MS TELS1</td><td>This paper</td><td>PRIDE: PXD066260</td></tr></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>Wild-type ES cells (XY)</td><td>Gift from Antoine Peters</td><td>N/A</td></tr><tr><td>Tels1<sup>-/-</sup></td><td>This paper</td><td>N/A</td></tr><tr><td>Trf2<sup>-/-</sup></td><td>This paper</td><td>N/A</td></tr><tr><td>Tels1<sup>-/-</sup>; Trf2<sup>-/-</sup></td><td>This paper</td><td>N/A</td></tr><tr><td>Tert<sup>-/-</sup></td><td>This paper</td><td>N/A</td></tr><tr><td>Tels1<sup>-/-</sup>; Tert<sup>-/-</sup></td><td>This paper</td><td>N/A</td></tr><tr><td>293T</td><td>ATCC</td><td>CRL-3216</td></tr><tr><td>Sf9 INSECT CELLS</td><td>ATCC</td><td>CRL1711</td></tr><tr><td>MEFs SV40T</td><td>ATCC</td><td>CRL-2907</td></tr><tr><td>MEFs Trf2<sup>F/-</sup> CreERT2</td><td>ATCC</td><td>CRL-3317</td></tr><tr><td colspan="3"><strong>Experimental models: organisms/strains</strong></td></tr><tr><td>C57BL/6J OlaHsd mouse</td><td>Envigo</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>Guide sequences for Cas9: Trf2 #1 (ATAGAACTTGAGCACCCAG)</td><td>This paper</td><td>N/A</td></tr><tr><td>Guide sequences for Cas9: Tels1 #1 (GCCACAGCCATCATCTGGCC)</td><td>This paper</td><td>N/A</td></tr><tr><td>Guide sequences for Cas9: Tels1 #2 (CGCGGGAGGGAAAGCGTTGT)</td><td>This paper</td><td>N/A</td></tr><tr><td>Guide sequences for Cas9: Tert #1 (TGACATCTACAGGACCTGG)</td><td>This paper</td><td>N/A</td></tr><tr><td>Guide sequences for Cas9: Tert #2 (GCTACGGGAGCTGTCAC)</td><td>This paper</td><td>N/A</td></tr><tr><td>siTrf1</td><td>Horizon Discovery</td><td>Cat#L-062394-01</td></tr><tr><td>siUbr5</td><td>Horizon Discovery</td><td>Cat#L-048858-01</td></tr><tr><td>siSCR</td><td>Horizon Discovery</td><td>Cat#D-001810-10</td></tr><tr><td>siTrf2</td><td>Horizon Discovery</td><td>Cat#L-048348-01</td></tr><tr><td>Primer for RT-qPCR</td><td>This Paper</td><td>See Table S1</td></tr><tr><td>PICh oligos</td><td>Gauchier et al.<sup>19</sup></td><td>N/A</td></tr><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>pcDNA3.1-EGFPpA</td><td>Gift from A. Peters</td><td>N/A</td></tr><tr><td>pCAG-GW-V5-Hygro</td><td>McHugh et al.,<sup>57</sup> gift from Guttman M</td><td>Cat# 68265; RRID: Addgene_68265</td></tr><tr><td>PB-TRE-EGFP-EF1a-rtTA</td><td>Sauter et al.<sup>58</sup></td><td>Cat#104454; RRID: Addgene_104454</td></tr><tr><td>pSpCas9(BB)-2A-Puro (PX459) V2.0</td><td>Ran et al.,<sup>59</sup> gift from Feng Zhang</td><td>Cat#62988; RRID: Addgene_62988</td></tr><tr><td>pBB</td><td>Bower et al.<sup>60</sup></td><td>Cat#53210; RRID: Addgene_53210</td></tr><tr><td>pFastBac-HTC plasmid</td><td>Invitrogen</td><td>Cat# 10712024</td></tr><tr><td>CRISPR Knockout Library</td><td>Doench et al.<sup>49</sup></td><td>Cat#: 73633; RRID: Addgene_73633</td></tr><tr><td>TRF1-FokI expression constructs</td><td>Gift from Roger Greenberg</td><td>N/A</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>RStudio 4.4.3 (2025-02-28 ucrt) – "Trophy Case"</td><td>Posit</td><td>https://posit.co/</td></tr><tr><td>CellProfiler 4.2.8</td><td>CellProfiler</td><td>https://cellprofiler.org/</td></tr><tr><td>MAGeCK-MLE 0.5.9</td><td>Wang et al.<sup>51</sup></td><td>https://github.com/liulab-dfci/MAGeCK</td></tr><tr><td>FIJI - ImageJ</td><td>ImageJ</td><td>https://imagej.net/</td></tr><tr><td>Imaris</td><td>OXFORD INSTRUMENTS</td><td>https://imaris.oxinst.com/</td></tr><tr><td>MaxQuant</td><td>Cox Lab</td><td>https://www.maxquant.org/</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>ImageQuant™ TL</td><td>Cytiva Europe GmbH</td><td>https://www.cytivalifesciences.com/en/us/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software/imagequant-tl-analysis-software-p-28619?psmenu=2</td></tr><tr><td>ZEN</td><td>ZEISS</td><td>https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html</td></tr><tr><td>Perseus 1.6.15.0</td><td>Perseus Framework</td><td>http://www.perseus-framework.org</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>OMX</td><td>Deltavision</td><td>N/A</td></tr><tr><td>Axioimager Z3 Apotome</td><td>Zeiss</td><td>N/A</td></tr><tr><td>Shandon Cytospin 4</td><td>Thermo Scientific</td><td>N/A</td></tr><tr><td>Misonix</td><td>Delta Labo</td><td>N/A</td></tr><tr><td>Bioruptor</td><td>Diagenode</td><td>N/A</td></tr><tr><td>Rotaphor System 6.0</td><td>Biometra</td><td>N/A</td></tr><tr><td>Typhoon Phosphorlmager</td><td>Amersham</td><td>N/A</td></tr><tr><td>LightCycler 480</td><td>Roche</td><td>N/A</td></tr><tr><td>Kodak BioMax</td><td>Sigma</td><td>N/A</td></tr><tr><td>ChemiDoc</td><td>BioRad</td><td>N/A</td></tr><tr><td>MACSQuant Analyzer 10</td><td>Miltenyi Biotec</td><td>N/A</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Animals and mouse embryo culture

Six-to eight-week-old female mice were superovulated by injection of pregnant mare serum gonadotropin (PMSG, 5U, Alcyon France) and of human chorionic gonadotropin 48 h later (hCG, 5U, Alcyon France). Mouse embryos were generated by natural fertilization. Fertilization occurred at 13–14 h after hCG injection, which was used as a reference time point for sub-staging of zygotes. Embryos were cultured in KSOM medium (Millipore, MR-106-D) covered by paraffin oil (Sigma, M5310) at 37°C with 5% CO2 and 5% O2 air as in.⁶¹

### Ethical statement

All animal experimental procedures were performed in accordance with French legislation, after approval from the local authorities (ethical committee CE036) and authorized by the French Ministry of Education and Research (CEEA-APAFIS: 2270–54384). Experiments including animals at the host laboratory are authorized by an agreement from the French authorities (B 34-172-41).

### Cell culture

Mouse embryonic fibroblasts (MEFs) were grown in Dulbecco's modified Eagle medium (DMEM, Gibco) with 10% (v/v) fetal bovine serum (FBS; Eurobio) supplemented with penicillin and streptomycin. Mouse embryonic stem cells (ESCs) were grown on 0.18% (w/v) gelatin-coated dishes in DMEM with 4.5 g/L glucose, 15% (v/v) FBS (Sigma), penicillin, streptomycin, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, non-essential amino acids, and 1 mM sodium pyruvate (Gibco) and leukemia inhibitory factor (LIF). Cells were transiently transfected with the indicated constructs using Lipofectamine 3000 following the manufacturer's instructions (Life Technologies, L3000001). Stable TELS1-V5 wild-type and mutant expressing mESCs were maintained in ES medium supplemented with 100µg/ml Hygromycin. Stable and inducible TELS1-V5-GFP wild-type were maintained in medium containing tetracycline free serum (SIGMA, F2442) and puromycin (2µg/ml for mouse fibroblasts and 1µg/ml for mouse stem cells). MEFs Trf2F/- CreERt2 were purchased from ATCC (CRL-3317). ATM inhibitor (AZD0156) was purchased from CliniSciences (B7822), ATR inhibitor (VE822) was a kind gift from Jihane Basbous. Inhibitor's concentration and incubation time are indicated in the corresponding figure legends.

## METHOD DETAILS

### Plasmids and mRNA generation

cDNAs encoding mouse *Tels1* was amplified from mESCs mRNA extracts and cloned into pcDNA3.1-EGFPpA plasmid (gift from A. Peters, FMI, Switzerland) by Gibson Assembly (New England Biolabs). Mutants of the SCAN, MYB and zinc finger domains of TELS1 were generated with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, E0554S). TELS1-V5 wild-type and mutant
constructs were cloned into pCAG-GW-V5-Hygro by Gateway cloning (Invitrogen, 11789020 and 11791020). pCAG-GW-V5-Hygro was a gift from Mitchell Guttman (Addgene plasmid # 68265). Doxycyclin inducible TELS1-V5-GFP wild-type and TELS1<sup>29-11</sup>-V5-GFP were cloned into PB-TRE-EGFP-EF1a-rtTA (gift from Volker Busskamp, Addgene plasmid # 104454). Zinc fingers 9 to 11 from Human TZAP (ZBTB48) were added by PCR from wild-type TELS1-V5-GFP plasmid. CRISPR/Cas9 guides for mouse *Tels1*, *Trf2* and *Tert* were generated using the pSpCas9(BB)-2A-Puro (PX459) V2.0 (gift from Feng Zhang; Addgene plasmid # 62988). Guide sequences are as follow: *Trf2* #1 (ATAGAACTTGAGCACCCAG), *Tels1* #1 (GCCACAGCCATCATCTGGCC), *Tels1* #2 (CGCGGGA GGGAAAGCGTTGT), *Tert* #1 (TGACATCTACAGGACCTGG), *Tert* #2 (GCTACGGGAGCTGTCAC). TRF1-FokI expression constructs were a kind gift from Roger Greenberg. Plasmids and mutations were verified by DNA sequencing (Eurofins Genomics).

*Tels1* mRNA generation for zygote injections: the *pcDNA3.1-Tels1-EGFPpA* plasmid was first linearized with XhoI-HF (New England Biolabs), and subsequently subjected to *in vitro* transcription using the mMessage mMachine T7 Kit (Ambion, AM1344).

## mRNA microinjection

*Tels1-EGFPpA* mRNA was diluted to the optimal concentration using nuclease-free water (Ambion, AM9937). 2–4 µL mRNA (100ng/µl) was microinjected into the cytoplasm of fertilized C57/Bl6 zygotes using a Leica DMI8 microscope equipped with micromanipulators (Eppendorf, TransferMan 4r) and FemtoJet 4i (Eppendorf). Zygotes were then cultured in KSOM medium plus amino acids (Millipore, MR-106-D) under a 5% CO2 atmosphere at 37°C and fixed at late zygotic stages (PN5).

## Immunofluorescence and microscopy

For mouse ES cells staining, transiently transfected cells were seeded on poly-/-lysine-coated coverslips prior to fixation with 2% PFA in PBS for 15min. Immunolabeling with the indicated antibodies was performed at room temperature for a minimum of 1h in a humid chamber. Then, slides were washed in PBS before incubation with the appropriate fluorescent secondary antibodies (Invitrogen) for 1h at room temperature. Slides were washed with PBS and DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Slides were washed again with PBS before mounting in Vectashield (Vector).

For fluorescence *in situ* hybridization (FISH) staining, slides were post-fixed in 3.7% PFA in PBS for 15min at room temperature. Then, slides were incubated for 20 min in 2x SSC at 72°C followed by (10 min for ES cells and 5min for MEFs) incubation in 0.1M NaOH and then dehydrated with ethanol. Air-dried. DNA was denatured at 82°C (2 min for ES cells and 4min for MEFs) and incubated with 500 nM of the appropriate probe overnight at 37°C. Cells were washed twice with 2x SSC for 20 min at 42°C. Last, slides were washed with PBS, stained with DAPI and mounted as described before.

Proximity ligation assays were conducted with the Duolink In Situ Red Starter Kit Goat/Rabbit (Sigma, DUO92105-1KT), following the manufacturer's instructions with minor modifications. Mouse Telomerase protein was detected with an anti-TERT IgM antibody (Novus Biologicals, NB100-317). For detection of the anti-TERT mouse IgM antibodies, an extra step using a goat anti-mouse IgM was performed (Abcam, ab102445). TRF1 was detected with specific antibodies raised against the mouse protein (this paper, Biologics Corps).

Slides were analyzed using a Zeiss Axiovert Apotome microscope equipped with a 63X Plan Apochromat 1.4 NA oil. Images were processed with the ZEN software (Zeiss). Z-stacks were acquired using a frame size of 1388 x 1040 pixels, a pixel depth of 16 bits, pixel size of 0.10µm and 1 µm z-step. Dapi, Alexa 488, Alexa 568 were imaged using following band-pass filter cubes: 365, 395, 445/50 for DAPI; 470/40, FT 495, 525/50 for Alexa 488, 545/25, 570, 605/70 for Alexa 568).

For mouse embryos imaging, image acquisition was performed with a Zeiss Axio imager Z1 apotome microscope equipped with a 63 x Plan-Apochromat objective (N.A. 1.4) and a Zeiss Axiocam MRM camera. Z-stacks were acquired using a frame size of 1388 x 1040 pixels, a pixel depth of 12 bits, pixel size of 0.10µm and 0.50 µm z-step. Dapi, Alexa 488, Alexa 568 were imaged using following band-pass filter cubes: 365, 395, 445/50 for DAPI; 470/40, FT 495, 525/50 for Alexa 488, 545/25, 570, 605/70 for Alexa 568).

High resolution 3D-SIM imagery was obtained with a Deltavision OMX (Model: 1-U2B836) equipped with a 100x Plan SuperApochromat 1.4 Oil (RI508 and RI512). Working distance is 0.13nm and ND filter (%) = 1. Alexa Fluor 568 (red) signal resolution is approximately 120nm, Alexa Fluor 488 (green) signal resolution is about 100nm.

## FISH on metaphase spreads

Cells were treated with 0.1µg/ml colcemid (Gibco, 15212012) for 3h before harvesting by trypsinization (for mouse ES cells) or shake-off (for mouse fibroblasts). After washing with PBS, cells were incubated in 75mM KCl at 37°C for 15min. 4–5 drops of fixative (3:1 methanol/acetic acid) before centrifugation at 200g for 5min (room temperature). Cells were washed 3 times in fixative solution before resuspension in 0.5-1mL final volume. 60µL were spotted on a microscope slide and air dried for at least 20min before post fixation with 4% paraformaldehyde in PBS for 5minutes. Slides were then washed twice in 2xSSC, 70% ethanol, 100% ethanol and air dried for 20min. DNA was then denatured at 82°C for 10 min and incubated with 500 nM of Cy3-labelled telomeric probe (PNA Bio, F1002) overnight at room temperature. Slides were washed twice with 50% formamide in 2xSSC for 15 min at room temperature. Last, slides were washed with PBS, stained with DAPI and mounted in Vectashield (VectorLabs, H-1000).

## Fluorescence-activated cell sorting

Cell cycle distribution of ESCs was analyzed with the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Life Technologies, C10425) following the manufacturer's instructions. Amount of the pluripotency factors OCT4 and NANOG were analyzed with the
Human and Mouse Pluripotent Stem Cell Analysis Kit (BD Biosciences, 560477) according to the manufacturer's instructions. Cell sorting was performed on a MACSQuant Analyzer 10 (Miltenyi Biotec).

## Immunoblotting

For immunoblot analysis, cells were washed with PBS before lysis in Laemml buffer for total extract. Nuclear extracts were prepared using the Nuclear Extract Kit (Active Motif, 40010) according to the manufacturer's protocol. After measuring protein concentrations with a Bradford assay, equal amounts of protein were resolved by SDS-PAGE, transferred to a nitrocellulose membrane (Biorad), and probed with a primary antibody overnight at 4°C. Membranes were then incubated with the appropriate HRP-conjugated secondary antibodies (1:10 000, Amersham). The immunoreactive bands were detected by chemiluminescence (Thermo Fisher Scientific) with autoradiography films (Kodak BioMax, Sigma) or with a Biorad Chemidoc.

## TELS1 pull-down assay

TELS1-V5-GFP expression was induced with 1µg/ml doxycycline for 24 h. Cells were harvested and nuclear extracts were made as described in the immunoblotting section. TELS1-V5-GFP and associated proteins were isolated with GFP-trap beads (Chromotek). Beads were washed twice with 300mM NaCl and 1% Triton and a final wash with 150mM NaCl. Beads were then resuspended in Laemml buffer and subjected to mass spectrometry for identification of TELS1 associated proteins.

## In vitro production of proteins

For production of TELS1 from insect cells, Tels1 was cloned into the pFastBac-HTC plasmid (Invitrogen) in frame with an N-terminal GFP coding sequence. Bacmids were produced with MultiBac DH10 competent cells (Invitrogen) and screened by PCR. TELS1 protein was expressed in SF9 insect cells using EX-CELL 420 media (Sigma) for insect cells and harvested 72 h post infection. Cell pellet was resuspended in lysis buffer (50 mM NaPi pH 7.5, 300 mM KCl, 1 mM MgCl2, 13% glycerol, 4 mM β-mercaptoethanol, 5 mM Imidazole). Cells were lyzed using a 15 mL Dounce homogenizer. The sample was then diluted at least 5 times and centrifuged at 15000 rpm for 20 min at 4°C. The collected supernatant was incubated for 2 h at 4°C with COBALT Talon Metal Affinity Resin beads (Clontech laboratories). The beads were pre-washed in distilled water and pre-equilibrated in a lysis buffer. Beads were pelleted by centrifugation and washed 3x in washing buffer (50 mM NaPi pH 7.5, 500mM NaCl, 1 mM MgCl2, 13% glycerol, 2 mM β-mercaptoethanol, 5 mM Imidazole, 1 mM PMSF, 0.08 mM Aprotinin, 2 mM Leupeptin, 1 mM Pepstatin A). After the washes, the beads were passed through a gravity column and allowed to settle to the bottom of the column. Beads-bound proteins were eluted in 500µL fractions with elution buffer containing 50 mM NaPi pH 4.5, 500 mM NaCl, 1 mM MgCl2, 13% glycerol, 2 mM β-mercaptoethanol, 40% glycerol, 300 mM Imidazole, 1 mM PMSF, 0.08 mM Aprotinin, 2 mM Leupeptin, 1 mM Pepstatin A. The recovered protein fraction was dialyzed overnight at 4°C against a final storage buffer (dialysis buffer) (50 mM NaPi pH, 150 mM NaCl, 1 mM MgCl2, 2 mM β-mercaptoethanol, 1 mM PMSF). TELS1-Flag was expressed in HEK293T by transient transfection with Lipofectamine 3000 twice. Then, cells were harvested and TELS1-Flag was purified with Flag agarose beads according to the manufacturer's protocol. TELS1-Flag was eluted by incubation with 3xFlag peptides. Human POT1 (NM_015450) produced in 293T cells was purchased from Origene (#TP316275M).

## Band shift and strand invasion assays

Oligonucleotide probes were labeled using a T4 Polynucleotide Kinase kit. A total of 50 pmol of DNA was labeled with γ [32P ATP] by the PNK enzyme (New England Biolabs) for 30 min at 37°C. Prior to use, probes were loaded through a microspin G25 column (GE Healthcare, 27-5325-01). The probes were diluted in HEPES 50mM pH 7.9 and 10 fmol of probes were used for each sample. The TELS1-GFP protein was diluted in a 2X buffer (50mM HEPES pH 7.9, 40mM KCl, 20% glycerol, 1mM ZnCl2, 0.1mg/ml BSA, 3mM DTT) and mixed with the radioactive probe 50/50. After an incubation of 20 min at room temperature, the samples were loaded on a 6% acrylamide gel and run for 40 min at 90V in TBE 0.5X. The gels were dried, exposed to a Phosphor screen and revealed by a Typhoon Phosphor Imager. Signals corresponding to the free oligos were quantified with the Fiji software. Strand invasions: T15G and T15C were labeled using T4PNK with γ [32P ATP], and then loaded on a G25 column. 3nM of radiolabeled probe was incubated in a strand invasion buffer (50mM HEPES, 20mM KCl, 0.5mM ZnCl2, 0.8mM DTT, 0.1mg/ml BSA) for 20 min at RT. In parallel, 0, 100, 200, 300nM of TELS1-FLAG was incubated with 10nM of negatively supercoiled pBB plasmid (Addgene plasmid #53210) during 20 min at RT in strand invasion buffer. Equal volumes of these mixtures were combined (to give a final concentration of TELS1-FLAG of 0, 50, 100, 150nM) and further incubated 15 min at RT. The reaction was then deproteinized with 0.5% of SDS and 1mg/ml of proteinase K during 15min at 37°C. Ficoll loading buffer (final concentration: 2% Ficoll, 1.67mM Tris pH7.5, 0.17mM EDTA, 0.017% SDS) was then added and the samples were separated for 40 min in a small format 1% agarose gel, 0.5X TBE at 100V. The gels were dried, exposed to a Phosphor screen and revealed by a Typhoon Phosphor Imager. The fractions were quantified with the Fiji software.

## Antibodies

Primary antibodies used were, anti-ATM (Cell Signaling, 2873), anti-phospho ATM (S1981) (R&D Systems, AF1655), anti-TRF1 (Biologics Corp, this paper, and Abcam, ab192629), anti-TRF2 (a kind gift from Titia de Lange and Cell Signaling, D1Y5D), anti-TELS1 (ZSCAN20) (SIGMA, AV33522), anti-FLAG M2 (SIGMA, F1804), anti-GFP (Roche, 1181440001), anti-γH2A.X (Millipore, 05-636 and Cell Signaling, 20E3), anti-Phospho P53 (Ser15) (Cell Signaling, D4S1H), anti-Phospho RPA (Ser4/8) (Abcam,
ab87277), anti-Phospho CHK1 (S345) (Cell Signaling, 2248), anti-Chk2 (Millipore, 05-649), anti-phospho CHK2 (T68) (Cell Signaling, 2661, a kind gift from Jihane Basbous), anti-phospho KAP1 (S824) (Bethyl, A300-767A), anti-UBR5 (Cell signaling, 65344), anti-OCT4 (Santa Cruz, sc-5279), anti-NANOG (Abcam, ab80892), anti-SOX2 (Abcam, ab15830, a kind gift from Francis Poulat), anti-PCNA (SIGMA, MABE288), anti-V5 tag (Invitrogen, R960-25), anti-p54NRB (Abcam, ab133574) and anti-αTubulin (T9026, Sigma).

Chromatin immunoprecipitation and slot blotting

Mouse ESCs were fixed with 1% (v/v) formaldehyde (Sigma) for 10 min at room temperature before quenching of the cross-linker with 125 mM glycine. Chromatin was prepared as described previously.⁶¹ Chromatin was sheared with a Bioruptor apparatus (Diagenode) to obtain DNA fragments with sizes ranging between 200 and 800 bp. The lysates were incubated overnight at 4°C with Dynabeads protein G (Life Technologies, 10004D) coupled with the appropriate antibody: anti-TRF1 (Biologics Corp), Control IgG (Abcam, ab171870). For ChIP with the TERT antibody (Novusbio, NB100-317), protein L magnetic beads were used to capture anti-TERT IgM (Life Technologies, 88849). DNA was purified using the MinElute PCR purification kit (Qiagen, Cat#28006) following the manufacturer's instructions.

For slot-blot, immunoprecipitated DNA was denatured at 99°C in 0.4 M NaOH and 10 mM EDTA for 10 min, and spotted on a positively charged nylon Hybond-XL membrane (Sigma, GERPN203B). DNA was then ultraviolet (UV)-cross-linked to the membrane at 0.14 J/cm² and labeled with a telomere radioactive probe (CCCTAA)₆ in PerfectHyb Plus Hybridization Buffer (Sigma, H7033) for 3 h at 42°C. After two washes with 2× SSC 0.1% SDS for 20 min at 42°C, the membrane was exposed for various times (2 h to overnight) to a Phosphor screen. The screen was read by a Typhoon PhosphorImager (Amersham), and the images were analyzed using the ImageJ software. Telomeric enrichment for each protein analyzed was normalized by the signal from the input material.

siRNA RNA knockdown

siRNA knock down was performed using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's instructions. Briefly, 1.10⁶ mouse ES cells were transfected twice with 60pmol of duplex siRNA and collected 24 h after the last transfection for subsequent analysis. siRNA duplexes were purchased from Horizon Discovery: siTrf1 (L-062394-01), siTrf2 (L-048348-01), siUbr5 (L-048858-01), siSCR (D-001810-10).

RNA extraction and RT-qPCR

Total RNA was isolated from mESC using the RNeasy Plus Mini Kit (Qiagen, 74134) following the manufacturer's instructions. RNA amounts were quantified with a Nanodrop and up to 5µg of RNA was used to generate cDNA with the Superscript III (Life Technologies, 18080051). Quantitative PCR were performed with LightCycler 480 SYBR Green I Master (Roche, 4887352001) on a Roche Light Cycler 480 apparatus. Primers were purchased from Integrated DNA Technologies (IDT).

PICh

PICh experiments were performed as described.³³ Purified chromatin samples were digested using S-Trap micro columns (Protifi) following the supplier's protocol. Briefly, after reduction (20 mM dithiothreitol for 10 min at 95°C) and alkylation (40 mM iodoaceta- mide for 30 min in the dark), proteins were digested using 1.5 µg of trypsin (Promega, Gold) for 2 h at 47°C. The peptides from each sample were fractionated (8 fractions) using High pH Reversed-phase Peptide Fractionation Kit (Thermo Fisher Scientific) following supplier's instruction.

Each fraction was analyzed using nano HPLC (Ultimate 3000-RSLC, Thermo Fisher Scientific) coupled to a mass spectrometer (Q Exactive-HFX, Thermo Fisher Scientific) equipped with a nanospray source. Peptides separation was carried out on a Pemap column (0.075 mm × 250 mm, reverse phase C18, Thermo Fisher Scientific) using a gradient from 2 to 25% buffer B (0.1% formic acid in 80% acetonitrile; A = 0.1% formic acid) for 100 min at a flow rate of 300 nL/min, then 25 to 40% in 20 min, and, lastly, 40 to 90% in 3 min. The spectra were acquired in "data-dependent acquisition" mode for wild-type and *Tels1* KO PICh, and in "data-independent acquisition" mode for PICh from rescue cell lines, each cycle composed of a full scan MS in the orbitrap (resolution 120,000) followed by MS/MS scans, for the 20 most abundant precursors, at a resolution of 30,000. Raw spectra were processed using the MaxQuant v2.0.3.0 software, using reference proteome for mouse entries (UniProt, 2022_01, taxon identifier 10029), 250 frequently observed contaminants, and reversed sequences for all entries. Enzyme specificity was set to trypsin/P, and the search included cysteine carbamidomethylation as a fixed modification and oxidation of methionine, acetylation (protein N terminus) as variable modifications. Up to two missed cleavages were allowed for protease digestion. The maximum false discovery rate for peptides and proteins was set to 0.01. Data were analyzed in Perseus (version 1.6.15.0).

Psoralen crosslinking and chromatin spreading for t-loop assays

Crosslinking, spreading of chromatin and detection of individual t-loops were performed as described in.⁶ T-loops were analyzed with a Deltavision OMX equipped with a 100x Plan SuperApochromat 1.4 Oil (RI508 and RI512). Samples were blinded to the scientist making acquisition and counting single molecules. Crosslinking, spreading of chromatin and detection of individual T-loops were performed as described in.⁶ T-loops were analyzed with a Deltavision OMX (Model: 1-U2B836) equipped with a 100x Plan SuperApochromat 1.4 Oil. Working distance was 0.13nm and ND filter (%) = 1. Alexa Fluor 488 (green) signal resolution is about 100nm A minimum of 100 molecules were counted in each replicate, ambiguous molecules were not counted.
## Probes radiolabeling

Telomeric oligonucleotide probes (C or G strands) were labeled using a T4 Polynucleotide Kinase kit. A total of 50 pmol of DNA was labeled with γ-[³²P ATP] by the PNK enzyme (New England Biolabs) for 1 h at 37°C. Prior to use, probes were loaded through a micro-spin G25 column (GE Healthcare, 27-5325-01).

## Analysis of ss telomeric 3' overhang and total telomeric DNA

Analysis of telomeric DNA was performed as described in⁶ with minor modifications. Briefly, 3x10⁶ cells were harvested and embedded in agarose plugs. Cells were digested with proteinase K (1mg/ml) overnight at 50°C. The next day, plugs were washed 3 times in TE with a final wash in TE supplemented with 1mM PMSF. For overhang assays, DNA was digested with 150U of Exol or 150U of RecJf (New England Biolabs) overnight at 37°C to digest 3'-G-rich or 5'-C-rich overhangs respectively. Buffer was replaced with fresh enzyme for an extended 4 h of digestion. Plugs were washed in H2O and twice in TE at room temperature for 1h each before digestion with Mbol (New England Biolabs) for 16h at 37°C. Pulse field gel electrophoresis (Rotaphor 8 Biometra) was used to separate the digested DNA according to the manufacturer's recommendations. Samples were loaded on 1% agarose gels in 0.3× tris-borate-EDTA (TBE) and run at 180 V (120° angle) for 24 h at 13°C. The gel with separated DNA fragments was processed for in-gel hybridization, or transferred on a positively charged nylon membrane by Southern blotting. For Southern blotting, the gel was treated with 0.25 N HCl for depurination for 10 min. The gel was incubated in denaturation solution (0.5 M NaOH and 1.5 M NaCl) for 30 min and in neutralization solution (0.5 M tris (pH 7) and 1.5 M NaCl) for 30 min. DNA was transferred by capillarity overnight in 20× SSC. DNA was UV-cross-linked to the membrane at 0.120 J, air-dried, and hybridized with a telomeric radiolabeled probe (CCCTAA)₆. The membrane was exposed to a Phosphor screen and revealed by a Typhoon Phosphor Imager.

For in-gel hybridization, the gels were dried and prehybridized in Church mix for 1 h at 50°C. Hybridization was performed overnight at 50°C in Church mix with a with a telomeric radiolabeled probes (CCCTAA)₆ to obtain the ss overhang signal. The gel was washed at 55°C three times for at least 30 min each in 4xSSC, once for 30 min in 4x SSC/0.1% SDS, and exposed to a Phosphor screen overnight. After the image was captured, the gel was denatured in 0.5 M NaOH, 1.5 M NaCl for 30 min, neutralized with two 15 min washes in 0.5 M Tris-HCl pH 7.5, 3 M NaCl, prehybridized in Church mix for 1 h at 55°C, and hybridized overnight with the same probe at 55°C to obtain the total telomeric DNA signal. The gel was washed and exposed as above. The ss overhang signal in the native gel was quantified with ImageQuant software and normalized to the total telomeric DNA quantified after the gel had been denatured and re-hybridized with the telomeric probe.

## CRISPR-Cas9 screen

Two independent genome-wide CRISPR knockout screens were conducted in wild-type and *Tels1* KO mouse ES cells constitutively expressing Cas9, using the genome-wide CRISPR Knockout Library (Addgene #73633, provided by the CRISPR'it platform, Institut Curie). The Cas9 expression vector was generously provided by François Dossin (Institut Pasteur). 12 h after puromycin selection, 50 x 10⁶ cells from each cell line were collected as the initial time point, and the remaining cells were cultured for an additional two weeks. During this period, 50 x 10⁶ cells were collected every two days for DNA sequencing. Genomic DNA was extracted, sgRNAs were PCR-amplified using Illumina adapter primers, and sequenced on an Illumina platform.

Analysis of the sequencing reads was performed with MAGeCK.⁵¹ Gene essentiality scores (β-scores) were determined through the MAGeCK-MLE algorithm. The average β-scores were calculated from two independent experiments. The genes are categorized into 3 groups: Neutral genes' β-score is close to 0 in all the cell lines; Essential genes have negative β-scores in all the conditions (-0.4); Synthetically lethal genes have negative β-scores in *Tels1KO* (-0.8), neutral β-scores in Wild-type (between -0.4 and 0.2). Data were plotted using Microsoft Excel.

## Clonogenic survival assay

After two rounds of siRNA transfection, 50 000 cells were seeded and cultivated in gelatin-coated 6 well-plates for 3 more days before fixation. Medium was removed, and plates were gently washed twice with water. After the remaining liquid was removed, cells were stained with 500μL of 0.5% crystal violet staining solution (0.5g crystal violet powder (Sigma-Alrich), 80 mL distilled water, 20 mL methanol) for 20 min at room temperature with gentle shaking. Plates were washed four times with water, then air-dried for 20 min before taking pictures.

## QUANTIFICATIONS AND STATISTICAL ANALYSES

Quantification of colocalizations were performed using the CellProfiler tool. Statistics were calculated with RStudio. Briefly, the process includes homogeneity of variances checks, normality checks as well as omnibus tests before proceeding to corrected (Bonferroni) two-sided *t* tests for multiple group comparisons, normal *t*-tests were used when *k* = 2. Results were considered significant at *p* < 0.05. Error bars are standard deviations.