Orphan nuclear receptors recruit TRIM28 to promote telomeric H3K9me3 for the alternative lengthening of telomeres pathway

  1. Chia-Tsen Tsai
  2. Venus Marie Gaela
  3. Hsuan-Yu Hsia
  4. Yu-Chen Huang
  5. Liuh-Yow Chen

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Abstract

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism deployed in embryonic stem cells and cancer cells. High levels of the heterochromatin mark H3 lysine 9 trimethylation (H3K9me3) at telomeres are critical for ALT, but how that is achieved remains unclear. Telomeric association of orphan nuclear receptors (NRs)—such as COUP-TF1, COUP-TF2, TR2, and TR4—has been shown previously to promote ALT activation. Here, we show that orphan NRs regulate telomeric H3K9me3 through TRIM28, a corepressor of ZNF transcription factors, to drive ALT. We report that H3K9me3 is induced by telomeric association of orphan NRs in cultured human fibroblast and ALT cancer cell lines. Moreover, TRIM28 is required for the orphan NR-induced H3K9me3 and ALT phenotypes. Importantly, physical interaction of TRIM28 with orphan NRs facilitates a telomeric localization of TRIM28. A TRIM28 variant defective in orphan NR interaction fails to localize to telomeres and is unable to promote H3K9me3 and ALT phenotypes. These findings indicate that telomeric orphan NRs recruit TRIM28, driving telomeric H3K9me3 and ALT activation, emphasizing the role of changes in chromatin structure in ALT activation.

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    Manuscript number: RC-2025-03091

    Corresponding author(s): Chia-Tsen, Tsai, Liuh-Yow Chen

    1. General Statements [optional]

    We thank the reviewers for their valuable time and constructive feedback on our study, which ultimately improved our manuscript. Herein, we provide a detailed response to each of the reviewers' comments, supported by new data that have been integrated into both the main text and the supplementary figures.

    2. Point-by-point description of the revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary This manuscript builds upon the authors' prior findings that targeting COUP-TF2 to TRF1 induces ALT-associated phenotypes and G2-mediated synthesis in telomerase-immortalised BJT human fibroblasts. In this study, the authors show that telomere-coupled COUP-TF2 promotes H3K9me3 enrichment in these cells, and that this effect is blocked by TRIM28 depletion. Furthermore, TRIM28 depletion also suppresses the formation of ALT phenotypes in VA13 ALT cells. Given that TRIM28 has been implicated in regulating H3K9me3 deposition via SETDB1, and has been reported to co-purify with TR2 and TR4 (though not previously in the context of ALT telomeres), these findings add mechanistic depth to how heterochromatin regulators contribute to ALT activity. Overall, the manuscript's conclusions are generally supported by the presented data, but several aspects require clarification or additional experimental validation.

    The authors report a modest reduction in telomeric H3K9me3 following COUP-TF2 and TR4 depletion in U-2 OS and VA13 cells (Figure 1B). To strengthen the claim that these orphan receptors specifically regulate H3K9me3, the authors should 1) Assess additional heterochromatic histone marks (e.g., H4K20me3) at telomeres, 2) Normalize telomeric signals to both parental histone levels and input, and 3) Evaluate whether global H3K9me3 levels also decrease upon receptor depletion

    Response: We appreciate the reviewer's suggestion. To address the concern regarding specificity, we assessed H3K27me3 and H4K20me3 levels upon COUP-TF2/TR4 depletion and found no significant changes (Supplementary Fig. 1C). Furthermore, we reprocessed the telomeric ChIP data, normalizing to both input DNA and parental histone levels (Figure 1B). This refined analysis reinforces our original conclusion. Finally, Western blot analysis showed no significant changes in global H3 or H3K9me3 levels upon COUP-TF2/TR4 depletion (Figure 1A). Altogether, these results further support the specificity of COUP-TF2/TR4 for H3K9me3 at telomeres. We have revised the main text (page 3) and updated Figure 1A, 1B, and Supplementary Figure 1C for these changes.

    Most experiments explore chromatin changes in telomerase-positive BJT fibroblasts (Figure 2, Figure 4D). It remains unclear whether similar manipulations in ALT cells yield consistent effects, which would give a broader context for ALT phenotype induction. Are ALT phenotypes similarly induced in ALT cells? Does altered chromatin status affect telomere length or telomerase recruitment/activity? Can these pathways drive ALT phenotypes in non-immortalised cells?

    Response: We appreciate the reviewer's suggestion and have explored chromatin changes in telomerase-negative BJ and IMR90 primary fibroblasts (Supplementary Fig. 2C, D). Consistent to the result in BJ-telomerase cells, we found that VP64-TRF1 decreased telomeric H3, H4, and H3K9me3 levels, whereas KRAB-TRF1 increased these marks. Moreover, expression of either VP64-TRF1 or KRAB-TRF1 was sufficient to induce APB formation and ATDs in BJ and IMR90 cells. These results indicate that the chromatin changes at telomeres can drive ALT phenotypes in both primary and telomerase-immortalized fibroblast cells.

        Additionally, regarding whether chromatin alteration affects telomere length or telomere regulation, we have explored telomere length changes in BJT cells expressing vector, TRF1, KRAB-TRF1 or VP64-TRF1. The result of telomere restriction fragment (TRF) assay showed that the cells of all conditions maintained static telomere lengths through 30 days in culture (data shown below), suggesting that the chromatin alterations may not impact telomerase recruitment or activity. As this result is beyond the scope of current study, this data is only shown here in the rebuttal letter for a reference and is not included in the revised manuscript.

    Moreover, according to the reviewer's suggestion, we also carried out VP64-TRF1 or KRAB-TRF1 expression experiments in WI38-VA13/2RA cells that express high TERRA and have altered chromatin structures. Our data revealed that VP64-TRF1 suppresses telomere H3K9me3 and ALT activity, while KRAB-TRF1 increases both (Supplementary Figure 2E), suggesting an association of heterochromatin state with ALT activation in WI38-VA13/2RA cells.

    The observation that VP64-TRF1 reduces ALT activity in WI38-2RA/VA13 cells contrasts with findings in BJT cells. It is worth noting that studies from the Azzalian and Linger groups demonstrated that experimentally induced TERRA expression promotes ALT activity in ALT and non-ALT cells (PMID: 36122232, PMID: 40624280). Therefore, we propose that TERRA upregulation by VP64-TRF1 may contribute to the ALT induction observed in BJT cells (Supplementary Figure 2A, B), whereas the ability of VP64-TRF1 to suppress ALT activity in WI38-2RA/VA13 cells could be attributed to the reduction of telomere H3K9me3 and heterochromatin loss. Importantly, KRAB-TRF1 concurrently enhanced histone H3, H4, and H3K9me3 occupancy and ATL activity in both human fibroblasts and ALT cells. Altogether, these results support the notion that heterochromatin formation triggers ALT.

    We also examined TRIM28 recruitment to telomeres by telomere-ChIP and found that COUP-TF2LBD-TRF1 promotes TRIM28 telomere enrichment in BJ, IMR90 and U2OS, similar to BJT cells (Supplementary Fig. 5A-D).  Moreover, in ALT cell lines WI38-2RA/VA13, U2OS, and Saos-2, depletion of COUP-TF2 or TR4 reduced TRIM28 telomeric association (Figure 4A, B). Together, the data from human fibroblasts and ALT cells supports a role of orphan NRs in recruiting TRIM28 to ALT telomeres.

    We acknowledge the reviewer's suggestions, which allow us to clarify and strengthen the conclusions. The corresponding data are presented in Figure 4A-B and Supplementary Figure 2B-D and 5E-F, and the main text has been modified on page 4-6 in the revised manuscript.

    When referring to Figure 3G, the authors state that that telomeric H3K9me3 was abolished upon depleting TRIM28 from the U2OS and WI38-VA13/2RA cells. Abolished is a strong word for a 50% decrease, and this sentence should be revised. The reduction appears greater than that seen with COUP-TF2/TR4 depletion. Are the effects additive? If so, might TRIM28 act, at least in part, independently of COUP-TF2/TR4?

    Response: We appreciate the reviewer's comments. We have revised the manuscript on page 5, replacing "abolished" with "significantly reduced" to better describe the effect of TRIM28 depletion on telomeric H3K9me3. To further investigate the interplay between TRIM28 and orphan NRs in regulating telomeric H3K9me3, we conducted single and combined knockdown experiments in U2OS and WI38-VA13/2RA cells, followed by telomere-ChIP analysis (Supplementary Figures 4D, E). Our results showed that single depletion of either orphan NRs or TRIM28 lead to a similar decrease in telomeric H3K9me3, and that combined knockdown do not result in any further reduction. These findings support an epistatic interaction between orphan NRs and TRIM28 in the regulation of telomeric H3K9me3. We have expanded on this interpretation in the main text (page 6) and included the relevant data in Supplementary Figures 4D, E.

    VA13 cells consistently exhibit stronger effects than U-2 OS (e.g., Figures 1 and 3). This discrepancy could be linked to the high content of variant repeats in VA13 cells. The authors should assess whether variant repeat content underlies the differential response. Repeating key experiments in additional ALT lines with varied repeat compositions would be informative.

    Response: We appreciate the reviewer's suggestion and have extended our analyses to two additional ALT osteosarcoma cell lines, SAOS-2 and G292. In both lines, depletion of orphan NRs resulted in a consistent decrease in telomeric H3K9me3 levels (Supplementary Figures 1A, B). We also examined the contribution of TRIM28 to telomeric H3K9me3 in these cells. siRNA-mediated depletion of TRIM28 in SAOS-2 and G292 cells similarly caused a significant reduction in telomeric H3K9me3 and ALT phenotypes (Supplementary Figure 4A-C). Together, these results from 4 ALT cell lines confirm that orphan NRs and TRIM28 promote telomeric H3K9me3 formation in ALT cells. We have modified the main text on page 3 and 5-6 for these results.

    In line with the previous point, it would be useful to show whether TRIM28 telomeric enrichment is affected by COUP-TF2/TR4 depletion in U2OS cells (Figure 4C). To improve confidence in these findings, the authors should perform telomeric ChIP assays, especially with the COUP-TF2^LBDΔAF2-TRF1 mutant construct.

    Response: Following the reviewer's suggestion, we performed telomere-ChIP assays to assess TRIM28 enrichment at telomeres upon expression of COUP-TF2LBD-TRF1 and its ΔAF2 mutant in U2OS cells. Consistent with our immunofluorescence results, telomere-ChIP revealed that COUP-TF2LBD-TRF1 expression promotes TRIM28 telomere enrichment, while the AF2 deletion mutant failed to recruit TRIM28 (Supplementary Figure 5D). We have modified the main text on page 6 for this result.

    The immunoprecipitation experiments showing TRIM28 association with orphan receptors should include benzonase treatment to rule out DNA-mediated co-association (Figure 4F-G).

    Response: We appreciate the reviewer's suggestion. To address the possibility of DNA-mediated interactions, we pre-incubated cell lysates with benzonase prior to Co-IP (Page 7). This treatment did not disrupt the association between TRIM28 and COUP-TF2 or TR4 in WI38-VA13/2RA and BJT cells (Supplementary Figures 5E-G), indicating a DNA-independent interaction. We have modified the main text on page 7 for this result.

    The study would benefit from a direct assessment of whether COUP-TF2LBDΔAF2-TRF1 fails to induce ALT phenotypes in BJTfibroblasts.

    Response: We thank the reviewer for this suggestion. As the role of the COUP-TF2 AF2 domain in ALT induction in BJT fibroblasts has recently been thoroughly investigated and published by our group (PMID: 38752489), we have directed the current study towards a more detailed mechanistic question. Specifically, we have carried out experiments to further demonstrate that COUP-TF2 recruits TRIM28 to telomeres via its AF2 domain in both human fibroblasts and ALT cells (Supplementary Figures 5A-D). On Page 6, we have modified the main text for these results and included a citation to our previous publication to provide the necessary background.

    The experiments performed in Figure 5E-H lack a vector-only + siCtrl control.• In Figure 5E, the observation that APB formation is restored in siTRIM28 + Vector-treated cells is unexpected. The authors should address this finding and clarify whether this reflects biological noise or a compensatory effect.

    Response: We thank the reviewer for this suggestion. We have repeated the experiments with a revised design, ensuring a consistent vector background across all groups (Vector + siCtrl, Vector + siTRIM28, TRIM28 WT + siTRIM28, and TRIM28 ΔRBCC + siTRIM28) (Figure 5E-H). This improved design confirms that expression of wild-type TRIM28, but not TRIM28 ΔRBCC, restores APB formation, ATDS, ssTeloC, and telomeric H3K9me3 levels in TRIM28-depleted cells. The updated dataset also resolves the previous unexpected increase in APB formation in the siTRIM28 + Vector condition, which is now excluded. We have modified the main text accordingly on page 8.

    Reviewer #1 (Significance (Required)):

    This work offers valuable mechanistic insight into how COUP-TF2 and TRIM28 coordinate to regulate heterochromatin deposition and ALT phenotype formation. It adds to the growing understanding of chromatin-mediated telomere regulation. What remains unclear is how important this interaction is for ALT maintenance, as H3K9me3 is only moderately altered upon TRIM28 depletion in ALT cells. Depletion of TRIM28 has been shown previously to induce APB formation and telomere elongation in U-2 OS ALT cells (Wang et al., 2021), the opposite to what the authors observed here in VA13 cells (Figure 5E-H). Clarifying whether these differences are variant repeat-dependent, or reflect intrinsic features of specific ALT cell lines, would substantially elevate the study's impact.

    Response: We appreciate the reviewer's recognition of the significance of our work in elucidating the molecular basis of ALT regulation through COUP-TF2-TRIM28-mediated heterochromatin formation. In response to the reviewer's insightful comment regarding the importance of this interaction for ALT maintenance, we have expanded our study. We now include data from three additional primary human fibroblasts and a total of four ALT cancer cell lines (Figure 4, Supplementary Figure 4). These new data further strengthen the conclusion that TRIM28 promotes telomeric H3K9me3 and ALT-associated features. Furthermore, our rescue experiments support the model that the ALT-promoting function of TRIM28 in both fibroblasts and ALT cell lines is mediated through its physical interaction with COUP-TF2 (Supplementary Figure 5). We believe these results provide a solid foundation for demonstrating a cooperative role of COUP-TF2 and TRIM28 in ALT maintenance, and address the reviewer's concern regarding the generalizability of our findings.

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

    Summary This manuscript investigates the role of orphan nuclear receptors (ORs), specifically COUP-TF2 and TR4, in promoting H3K9me3 enrichment at ALT telomeres via recruitment of TRIM28 (KAP1). The authors propose that the AF2 domain of COUP-TF2, located in its ligand-binding domain (LBD), is sufficient to recruit TRIM28 to telomeres. This, in turn, promotes heterochromatinization and induces hallmarks of the Alternative Lengthening of Telomeres (ALT) pathway, including APB formation and telomeric DNA synthesis outside of S-phase. This study addresses one important and unresolved question in the field: by what mechanism is the heterochromatic state established at ALT telomeres? Another timely question, not addressed here is: how is heterochromatin (specifically H3K9me3) functionally linked to ALT? The findings are potentially novel and mechanistically insightful. However, key elements of the study, particularly the central tethering experiments, require stronger quantification and clarity. Additional mechanistic tests and literature adjustments would also improve the manuscript.

    Major Concerns

    Central TRF1-COUP-TF2-LBD result lacks quantification and clarity: the tethering of COUP-TF2's LBD to telomeres via TRF1 is a core result of the paper. This experiment demonstrates that this domain is sufficient to induce weak H3K9me3 enrichment and ALT features (APBs and ATDS). However, the supporting ALT data are presented only in Supplementary Figures S1A and S1B, and are not quantified. These data should be quantified with appropriate statistics and moved to a main figure.

    Response: The current study builds upon our recent publication (PMID: 38752489), which comprehensively analyzed ALT induction (APBs, ATDS, C-circles, T-SCEs) by orphan NR-TRF1 expression (COUP-TF1, COUP-TF2, TR2, and TR4; full-length and LBD) in various human fibroblast cell lines. To avoid potential duplicate publication concerns, particularly regarding APB and ATDS results for COUP-TF2LBD-TRF1 in BJT cells, we have put the data with revised quantification results in Supplementary Figure 1D-E. We will follow the reviewer's suggestion and move this data to the main figures if the editors agree.

    Furthermore, the broader functional implication is not explored. Does this tethering induce a fully functional ALT pathway? For example, can telomerase knockout cells expressing TRF1-COUP-TF2-LBD maintain long-term proliferation? Such evidence would significantly strengthen the impact of the study.

    Response: While COUP-TF2LBD-TRF1 expression rapidly induces key ALT phenotypes, we acknowledge that this alone is insufficient to directly promote telomere lengthening and long-term proliferation of primary fibroblasts, as discussed in Gaela et al., 2024 (PMID: 38752489). However, our ongoing, unpublished studies indicate that COUP-TF2LBD-TRF1 can drive immortalization of primary BJ fibroblasts expressing SV40LT by promoting ALT-mediated telomere elongation (Attached Figure A-C; additional data not shown). These findings suggest that COUP-TF2 may cooperate with additional genetic or epigenetic alterations to facilitate ALT development. We appreciate the reviewer's recognition of this critical aspect. As our immortalization study is still in progress and will be the subject of a separate manuscript, we hope the reviewer understands that the data shown in this letter will not be included in the revised manuscript.

    Chromatin manipulation experiments lead to ambiguous conclusions: the authors propose that telomeric heterochromatin promotes ALT activity, but their own experiments (e.g., Figure 2) show that both heterochromatin-inducing (KRAB-TRF1) and euchromatin-inducing (VP64-TRF1) tethering can trigger ALT-like features. This makes it difficult to conclude that heterochromatin is specifically required.

    To clarify:

    -Did the authors express TRF1-VP64 in an ALT cell line? According to their model, this should suppress ALT activity.

    -More broadly, do chromatin alterations per se (regardless of direction) trigger ALT features? Clarifying these points is important for interpretation.

    Response: In response to the reviewer's suggestion, we expressed VP64-TRF1 and KRAB-TRF1 in WI38-2RA/VA13 cells to investigate telomere chromatin changes and ALT activity. Our data indeed revealed that VP64-TRF1 suppresses telomere H3K9me3 and ALT activity, while KRAB-TRF1 increases both (Supplementary Figure 2E), suggesting that heterochromatin triggers ALT activation.

    The observation that VP64-TRF1 reduces ALT activity in WI38-2RA/VA13 cells contrasts with findings in BJT cells. Of note, studies from the Azzalian and Lingner groups demonstrated that experimentally induced TERRA expression promotes ALT activity in ALT and non-ALT cells (PMID: 36122232, PMID: 40624280). Therefore, we propose that TERRA upregulation may contribute to the ALT induction observed in BJT cells (Figure 2A, Supplementary Figure 2A, B). Given the high basal TERRA expression, expression of VP64-TRF1 and KRAB-TRF1 did not result in a consistent change in TERRA levels (Supplementary Figure 2F). Thus, the ability of VP64-TRF1 to suppress ALT activity in WI38-2RA/VA13 cells could be attributed to the reduction of telomere H3K9me3 and heterochromatin loss. Altogether, our results support the hypothesis that heterochromatin formation, rather than euchromatin triggers ALT.

    We thank the reviewer's insightful comments, which have allowed us to resolve the ambiguity of our results and strengthen the notion that heterochromatin formation promotes ALT. We think that the heterochromatin features and high TERRA expression represent two independent, coexisting mechanisms within ALT cancer cells to guarantee ALT activation. We have modified the main text on page 4-5 accordingly.

    TERRA downregulation contradicts current models: while TERRA upregulation is often observed in ALT cells and is thought to contribute to replication stress and recombination at telomeres, the authors show that TRF1-KAP1 expression induces ALT features while TERRA is downregulated. This observation is not addressed in the manuscript. The authors should at least discuss this discrepancy and propose whether this reflects a cell line-specific phenomenon or a decoupling between TERRA levels and ALT induction in this context.

    Response: We thank the reviewer for the comments. As mentioned above (Major Concerns 2), heterochromatin formation and TERRA expression are two mechanisms that can independently promote ALT. Unlike ALT cell lines that have high TERRA levels, human fibroblasts BJ cells have low TERRA that does not induce ALT phenotypes. Thus, the effect of KRAB-TRF1 on ALT induction in BJ cells could be attributed to the heterochromatin formation, but not reduction of TERRA. We have modified the main text on page 5 to clarify the result.

    Minor Comments

    Introduction (p. 3): The authors cite Episkopou et al. as showing increased H3K9me3 at ALT telomeres. This is incorrect; that paper suggests the opposite. The first study to clearly demonstrate H3K9me3 enrichment at ALT telomeres is Cubiles et al., 2018 and should be cited instead. Results (p. 5, first paragraph): The manuscript should cite Déjardin and Kingston, 2009 as the first to report COUP-TF2 and TR4 localization at ALT telomeres. The studies by Conomos et al., 2012 and Gaela et al., 2024 build on this prior evidence. Please also include this citation in the bibliography.

    Response: We appreciate the reviewer's careful reading and for pointing out these errors. The citation errors on pages 2 and 3 have now been corrected.Broader relevance of TRIM28-OR interaction: TRIM28 is a complex protein with roles in SUMOylation, heterochromatin formation, and transcriptional initiation/elongation regulation.

    The authors should explore whether similar COUP-TF2/TRIM28 interactions occur at other genomic loci. Public ChIP-seq data for COUP-TF2, TR4, and TRIM28 could be mined to investigate whether these factors co-occupy regulatory regions elsewhere in the genome, and how this relates to gene expression states.

    Response: We appreciate the reviewer's insightful suggestion regarding a potential genome-wild functional interaction between TRIM28 and COUP-TF2. To address this, we analyzed public ENCODE ChIP-seq data from K562 cells (TRIM28: ENCSR000BRW; COUP-TF2: ENCSR000BRS). This analysis revealed 3,326 co-binding sites for TRIM28 and COUP-TF2 (Attached Figure A). Interestingly, these co-binding sites were preferentially located within gene bodies (70.7%) and promoter regions (4.3%) (Attached Figures B-D), suggesting a potential cooperative role in gene regulation that aligns with our observation of physical interaction. While the finding is intriguing, a full exploration is beyond the scope of this manuscript, which focuses on ALT telomere regulation. We consider this is an important insight and have briefly noted it in the discussion (p. 9), although the corresponding analyses are not included in the revised manuscript.

    Reviewer #2 (Significance (Required)):

    This work contributes mechanistic insight into how heterochromatin is established at ALT telomeres-an important and timely question in telomere biology and cancer research. It offers a noncanonical recruitment mechanism for TRIM28, independent of KRAB-ZNFs, and highlights the functional role of orphan nuclear receptors in telomeric chromatin regulation. The study has potential implications for understanding ALT regulation and for identifying new intervention points in ALT-positive cancers. The work is conceptually interesting, but the conclusions are currently limited by insufficient quantification, some interpretative ambiguities, and a few overlooked references. Addressing the concerns listed above would significantly enhance the rigor and impact of the manuscript.

    Response: We appreciate the reviewer's recognition of the significance of our work in elucidating the molecular basis of ALT regulation through COUP-TF2-TRIM28-mediated heterochromatin formation. We also thank the reviewer for the valuable feedback, which has significantly strengthened our manuscript.

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This manuscript investigates the role of orphan nuclear receptors (ORs), specifically COUP-TF2 and TR4, in promoting H3K9me3 enrichment at ALT telomeres via recruitment of TRIM28 (KAP1). The authors propose that the AF2 domain of COUP-TF2, located in its ligand-binding domain (LBD), is sufficient to recruit TRIM28 to telomeres. This, in turn, promotes heterochromatinization and induces hallmarks of the Alternative Lengthening of Telomeres (ALT) pathway, including APB formation and telomeric DNA synthesis outside of S-phase. This study addresses one important and unresolved question in the field: by what mechanism is the heterochromatic state established at ALT telomeres? Another timely question, not addressed here is: how is heterochromatin (specifically H3K9me3) functionally linked to ALT? The findings are potentially novel and mechanistically insightful. However, key elements of the study, particularly the central tethering experiments, require stronger quantification and clarity. Additional mechanistic tests and literature adjustments would also improve the manuscript.

    Major Concerns

    1. Central TRF1-COUP-TF2-LBD result lacks quantification and clarity: the tethering of COUP-TF2's LBD to telomeres via TRF1 is a core result of the paper. This experiment demonstrates that this domain is sufficient to induce weak H3K9me3 enrichment and ALT features (APBs and ATDS). However, the supporting ALT data are presented only in Supplementary Figures S1A and S1B, and are not quantified. These data should be quantified with appropriate statistics and moved to a main figure. Furthermore, the broader functional implication is not explored. Does this tethering induce a fully functional ALT pathway? For example, can telomerase knockout cells expressing TRF1-COUP-TF2-LBD maintain long-term proliferation? Such evidence would significantly strengthen the impact of the study.
    2. Chromatin manipulation experiments lead to ambiguous conclusions: the authors propose that telomeric heterochromatin promotes ALT activity, but their own experiments (e.g., Figure 2) show that both heterochromatin-inducing (KRAB-TRF1) and euchromatin-inducing (VP64-TRF1) tethering can trigger ALT-like features. This makes it difficult to conclude that heterochromatin is specifically required. To clarify:
    • Did the authors express TRF1-VP64 in an ALT cell line? According to their model, this should suppress ALT activity.
    • More broadly, do chromatin alterations per se (regardless of direction) trigger ALT features? Clarifying these points is important for interpretation.
    1. TERRA downregulation contradicts current models: while TERRA upregulation is often observed in ALT cells and is thought to contribute to replication stress and recombination at telomeres, the authors show that TRF1-KAP1 expression induces ALT features while TERRA is downregulated. This observation is not addressed in the manuscript. The authors should at least discuss this discrepancy and propose whether this reflects a cell line-specific phenomenon or a decoupling between TERRA levels and ALT induction in this context.

    Minor Comments

    Introduction (p. 3): The authors cite Episkopou et al. as showing increased H3K9me3 at ALT telomeres. This is incorrect; that paper suggests the opposite. The first study to clearly demonstrate H3K9me3 enrichment at ALT telomeres is Cubiles et al., 2018 and should be cited instead. Results (p. 5, first paragraph): The manuscript should cite Déjardin and Kingston, 2009 as the first to report COUP-TF2 and TR4 localization at ALT telomeres. The studies by Conomos et al., 2012 and Gaela et al., 2024 build on this prior evidence. Please also include this citation in the bibliography. Broader relevance of TRIM28-OR interaction: TRIM28 is a complex protein with roles in SUMOylation, heterochromatin formation, and transcriptional initiation/elongation regulation. The authors should explore whether similar COUP-TF2/TRIM28 interactions occur at other genomic loci. Public ChIP-seq data for COUP-TF2, TR4, and TRIM28 could be mined to investigate whether these factors co-occupy regulatory regions elsewhere in the genome, and how this relates to gene expression states.

    Significance

    This work contributes mechanistic insight into how heterochromatin is established at ALT telomeres-an important and timely question in telomere biology and cancer research. It offers a noncanonical recruitment mechanism for TRIM28, independent of KRAB-ZNFs, and highlights the functional role of orphan nuclear receptors in telomeric chromatin regulation. The study has potential implications for understanding ALT regulation and for identifying new intervention points in ALT-positive cancers.

    The work is conceptually interesting, but the conclusions are currently limited by insufficient quantification, some interpretative ambiguities, and a few overlooked references. Addressing the concerns listed above would significantly enhance the rigor and impact of the manuscript.

  3. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    This manuscript builds upon the authors' prior findings that targeting COUP-TF2 to TRF1 induces ALT-associated phenotypes and G2-mediated synthesis in telomerase-immortalised BJT human fibroblasts. In this study, the authors show that telomere-coupled COUP-TF2 promotes H3K9me3 enrichment in these cells, and that this effect is blocked by TRIM28 depletion. Furthermore, TRIM28 depletion also suppresses the formation of ALT phenotypes in VA13 ALT cells. Given that TRIM28 has been implicated in regulating H3K9me3 deposition via SETDB1, and has been reported to co-purify with TR2 and TR4 (though not previously in the context of ALT telomeres), these findings add mechanistic depth to how heterochromatin regulators contribute to ALT activity. Overall, the manuscript's conclusions are generally supported by the presented data, but several aspects require clarification or additional experimental validation.

    • The authors report a modest reduction in telomeric H3K9me3 following COUP-TF2 and TR4 depletion in U-2 OS and VA13 cells (Figure 1B). To strengthen the claim that these orphan receptors specifically regulate H3K9me3, the authors should 1) Assess additional heterochromatic histone marks (e.g., H4K20me3) at telomeres, 2) Normalize telomeric signals to both parental histone levels and input, and 3) Evaluate whether global H3K9me3 levels also decrease upon receptor depletion
    • Most experiments explore chromatin changes in telomerase-positive BJT fibroblasts (Figure 2, Figure 4D). It remains unclear whether similar manipulations in ALT cells yield consistent effects, which would give a broader context for ALT phenotype induction. Are ALT phenotypes similarly induced in ALT cells? Does altered chromatin status affect telomere length or telomerase recruitment/activity? Can these pathways drive ALT phenotypes in non-immortalised cells?
    • When referring to Figure 3G, the authors state that that telomeric H3K9me3 was abolished upon depleting TRIM28 from the U2OS and WI38-VA13/2RA cells. Abolished is a strong word for a 50% decrease, and this sentence should be revised. The reduction appears greater than that seen with COUP-TF2/TR4 depletion. Are the effects additive? If so, might TRIM28 act, at least in part, independently of COUP-TF2/TR4?
    • VA13 cells consistently exhibit stronger effects than U-2 OS (e.g., Figures 1 and 3). This discrepancy could be linked to the high content of variant repeats in VA13 cells. The authors should assess whether variant repeat content underlies the differential response. Repeating key experiments in additional ALT lines with varied repeat compositions would be informative.
    • In line with the previous point, it would be useful to show whether TRIM28 telomeric enrichment is affected by COUP-TF2/TR4 depletion in U-2 OS cells (Figure 4C). To improve confidence in these findings, the authors should perform telomeric ChIP assays, especially with the COUP-TF2^LBDΔAF2-TRF1 mutant construct.
    • The immunoprecipitation experiments showing TRIM28 association with orphan receptors should include benzonase treatment to rule out DNA-mediated co-association (Figure 4F-G).
    • The study would benefit from a direct assessment of whether COUP-TF2LBDΔAF2-TRF1 fails to induce ALT phenotypes in BJT fibroblasts.
    • The experiments performed in Figure 5E-H lack a vector-only + siCtrl control.
    • In Figure 5E, the observation that APB formation is restored in siTRIM28 + Vector-treated cells is unexpected. The authors should address this finding and clarify whether this reflects biological noise or a compensatory effect.

    Significance

    This work offers valuable mechanistic insight into how COUP-TF2 and TRIM28 coordinate to regulate heterochromatin deposition and ALT phenotype formation. It adds to the growing understanding of chromatin-mediated telomere regulation. What remains unclear is how important this interaction is for ALT maintenance, as H3K9me3 is only moderately altered upon TRIM28 depletion in ALT cells. Depletion of TRIM28 has been shown previously to induce APB formation and telomere elongation in U-2 OS ALT cells (Wang et al., 2021), the opposite to what the authors observed here in VA13 cells (Figure 5E-H). Clarifying whether these differences are variant repeat-dependent, or reflect intrinsic features of specific ALT cell lines, would substantially elevate the study's impact.

  4. Version published to 10.1101/2025.06.16.658187 on bioRxiv