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