TRF2 Non-Telomeric Function is Indispensable for Neural stemness

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   Oct 20, 2025

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   Reviewer #1

   Major comments:

   (comment #1)- It is interesting that TRF2 loss not only fails to increase γH2AX/53BP1 levels but may even slightly reduce them (e.g., Fig. S2c and the IF images). While the main hypothesis is that TRF2 loss does not trigger telomere dysfunction in NSCs, this observation raises the possibility that TRF2 itself contributes to DDR signaling (ATM-P, γH2AX, 53BP1) in these cells and that in its absence, cells are not able to form those foci. To exclude the possibility that telomere-specific DDR is being missed due to an overall dampened DDR response in the absence of TRF2, it would be informative to induce exogenous DSBs in …

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   Reply to the reviewers

   Reviewer #1

   Major comments:

   (comment #1)- It is interesting that TRF2 loss not only fails to increase γH2AX/53BP1 levels but may even slightly reduce them (e.g., Fig. S2c and the IF images). While the main hypothesis is that TRF2 loss does not trigger telomere dysfunction in NSCs, this observation raises the possibility that TRF2 itself contributes to DDR signaling (ATM-P, γH2AX, 53BP1) in these cells and that in its absence, cells are not able to form those foci. To exclude the possibility that telomere-specific DDR is being missed due to an overall dampened DDR response in the absence of TRF2, it would be informative to induce exogenous DSBs in TRF2-depleted cells and test DDR competence (e.g., IF for γH2AX/53BP1). In other words, are those NSC lacking TRF2 even able to form H2AX/53BP1 foci when damaged? In addition, it would be interesting to perform telomere fusion analysis in TRF2 silenced cells (and TRF1 silenced cells as a positive control).

   We acknowledge a slight reduction; however, this difference is not statistically significant (Fig S2c,e). We will quantify the levels of DDR markers upon TRF2 loss and exogenous DSBs and include it in the subsequent revision.

   (comment #2)-A TRF2 ChIP-seq should be performed in NSC as this list of genes (named TAN genes in the text) was determined using a ChIP performed in another cell line (HT1080). For the ChIP-qPCR in the various conditions, primers for negative control regions should be included to show the specific binding of TRF2 to the promoter of the genes associated with neuronal differentiation. For example, an intergenic region and/or promoters of genes that are not associated with neuronal differentiation (or don't contain a potential G4). The same comment goes true for the gene expression analysis: a few genes that are not bound by TRF2 should be included as negative controls to exclude a potential global effect of TRF2 loss on gene expression (ideally a RNA-seq would be performed instead). We have performed NSC-specific TRF2 ChIP-seq for an upcoming manuscript, which confirms TRF2 occupancy at multiple promoters of differentiation-associated genes. These data are provided solely for confidential evaluation by the designated reviewers.

   Regarding the ChIP-qPCR control experiments: We thank reviewer for pointing this out, indeed we included controls in our PCR assays as positive (telomeric) and TRF2-nonbinding loci (GAPDH, RPS18, and ACTB, based on HT1080 TRF2 ChIP-seq data) as negative controls. These results were not included earlier for clarity given that we were presenting several ChIP-PCR figures - in response to the comment we have included this now in the revised version (Fig. S3d,e). Gene expression analyses show selective upregulation of the TAN genes upon TRF2 loss (data normalised to GAPDH); whereas negative control genes lacking TRF2 binding (RPS18, ACTB) remain unchanged, ruling out non-specific effects. (Fig S3f,g,j,k).

   -(comment #3) A co-IP should be performed between the TRF2 PTM mutant K176R or WT TRF2 and REST and PRC2 components to directly show a defect of interaction between them when TRF2 is mutated (a co-IP with DNase/RNase treatment to exclude nucleic-acid bridging). The TRF2 PTM mutant T188N also seems to lead to an increased differentiation (Fig. S5a). Could the author repeat the measure of gene expression and co-IP with REST upon the overexpression of this mutant too?

   We confirm that DNase/RNase is routinely included in our pull-down experiments to exclude nucleic-acid bridging, with detailed methodology now elaborated in the Methods section. Not including this in the manuscript Methods was an oversight from our side. Our data demonstrate that only REST directly interacts with TRF2, while TRF2 engages PRC2 indirectly via REST, as also previously shown by us and others (page 6; ref. [62]; Sharma et al., ref. [15]).

   We thank the reviewer for noting the apparent differentiation in Fig. S5a. However, this observation represents rare spontaneous differentiation event and is not statistically significant (as shown in Fig S5b). Consistently, gene expression analysis of the TRF2-T188N mutant shows no significant change in TRF2-associated neuronal differentiation (TAN) genes. Therefore, Co-IP for TRF2-T188N with REST was not done.

   (comment #4) - The authors show that the G4 ligands SMH14.6 and Bis-indole carboxamide upregulate TAN genes and promote neuronal differentiation, but the underlying mechanism remains unclear. Bis-indole carboxamide is generally considered a G4 stabilizer, while SMH14.6 is less characterized and should be better introduced. The authors should clarify how G4 stabilization would interfere with TRF2 binding, it seems that it would likely be by blocking access. A more detailed discussion, and ideally TRF2 ChIP after ligand treatment and/or G4 helicase treatment, would strengthen the model.

   We clarify that Bis-indole carboxamide acts as a G4 stabilizer, while SMH14.6 is also a noted G4-binding ligand that stabilizes G4s (ref. [15]). The exclusion of TRF2 from G4 motifs in gene promoters by G4-binding ligands has also been documented previously (ref. [18]). In line with these findings, ChIP experiments performed following ligand treatment revealed a decreased occupancy of TRF2 at TAN gene promoters, supporting the proposed mechanism (added Fig. 6h).

   Minor comments:

   • Supp Figures related to the scRNA-seq are difficult to read (blurry).

   Corrected

   • Fig S1h: The red box mentioned in the legend is not visible

   Corrected

   • In the text, the Figures 1 f-g are misannotated as Fig 1m and l

   Corrected

   • The symbol γ of γH2AX is missing in the text

   Corrected

   • Fig.3d, please indicate in the legend that it is done in SH-SY5Y.

   Added SH-SY5Y in the legend of Fig. 3d.

   • Fig. S3b: Please consider replotting this panel with an increased y-axis scale. As currently presented, the TRF2 ChIP-seq peaks at several promoters appear truncated by the scaling.

   Corrected

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   1. For most of the data graphs in the manuscript, there is no indication of the number of independent biological replicates carried out (which should ideally be plotted as individual dots overlaying the column graphs), or what the error bars represent, or what statistical test was used. All the figure legends and methods have now been updated with the corresponding biological replicates per experiment, with error bars as SD/SEM and the corresponding statistical test along with p values.

   Figure S1.1a: needs a marker to show that the tissue is dentate gyrus.

   We acknowledge the reviewers' concern that high-magnification images alone make it difficult to verify whether the fields are taken from the correct anatomical location. The dentate gyrus (DG) of the hippocampus is a well-defined structure. In the revised figure (Fig S1.1a), we now include a low-magnification image showing the entire hippocampus, including the CA fields, along with two high-magnification fields specifically from the DG region. Consistent with our claim, the co-immunostaining demonstrates that Sox2-positive neural stem cells in the DG are also positive for TRF2.

   Figure 1c (and all other flow cytometry panels throughout the manuscript): it is not clear if the expression of any of these proteins, except maybe MAP2, are significantly different in the presence or absence of TRF2. These differences need to be presented more quantitatively, with the results compiled from multiple biological replicates and analysed statistically. I am not sure that flow cytometry is the best way to determine differences in protein expression levels for non-surface proteins, because many of the reported differences are not at all convincing.

   To detect intracellular/nuclear proteins by flow cytometry, cells were permeabilized using pre-chilled 0.2% Triton X-100 for 10 minutes, as described in the Methods section.

   We have revised the figures (Fig 1c,e) and now included statistical analysis from three independent biological replicates for these experiments.(Fig S1.4h-j, S2e, S6d)

   Fig 1d: has TRF2 been effectively silenced in this experiment? There appears to be just as many TRF2+ nuclei in the "TRF2 silenced" panel vs the control, including in the cells with neurite outgrowths.

   Quantification of nuclear levels of TRF2 showing decrease in nuclear TRF2 has been included in supplementary Fig S1g.

   Fig 2a-c: these experiments need a positive control, showing increased expression of these proteins in mNSC and SH-SY5Y cells in response to a DNA damaging agent. Again, flow cytometry may not be the best method for this; immunofluorescence combined with telomere FISH would be more convincing.

   We confirm that doxorubicin induces 53BP1 foci (IF-FISH Sup Fig. S2b) and TRF1 silencing elevates γH2AX (Sup Fig. S2c) validating DDR sensitivity. Unlike TRF2 loss (Fig. 2a-c), no TIFs appear with IF and telomere probes (Fig. 2d, Sup Fig. 2a), and without TIFs, there is no telomeric fusion. Flow cytometry was performed with Triton X- 100 to target nuclear protein. These findings adequately address the concern; therefore, further IF-FISH experiments were not included in the present study.

   To conclude that telomere damage is not occurring, an independent marker of such damage, such as telomere fusions, should also be measured.

   In response to uncapped telomeres, ATM kinase activates the DNA damage response (DDR), recruiting γH2AX and 53BP1 to telomeres, which precedes the end-to-end fusions (Takai et al., 2003; Maciejowski & de Lange, 2015; Takai et al., 2003; d'Adda di Fagagna et al., 2003; Cesare & Reddel, 2010; Hayashi et al., 2012; Sarek et al., 2015). We observe no DDR activation or foci (Fig. 2; Sup. Fig. 2). This absence of a DDR response and TIFs indicates no telomere uncapping, negating the need for direct telomere fusion analysis.

   Figure S2b is lacking a no-doxorubicin control.

   Untreated control has been included Fig. S2b.

   Figures 3a and 3b need a positive control (e.g. TRF2 binding to telomeric DNA) and a negative control (e.g. a promoter that did not show any TRF2 binding in the HT1080 ChiP-seq experiment in Fig S3).

   We have included positive (telomere) and negative (GAPDH) controls (based on HT1080 TRF2 ChIP-seq data) for the TRF2 ChIP assay in Supplementary Fig. S3d,e. Additionally, positive and negative controls for all ChIP experiments conducted in this study are presented in Supplementary Figs. S3d, S3e, S3h, S3i, S4c-h, and S5c-e

   The data in Figure 3 would be more compelling if all experiments were also performed in fibroblasts to confirm the cell-type specificity of the effect.

   Our HT1080 fibrosarcoma ChIP-seq data (ref. [18]; Sup. Fig. 3a,b) show TRF2 binding to TAN gene promoters in a fibroblast-derived model, with enrichment in neurogenesis-related genes (refs. [19,20]). In fibroblasts TRF2 depletion, as expected, induce telomere dysfunction and DDR (Fig. 2d; Sup. Fig. 2a), and eventually cell-cycle arrest and cell death as also reported earlier (van Steensel et al., 1998; Smogorzewska & de Lange, 2002). Therefore, the suggested experiments which would require sustained TRF2-depletion are not possible to perform in fibroblasts. TRF2 occupancy on the promoter of the genes in question in cells other than NSC was noted in HT1080 cells (ref. [18]; Sup. Fig. 3a,b).

   No references are provided for the TRF2 posttranslational modifications on R17, K176, K190 and T188. What is the evidence for these modifications, and is it known if they participate in the telomeric role of TRF2?

   These lines with references have been included in the manuscript (highlighted in blue).

   R17 methylation enhances telomere stability (66). K176/K190 acetylation stabilizes telomeres and is deacetylated by SIRT6 (67). T188 phosphorylation facilitates telomere repair after DSBs(68). These PTMs primarily support telomeric roles.

   The experiments in Fig 5 should also be performed with WT TRF2, to confirm that effects are not due to the overexpression of TRF2.

   WT TRF2 shows no differentiation phenotype and change in TAN gene expression (Fig. 1f,g; 3h, Sup Fig. 5a). Confirming effects are not due to TRF2 overexpression.

   Fig 5c has not been described in the text, and there are multiple technical problems with the TRF2 WT experiment: i) There appears to be significant background binding of REST to the IgG beads, though this blot has such high background it is hard to tell (the REST blot in Fig S4b is also of poor quality), ii) TRF2 is migrating at two different positions in the Input and IP lanes, and the TRF2 band in the K176R blot is at a different position to either, and iii) the relative loading of the Input and IP lanes is not indicated, so it's not clear why K176R appears to be so enriched in the IP.

   We acknowledge the oversight in not citing Fig 5c in the manuscript. This has been corrected, and, highlighted in blue in the revised manuscript.

   i) Multiple optimization attempts were made for the Co-IP experiments, and the presented figure reflects the best achievable result despite REST blot smearing, a pattern also reported previously (Ref. 65). The TRF2-REST interaction is well established, and a similar background was also observed in the cited study

   ii)Variable migration patterns of TRF2 were also noted in the cited study (Ref. 65), consistent with our observations. Our primary emphasis, however, is on the TRF2 K176R mutant, which clearly disrupts its interaction with REST.

   iii)The input loading corresponds to 10% of the total lysate. As the experiments were conducted independently, variations in transfection and pull-down efficiencies may account for observed differences.

   To rule out indirect effects of the G4 ligands on the results in Fig 6g, the binding of BG4 and TRF2 at the promoters of these genes should be measured by ChIP.

   To confirm that G4 ligand effects on TAN gene promoters are direct, TRF2 occupancy was assessed using ChIP. Significantly decreased occupancy of TRF2 was noted at TAN gene promoters, (added Fig. 6h). This implies that ligand-induced changes in TRF2 binding are directly linked to promoter-level G4 stabilization.

   Minor comments:

   1. The size of all the size markers in western blots should be added to the figures. Size has been included in all the western blots

   2. There are several figure panels that are incorrectly referenced in the text, e.g. Fig S1.1 (e-f) should be Fig S1.1 (e-h); Fig. 1m should be Fig. 1f; Figs 5e and 5f have been swapped.

   Corrected.

   1. Fig S1.4 is not referred to in the text. It is not clear what the purpose of Fig S1.4a is.

   The following line has been included in the manuscript highlighted in blue.

   Neurospheres were characterized using PAX6, a NSC marker (Fig S1.4a).

   Are the experiments in Figs 3e, 4a, 4c and 4e using 4-OHT treatment, or siRNA? If the latter, I don't think a control for the effectiveness of the knockdown in this cell type has been included anywhere in the manuscript.

   It is using siRNA, a western blot showing the effectiveness of knockdown is presented in supplementary figure S4c (now S4a).

   The lanes of the western blots in Fig S4c are not labelled.

   Corrected.

   1. Given that the experiments in Fig 5 were carried out on a background of endogenous WT TRF2 expression, presumably the K176R mutant is having a dominant negative effect. To understand the mechanism of this effect (e.g, is it simply due to replacement of endogenous WT TRF2 at its genomic binding sites by a large excess of exogenous K176R, or is dimerisation with WT TRF2 needed?) it would be helpful to know the relative expression levels of endogenous and K176R TRF2.

   To address the query, qRT-PCR with 3′ UTR-specific primers showed no change in endogenous TRF2 mRNA upon K176R expression in SH-SY5Y cells, while primers detecting total TRF2 revealed ~10-fold higher expression of K176R compared to control (Figure below). This indicates the absence of suppression of endogenous TRF2 mRNA. Given that the mutant's DNA binding is intact (Fig. 5f), the dominant-negative effect of K176R likely arises from overexpression of the exogenous mutant.

   For the sentence "...and critical for transcription factor binding including epigenetic functions that are G4 dependent" (bottom of page 3 of the PDF), the authors cite only their own prior papers, but there are examples from others that could be cited.

   We have incorporated citations from other research groups, now included as references 23-26.

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   Oct 20, 2025

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   Referee #2

   Evidence, reproducibility and clarity

   This manuscript examines the effects of depletion of the telomeric protein TRF2 in mouse neural stem cells, using mice carrying a floxed allele of TRF2 and inducible Cre recombinase under the control of the stem cell-specific Nestin promoter. The results are also backed up in a human neuroblastoma cell line that has progenitor-like properties. There is no apparent induction of telomere damage in either of these cell types, but there is an increase in expression of neurogenesis genes. This is accompanied by an increase in binding of TRF2 to the relevant promoters, and evidence is provided that this binding involves G-quadruplexes in …

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   Referee #2

   Evidence, reproducibility and clarity

   This manuscript examines the effects of depletion of the telomeric protein TRF2 in mouse neural stem cells, using mice carrying a floxed allele of TRF2 and inducible Cre recombinase under the control of the stem cell-specific Nestin promoter. The results are also backed up in a human neuroblastoma cell line that has progenitor-like properties. There is no apparent induction of telomere damage in either of these cell types, but there is an increase in expression of neurogenesis genes. This is accompanied by an increase in binding of TRF2 to the relevant promoters, and evidence is provided that this binding involves G-quadruplexes in the promoters.

   On the whole, these core findings of this study are interesting, and reasonably robust. However, the study as a whole is marred by a large number of technical issues and missing controls which should be addressed prior to publication:

   1. For most of the data graphs in the manuscript, there is no indication of the number of independent biological replicates carried out (which should ideally be plotted as individual dots overlaying the column graphs), or what the error bars represent, or what statistical test was used.
   2. Figure S1.1a: needs a marker to show that the tissue is dentate gyrus.
   3. Figure 1c (and all other flow cytometry panels throughout the manuscript): it is not clear if the expression of any of these proteins, except maybe MAP2, are significantly different in the presence or absence of TRF2. These differences need to be presented more quantitatively, with the results compiled from multiple biological replicates and analysed statistically. I am not sure that flow cytometry is the best way to determine differences in protein expression levels for non-surface proteins, because many of the reported differences are not at all convincing.
   4. Fig 1d: has TRF2 been effectively silenced in this experiment? There appears to be just as many TRF2+ nuclei in the "TRF2 silenced" panel vs the control, including in the cells with neurite outgrowths.
   5. Fig 2a-c: these experiments need a positive control, showing increased expression of these proteins in mNSC and SH-SY5Y cells in response to a DNA damaging agent. Again, flow cytometry may not be the best method for this; immunofluorescence combined with telomere FISH would be more convincing.
   6. To conclude that telomere damage is not occurring, an independent marker of such damage, such as telomere fusions, should also be measured.
   7. Figure S2b is lacking a no-doxorubicin control.
   8. Figures 3a and 3b need a positive control (e.g. TRF2 binding to telomeric DNA) and a negative control (e.g. a promoter that did not show any TRF2 binding in the HT1080 ChiP-seq experiment in Fig S3).
   9. The data in Figure 3 would be more compelling if all experiments were also performed in fibroblasts to confirm the cell-type specificity of the effect.
   10. No references are provided for the TRF2 postranslational modifications on R17, K176, K190 and T188. What is the evidence for these modifications, and is it known if they participate in the telomeric role of TRF2?
   11. The experiments in Fig 5 should also be performed with WT TRF2, to confirm that effects are not due to the overexpression of TRF2.
   12. Fig 5c has not been described in the text, and there are multiple technical problems with the TRF2 WT experiment: i) There appears to be significant background binding of REST to the IgG beads, though this blot has such high background it is hard to tell (the REST blot in Fig S4b is also of poor quality), ii) TRF2 is migrating at two different positions in the Input and IP lanes, and the TRF2 band in the K176R blot is at a different position to either, and iii) the relative loading of the Input and IP lanes is not indicated, so it's not clear why K176R appears to be so enriched in the IP.
   13. To rule out indirect effects of the G4 ligands on the results in Fig 6g, the binding of BG4 and TRF2 at the promoters of these genes should be measured by ChIP.

   Minor comments:

   1. The size of all the size markers in western blots should be added to the figures.
   2. There are several figure panels that are incorrectly referenced in the text, e.g. Fig S1.1 (e-f) should be Fig S1.1 (e-h); Fig. 1m should be Fig. 1f; Figs 5e and 5f have been swapped.
   3. Fig S1.4 is not referred to in the text. It is not clear what the purpose of Fig S1.4a is.
   4. Are the experiments in Figs 3e, 4a, 4c and 4e using 4-OHT treatment, or siRNA? If the latter, I don't think a control for the effectiveness of the knockdown in this cell type has been included anywhere in the manuscript.
   5. The lanes of the western blots in Fig S4c are not labelled.
   6. Given that the experiments in Fig 5 were carried out on a background of endogenous WT TRF2 expression, presumably the K176R mutant is having a dominant negative effect. To understand the mechanism of this effect (e.g is it simply due to replacement of endogenous WT TRF2 at its genomic binding sites by a large excess of exogenous K176R, or is dimerisation with WT TRF2 needed?) it would be helpful to know the relative expression levels of endogenous and K176R TRF2.
   7. For the sentence "...and critical for transcription factor binding including epigenetic functions that are G4 dependent" (bottom of page 3 of the PDF), the authors cite only their own prior papers, but there are examples from others that could be cited.

   Significance

   The protein TRF2 was first identified as one of the core proteins that bind to the double-stranded region of telomeric DNA, and its many-faceted role in telomere protection has been well studied over the last 3 decades. More recent data from several labs indicate that TRF2 has additional roles outside the telomere, including in regulating gene expression, but these roles are so far much less characterised. Also, it has recently been shown that mouse ES cells, unexpectedly, do not require TRF2 for telomere protection (references 3 and 4 in this paper).

   The findings of the current findings expand the type of stem cells in which TRF2 is likely to be playing more of a role elsewhere in the genome, and not at telomeres, and hence is likely to be of high interest to both researchers of telomere biology, and those interested in the regulation of stem cell biology and neurogenesis.

   The strengths of the study are its novelty, its use of an inducible system to knock out TRF2 in the mouse neural stem cells of interest, and a thorough analysis of changes in gene expression and promoter occupancy across a range of genes of relevance to neurogenesis. The major weakness of the study, as descibed above, is the large number of technical problems, missing controls and missing indications of biological reproducibility.

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   Oct 20, 2025

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   Referee #1

   Evidence, reproducibility and clarity

   In this study, the authors show that TRF2 binds non-telomeric G-quadruplexes in promoters of a set of genes ("TAN" genes for TRF2-associated neuronal differentiation) and recruits REST/chromatin remodelers to repress those genes in neural stem cells, thereby maintaining the NSC state in a telomere-independent manner. They show that the loss of TRF2 derepresses TAN genes and promotes neuronal differentiation.

   However, key experiments are missing to fully support the claims: a genome-wide TRF2 ChIP-seq in NSC to validate binding beyond a restricted set of TAN genes, more robust evidence confirming the absence of telomeric dysfunction, …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   In this study, the authors show that TRF2 binds non-telomeric G-quadruplexes in promoters of a set of genes ("TAN" genes for TRF2-associated neuronal differentiation) and recruits REST/chromatin remodelers to repress those genes in neural stem cells, thereby maintaining the NSC state in a telomere-independent manner. They show that the loss of TRF2 derepresses TAN genes and promotes neuronal differentiation.

   However, key experiments are missing to fully support the claims: a genome-wide TRF2 ChIP-seq in NSC to validate binding beyond a restricted set of TAN genes, more robust evidence confirming the absence of telomeric dysfunction, and mechanistic clarification of the effects of G4 ligands on TRF2 binding.

   Major comments:

   • It is interesting that TRF2 loss not only fails to increase γH2AX/53BP1 levels but may even slightly reduce them (e.g., Fig. S2c and the IF images). While the main hypothesis is that TRF2 loss does not trigger telomere dysfunction in NSCs, this observation raises the possibility that TRF2 itself contributes to DDR signaling (ATM-P, γH2AX, 53BP1) in these cells and that in its absence, cells are not able to form those foci. To exclude the possibility that telomere-specific DDR is being missed due to an overall dampened DDR response in the absence of TRF2, it would be informative to induce exogenous DSBs in TRF2-depleted cells and test DDR competence (e.g., IF for γH2AX/53BP1). In other words, are those NSC lacking TRF2 even able to form H2AX/53BP1 foci when damaged? In addition, it would be interesting to perform telomere fusion analysis in TRF2 silenced cells (and TRF1 silenced cells as a positive control).
   • A TRF2 ChIP-seq should be performed in NSC as this list of genes (named TAN genes in the text) was determined using a ChIP performed in another cell line (HT1080). For the ChIP-qPCR in the various conditions, primers for negative control regions should be included to show the specific binding of TRF2 to the promoter of the genes associated with neuronal differentiation. For example, an intergenic region and/or promoters of genes that are not associated with neuronal differentiation (or don't contain a potential G4). The same comment goes true for the gene expression analysis: a few genes that are not bound by TRF2 should be included as negative controls to exclude a potential global effect of TRF2 loss on gene expression (ideally a RNA-seq would be performed instead).
   • A co-IP should be performed between the TRF2 PTM mutant K176R or WT TRF2 and REST and PRC2 components to directly show a defect of interaction between them when TRF2 is mutated (a co-IP with DNase/RNase treatment to exclude nucleic-acid bridging). The TRF2 PTM mutant T188N also seems to lead to an increased differentiation (Fig. S5a). Could the author repeat the measure of gene expression and co-IP with REST upon the overexpression of this mutant too?
   • The authors show that the G4 ligands SMH14.6 and Bis-indole carboxamide upregulate TAN genes and promote neuronal differentiation, but the underlying mechanism remains unclear. Bis-indole carboxamide is generally considered a G4 stabilizer, while SMH14.6 is less characterized and should be better introduced. The authors should clarify how G4 stabilization would interfere with TRF2 binding, it seems that it would likely be by blocking access. A more detailed discussion, and ideally TRF2 ChIP after ligand treatment and/or G4 helicase treatment, would strengthen the model.

   Minor comments:

   • Supp Figures related to the scRNA-seq are difficult to read (blurry).
   • Fig S1h: The red box mentioned in the legend is not visible
   • In the text, the Figures 1 f-g are misannotated as Fig 1m and l
   • The symbol  of H2AX is missing in the text
   • Fig.3d, please indicate in the legend that it is done in SH-SY5Y.
   • Fig. S3b: Please consider replotting this panel with an increased y-axis scale. As currently presented, the TRF2 ChIP-seq peaks at several promoters appear truncated by the scaling.
   • Fig S4b: the legends should be fixed, the figure shows TRF2 occupancy upon REST silencing and not the other way around.

   Significance

   Non-telomeric roles of TRF2 have been reported before: in repressing neuronal genes and promoting a stem-like state by stabilizing REST (PMID: 18818083), in promoter G4 binding and recruitment of chromatin repressors (previous studies from the same lab), and TRF2 was shown to be dispensable for telomere protection in pluripotent stem cells (ES). The novelty of the current study lies primarily in extending/combining these mechanisms to NSCs.

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   Version published to 10.1101/2025.02.28.640739 on bioRxiv

   Mar 3, 2025