Peer review of: Persistent telomere cohesion protects aged cells from premature senescence (2020)


Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This manuscript details a robust study by the Smith lab focusing on the intersection between telomere 
length and telomere cohesion in aging and ALT cells. The presented experimentation probes how 
altering telomere cohesion through multiple methods leads to different molecular outcomes at 
chromosome ends and the resulting functional consequences in pre-senescent/senescent or ALT-
positive human cells. 

In summary, the authors identify that decreased TRF1 density at shortened telomeres in aged cells 
results in persistent telomere cohesion. TRF1 over-expression in aged cells promotes resolution of 
telomere cohesion through tankyrase recruitment (nicely demonstrated through endogenous TRF1 
gene editing and expression of mutant TRF1 alleles). Promoting resolution of telomere cohesion in 
aged cells through TRF1 over-expression conferred an additional sub-telomere recombination 
phenotype. Critically, the sub-telomere recombination phenotype was suppressed at cohesion resolved 
telomeres by telomerase-dependent telomere elongation. This key result defines that it is specifically 
shortened telomeres that are subjected to sub-telomeric recombination when telomere cohesion is 
resolved. Thereby identifying why similar recombination does not occur at the cohesion resolved 
telomeres in young or telomerase positive cells. Further, the team identifies sub-telomere 
recombination in this context is regulated by ATR, CHK1 and RAD51, and independent of RAD52, 
CHK2 and POLD3. Thus distinguishing the sub-telomere recombination identified here from break-
induced telomere synthesis. The surprising result to this reviewer is that the observed sub-telomere 
recombination drives DNA damage response activation. Recombination typically resolves DNA damage 
response foci. Regardless, the data demonstrate that suppressing recombination reduces rescues 
growth arrest in p53-competent U2OS ALT cells and suppresses senescence phenotypes in primary 
fibroblasts. Finally, in the cell division immediately preceding senescence an increase in telomere 
cohesion resolution in observed, coinciding with increased sub-telomere recombination. This may 
contribute to telomere-dependent senescence. 

Overall the experiments are well done and the data convincing. The topic is pertinent to those 
interested in telomere biology, genome maintenance, cohesion, homologous recombination, and cell 
aging. I have comments that requires addressing in relation to the “telomere copying” and DDR 
phenotypes. Other comments are cosmetic or clarifying statements that address minor details in the 
manuscript. Upon revision the manuscript is suitable for publication in Nature Communications. 

Major Comments. 
1. The readout of sub-telomere recombination phenotype is clear. Further, it is clear this phenotype is 
dependent on ATR/CHK1/RAD51. However, it is not clear to this reviewer that the authors are 
observing “telomere copying”. Telomere copying would result in duplication of the sub-telomeric 
region from one chromosome to another. This is suggestive of break-induced telomere synthesis, 
which is inconsistent with pathway analysis in figure 2A, B. To me, the phenotype looks similar to 
“telomere fragility”. With telomere fragility (still a poorly defined phenotype) the telomere focus 
becomes spread out and ill-defined, or multiple dots occur at a single chromosome end, but the 
intensity of the entirety of the telomere signal at that chromosome end is not necessarily diminished. 
This is like the sub-telomere phenotype shown here (most notably the 13q telomere in Figure 2F and 
16p in Fig 4I). What exactly does these probes hybridize to? Is it a repetitive sequence? To show 
telomere copying occurs it is necessary to perform cytogenetic experiments and demonstrate 
movement of the sub-telomere tag to another chromosome on a metaphase spread under conditions 
where sub-telomere recombination is promoted. This experiment will determine if the outcome is 
inter-chromosome recombination (i.e. copying) or potentially intra-chromosome recombination (sub-
telomere fragility?). This may be significant, as it could indicate that an intra-chromosomal telomere 

recombination event leads to the unexpected recombination-dependent damage response. Unlike 
classical HR which resolves DDR foci. 

2. Similarly, I do not think it is possible to conclude in Figure 4J that the observed increase is sub-
telomere recombination does not the result of supernumerary chromosomes. A common outcome in 
aged cells after p53 is suppressed is genome duplication and tetraploid cells. Tetrapliody also occurs in 
SV40 lgT expressing cells. In Supplementary Figure S3 the 2N cells are at an X-axis value of ~ 275 
and the 4N cells ~ 550. If genome duplication occured, the 4N peak represents both G2/M phase 
diploid cells and G1-phase tetraploid cells. The resulting G2/M tetraploid cells would have an 8N DNA 
peak which in this instance would be an X-axis value twice that of the 4N cells, or ~ 1100. This value 
is off the chart. However, there is a peak stacked on the edge of the graph that may be that tetraploid 
G2/M phase cells and the “polyploid” measurement represents S-phase tetraploid cells. It is necessary 
to redo this flow cytometry with an altered voltage to move the 2N peak to ~ 150, so the resulting 
DNA peaks for 2N, 4N and 8N are 150, 300 and 600. This will indicate if the observed increase is 
recombined telomeres are due to genome duplication or more recobination events. If the 8N peak is 
not observed, a cytogenetic experiment as described in point 1 will definitively demonstrate telomere 
copying. 

3. Is the recombination-dependent DDR observed in figure 3E occurring at the telomeres If so, this 
would nicely demonstrate that the recombination phenotype at telomeres drives the DDR. 

Minor comments. 
1. While the data all appear significant, providing stats would be useful for Fig 2A, D, E, G, H; Fig 5H, 
I; Fig 6G 

2. Describing the phenotype as “telomere copying” requires verification as described above. If this 
proves impossible, “recombination” or some other general term should be applied. 

3. Page 8 2nd paragraph. Figure 4B is accidentally called out in place of 5B. 

4. It is worth clarifying for the reader that U2OS cells are unique as the only p53-competent ALT cell 
line. Unless data are presented that demonstrate otherwise, I expect altering telomere cohesion only 
induces growth arrest in the U2OS ALT line. This will likely not be a universal attribute of ALT which 
almost always stratifies with a compromised p53 pathway. 

5. Page 10, top paragraph. I suggest adding the clarifier to assist reader comprehension putting 
emphasis that it is the telomere elongation that matter: “Thus, similar to the results described above 
for ALT cells, telomerase forced resolution of telomere cohesion, but at the same time suppressed 
subtelomere recombination between the resolved and extended telomeres.” 

Reviewer #2 (Remarks to the Author): 

In this manuscript, Azarm et al. investigated the effect of overexpression of TRF1WT and TRF1 
mutants on resolution of persistent telomere cohesion and subtelomere recombination in several cell 
types. They demonstrated that overexpression of TRF1WT increases resolution of telomere cohesion 
and subtelomere recombination in aging cells, which is associated with increased DNA damage, RAD51 
foci as well as drastic growth arrest. They went on to show that overexpression of TRF1WT also 
increases resolution of telomere cohesion and subtelomere recombination in ALT cells. In addition, 
they showed that overexpression of TRF1WT in telomerase-inhibited HTC75 cells rescues resolution of 
telomere cohesion and increases subtelomere recombination. Furthermore, they showed that loss of 
persistent telomere cohesion and subtelomere recombination accompany an onset of senescence. The 
authors propose that telomere erosion that occurs in the absence of telomerase induces persistent 

telomere cohesion, which serves as a protective mechanism against subtelomere recombination, DNA 
damage and premature activation of senescence. 

The manuscript presents several major issues as detailed below that raise questions about their 
model. 

1. Although the authors have shown that persistent telomere cohesion is positively correlated with 
telomere shortening, they have fallen short of demonstrating a direct link that resolution of persistent 
telomere cohesion causes subtelomere recombination. Work from several groups has implicated TRF1 
in regulating homologous recombination (Wang et al 2018 Cell Death Differ; Bower and Griffith 2014 
Biochem; McKerlie et al 2013 NAR). A possible scenario is that resolution of persistent telomere 
cohesion and subtelomere recombination as well as increased DNA damage foci and RAD51 foci in 
aged cells overexpressing TRF1WT are individual consequences of overexpression of TRF1WT and that 
persistent telomere cohesion is an independent event from subtelomere recombination. 

2. The authors showed that overexpression of TRF1WT causes rapid entry into senescence in U2OS 
cells, which conflicts with a recent report that overexpression of TRF1WT does not affect cell cycle 
progression in U2OS cells (Silva et al 2019 Nat Commun). This discrepancy is concerning given that 
many of key experiments were performed with overexpression of TRF1WT. The authors need to 
address whether an excessive amount of overexpressed TRF1WT (rather than resolution of persistent 
telomere cohesion per se) is responsible for causing subtelomere recombination, DNA damage and 
rapid growth arrest. 

3. Presumably the number of TRF1 binding sites in aged WI38 PD52 cells is very limited due to 
shortened telomeres. One might assume that a vast majority of overexpressed TRF1WT and TRF1AA 
are not bound to telomeres. Presumably overexpressed unbound TRF1WT would bind tankyrase and 
soak tankyrase away from telomeres whereas overexpressed unbound TRF1AA would not do so. This 
scenario would raise questions about their model that TRF1WT aged cells have more tankyrase at 
telomeres than TRF1AA aged cells. Is tankyrase responsible for the resolution of persistent telomere 
cohesion in TRF1WT aged cells? 

4. TRF1 is essential for ALT activity. Does overexpression of TRF1 affect hallmarks of ALT cells (APBs, 
C-circles, telomere heterogeneity and T-SCE)? Could misregulation in ALT activity as a result of 
overexpressed TRF1WT (rather than resolution of persistent telomere cohesion) account for rapid 
growth arrest seen in ALT U2OS cells? 

Other specific issues: 

1. In Fig. 1, the authors showed that TRF1G18P, a knockin mutation, leads to persistent telomere 
cohesion. The authors need to use these TRF1WT and TRF1G18P 293T cell lines to address whether 
persistent telomere cohesion induced by TRF1G18P expressed at a physiological level serves a 
protective mechanism against subtelomere recombination, DNA damage and RAD51 foci. Does 
TRF1G18P affect telomere length and prevent subtelomere recombination, DNA damage foci and 
RAD51 foci? In addition, does telomerase inhibition cause growth arrest in 293T TRF1WT cells but not 
in TRF1G18P cells? 
Regarding Fig. 1A and 1B, given that the authors suggest that TRF1-mediated tankyrase controls 
resolution of telomere cohesion, they should demonstrate via ChIPs whether the level of tankyrase at 
telomeres is affected by TRF1G18P in 293T cells. 

2. The authors showed a single cell image in Fig. 1F, claiming that TRF1AA does not recruit tankyrase 
to telomeres in WI38 PD52 cells. This data from a single cell image is insufficient. A quantitative 
method such as ChIPs should be used to demonstrate if TRF1AA affects the level of tankyrase at 
telomeres in aged WI38 PD52 cells. 

3. Fig. 3A-3D showed that over 15% of aged WI38 cells overexpressing TRF1WT exhibit >5 RAD51 
foci overall whereas less than 8% of them exhibit RAD51 foci at telomeres. Given that a lower 
threshold was used for scoring cells with RAD51 foci at telomeres (≥2 RAD51/TRF2 colocalizing foci vs 
>5 RAD51 foci overall), their data suggest that at least half of RAD51 foci as a result of 
overexpression of TRF1WT in aged WI38 are not associated with telomeres. How specific is the effect 
of overexpression of TRF1WT on subtelomere recombination in aged WI38 cells? Does overexpression 
of TRF1WT in aged WI38 cells lead to an increase in overall recombination? 
Fig. 3E and 3F showed that overexpression of TRF1WT increased damage foci of gammaH2AX/53BP1 
in aged WI38 PD51 cells. Are these damage foci at telomeres or elsewhere in the genome? Is there an 
increase in chromosome abnormalities, e.g. breaks, fusions etc. in WI38 PD51 cells overexpressing 
TRF1 WT? Could increased DNA damage be a consequence of increased overall recombination in aged 
WI38 TRF1WT cells? The authors need to address if overexpression of TRF1 WT causes genome wide 
DNA damage or telomere-specific DNA damage. In the absence of answers to these questions, it 
seems premature for them to state “… subtelomere recombination drives the DNA damage”. 

4. Fig. 4C and 4D showed that overexpression of SV40-LT does not affect persistent telomere 
cohesion, but increases subtelomere recombination in aged WI38 cells. This data seems to argue 
against the authors’ claim that persistent telomere cohesion affects subtelomere recombination. 
In Fig. 4D and 4H, the authors showed that overexpression of SV40-LT increase subtelomere 
recombination and abrogates growth arrest of TRF1WT WI38 cells. Based on these data, they 
concluded that loss of p53 permits subtelomere copying and abrogates growth arrest. However, SV40-
LT has many targets in addition to p53. The authors need to show if targeting p53 alone increases 
subtelomere copying and abrogates growth arrest. 

5. Fig. 4J showed a further increase in subtelomere coping on Day 4. Is this increase associated with a 
further reduction in telomere singlets in TRF1 WT WI38 SV40-LT cells on Day 4? In addition, is this 
increase in subtelomere copying associated with an increase in RAD51 foci overall? 

6. The authors need to use a quantitative method such as ChIPs to show that TRF1WT but not TRF1AA 
increases the level of tankyrase at telomeres in ALT cells. A single cell image shown in Fig. 5B is 
insufficient. 

7. In Fig. 5F, without POLD3 knockdown, about 5% of GM847-TRF1AA cells showed >3 16p foci. In 
Fig. 5I, knockdown of POLD3 resulted in 30% of GM847-TRF1AA mitotic cells with >3 16p foci (Fig. 
5I). Their data suggest that loss of POLD3 increases subtelomere recombination, which contradicts 
their statement “Subtelomere recombination was independent of POLD3…” on Pg8. 

8. In Fig. 5M-5O, overexpression of TRF1WT causes rapid growth arrest in U2OS cells. Does 
overexpression of TRF1WT cause growth arrest in other ALT cell lines? In addition, does knockdown or 
knockout of p53 abrogate growth arrest in U2OS TRF1WT cells? 

9. The authors claimed that telomerase expression abrogates subtelomere recombination. This 
statement is not supported by their data shown in Fig. 6D and 6L that telomerase expression or 
inhibition has no impact on the percentage of mitotic cell with >3 16p loci. 

10. In Fig. 6D, introduction of telomerase into GM847 leads to resolution of persistent telomere 
cohesion but has no effect on subtelomere recombination. This data does not support the idea that 
persistent telomere cohesion protects against subtelomere recombination. 

11. Does overexpression of TRF1WT cause increased DNA damage, RAD51 foci and growth arrest in 
BIBR-treated HTC75 PD245 cells? 

12. The authors suggest that telomere erosion at the last few PDs prior to senescence onset results in 
loss of telomeres cohesion (singlets). Could this sudden loss of singlets be caused by a sudden 

increase in chromosome missegregation? Is there any increase in micronuclei with 16p telo or 13q telo 
foci at senescence onset? 

Reviewer #3 (Remarks to the Author): 

This manuscript by Azarm et al. investigates the role of the shelterin component TRF1 in senescence 
induction. They demonstrate that insufficient binding of TRF1 at shortened telomeres plays a 
protective role, preventing a recombination induced DNA damage response. Importantly, they show in 
every cellular context tested – ageing cells, ALT cells, or telomerase positive cells where telomerase is 
inhibited – that shortened telomeres induce persistent telomere cohesion due to reduced TRF1 binding 
at telomeres, thereby delaying senescence. 

The manuscript is logically laid out and the data are clearly presented. Additionally, the data fully 
support the conclusions reached except for a couple minor exceptions noted below. The impact and 
significance of the findings are appropriate for publication for Nature Communications in my opinion. 

Only a few minor issues remain to be addressed in my opinion before publication: 

1. In the introduction, the sentence “Telomere function is regulated by shelterin, which is recruited to 
telomeres by the TTAGGG doubled-stranded repeat binding proteins TRF1 and TRF2.” needs to be 
modified, as TRF1 and TRF2 are actually part of the shelterin complex. As currently stated, it suggests 
that the 6-member shelterin complex is recruited by TRF1 and TRF2. 

2. The authors hypothesize that insufficient TRF1 loading at telomeres in ageing cells results in 
attenuated recruitment of endogenous tankyrase and a lack of resolution of telomere cohesion. While 
the authors demonstrate that overexpression of an epitope-tagged version of TRF1 leads to TRF1 
loading at telomeres and consequent tankyrase recruitment (Fig. 1F), they never directly demonstrate 
a decrease in endogenous TRF1 loading at aging cell telomeres. I think this should be addressed 
experimentally before publication. 

3. The data in Fig. 2D, E, G and H appear to be from a single experiment. Replicates are needed for 
these experiments, and the graphs need to show error bars. 

4. Methods under the section Plasmids: “Telomerase was expressing using the pBABE hTERT-hTR 
plasmid.” Change to “Telomerase was expressed…”.