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

Response to Reviewer 2 
 
 
Reviewer #2 (Remarks to the Author):  
 
The authors have made efforts to address some of the concerns raised previously, however several 
main issues remain outstanding. This dampens one’s enthusiasm to endorse it for publication as it 
currently stands.  
 
1. The TRF1AA work cited by the authors demonstrates that the tankyrase binding site of TRF1 is 
responsible for the observed phenotypes (resolution of persistent telomere cohesion, subtelomere 
recombination) in aged cells overexpressing TRF1WT, but it does not demonstrate the causal 
relationship between the phenotypes. As a result, the authors have not convincingly shown that there 
is a causal relationship among phenotypes caused by overexpression of TRF1WT in aged cells. In the 
absence of experimental evidence for a demonstrated causal relationship between persistent telomere 
cohesion and subtelomere recombination, their conclusion that persistent telomere cohesion protects 
against inappropriate recombination is not sufficiently supported.  
 
We have now “toned down” our conclusion “that persistent telomere cohesion protects against 
inappropriate recombination” throughout the manuscript, as indicated below. 
 
In the abstract in line 8. We changed “The persistent cohesion protects short telomeres from 
inappropriate recombination.” to “Our findings suggest that the persistent cohesion protects short 
telomeres from inappropriate recombination.” 
 
On the bottom of page 8 we already say “suggest” – “Our studies thus far in normal primary cells 
suggest that telomere shortening that occurs in the absence of telomerase induces persistent telomere 
cohesion (resulting from the inability of limiting TRF1 to recruit tankyrase 1), which serves as a 
protective mechanism against subtelomere recombination, DNA damage, and premature activation of 
senescence.” 
 
In the last paragraph of page 10 we already say “suggests”- “The absence of subtelomere 
recombination upon forced resolution by telomerase further suggests that it is the critically short 
telomeres that are responsible for the subtelomere recombination.” 
 
On page 11 first paragraph we changed “Our data indicate that as telomeres shorten, persistent 
telomere cohesion driven by insufficient TRF1 (and lack of tankyrase 1 recruitment), protects cells 
from subtelomere recombination and premature activation of senescence.” to  “Our data suggest that 
as telomeres shorten, persistent telomere cohesion driven by insufficient TRF1 (and lack of tankyrase 
1 recruitment), protects cells from subtelomere recombination and premature activation of 
senescence.” 
 
On the top of page 12 we changed “Together these data indicate that as telomeres shorten and cells 
approach senescence, transient persistent telomere cohesion protects cells from subtelomere 
recombination.” to “Together these data suggest that as telomeres shorten and cells approach 
senescence, transient persistent telomere cohesion protects cells from subtelomere recombination.” 
 
In the first paragraph of the discussion on page 12 we changed “As telomeres shorten, the inability to 
recruit sufficient TRF1 and as a result tankyrase 1 leads to persistent telomere cohesion in mitosis that 
protects shortened telomeres from inappropriate recombination and DNA damage-induced 
senescence” to “As telomeres shorten, the inability to recruit sufficient TRF1 and as a result tankyrase 
1 leads to persistent telomere cohesion in mitosis, which we suggest, protects shortened telomeres 
from inappropriate recombination and DNA damage-induced senescence. 
 
On the bottom of page 12 we changed “We showed that persistent cohesion protects cells from 
illegitimate subtelomere recombination.”  to  “Our data suggest that persistent cohesion protects cells 
from illegitimate subtelomere recombination.” 
 
In the middle of page 13 we changed “Thus, persistent cohesion (in any cell type) likely serves a 

protective role by providing shortened (perhaps endogenously damaged) telomeres with a sister for 
DNA repair and by preventing damage-inducing subtelomere recombination with non-sisters.” to 
“Thus, persistent cohesion (in any cell type) may serve a protective role by providing shortened 
(perhaps endogenously damaged) telomeres with a sister for DNA repair and by preventing damage-
inducing subtelomere recombination with non-sisters.” 
 
 
 
2. The authors showed that overexpression of TRF1WT in U2OS cells induces DNA damage and cellular 
senescence, which is in stark contrast to the published report by Silva et al that showed that 
overexpression of TRF1WT in U2OS does not affect cell cycle profile. The authors attributed this 
discrepancy to the difference in their ways of doing experiments, e.g. through Day 5 vs Day 7 by Silva 
et al. It is rather inadequate that the authors just stated that “we cannot say what the FACS analysis 
would look like at Day 7”. Given that their data in Fig. 5O showed that 30% of TRF1WT-overexpressed 
cells are senesced, it is difficult to imagine that the cell cycle profile of these cells on Day 7 would be 
similar to control cells. The authors should at least make the effort to perform FACS analysis (a 
method that they have used in the manuscript) on Day 5 and Day 7 to clarify this discrepancy.  
The discrepancy between their results and the published results begs a concern as to whether an 
excessive amount of overexpressed TRF1WT in U2OS cells (rather than resolution of persistent 
telomere cohesion per se) is responsible for causing subtelomere recombination, DNA damage and 
rapid growth arrest. This concern was not addressed by the authors but needs to be experimentally 
addressed (e.g. knocking down endogenous TRF1 but not overexpressed TRF1WT in U2OS cells), 
especially given that the authors have not demonstrated a direct link that resolution of persistent 
telomere cohesion causes subtelomere recombination.  
 
In the published report by Silva et al, TRF1 or an empty vector control was overexpressed in U2OS 
cells by retroviral infection in order to determine if it would influence the FACS profile upon FANCM 
depletion. Fig. 1h in the paper shows a FACS profile of an empty vector versus TRF1 after 5 days of 
retroviral infection followed by 2 days of control siRNA transfection. The result shows that the FACS 
profile between the empty vector and TRF1 is similar. We see no discrepancy between this piece of 
data and our work. The experiment in Fig. 1h is completely different from what we have done in our 
paper. We infect U2OS cells with TRF1 lentivirus and immediately (following 48 hours of infection) 
seed cells at low density for growth curve analysis. We are taking a snapshot at the earliest time 
possible after infection before cells have a chance to compensate and overgrow. We seed at very low 
density so that we can monitor growth over time. This is completely different from the experiment in 
Fig. 1h. In fact, if we allow the cells to sit in the tissue culture dish at higher (more typical) density for 
several days, the non-senescing cancer cells eventually take over the population. Thus, unlike aged 
fibroblasts infected with TRF1.WT, the non-senescing U2OS cancer cells eventually continue growing. 
We now state this in the paper at the bottom of page 9. The result is specific and not due to 
overexpression because the TRF1.AA allele is expressed to the same extent as WT and has no effect. 
Reviewer 2 cites this specificity in the first line of point 1 above: “The TRF1AA work cited by the 
authors demonstrates that the tankyrase binding site of TRF1 is responsible for the observed 
phenotypes..”. 
 
 
 
3. The authors provided their measurement on the frequency of APBs in their rebuttal. They should 
also cite these APB data and discuss the associated concern in the manuscript.  
 
On the bottom of page 9 we added “The growth arrest was unlikely due to misregulation of ALT 
activity, as there was no change in the frequency of ALT-associated PML-bodies (a hallmark of the ALT 
pathway).” 
 
 
 
4. The authors declined to address whether overexpression of TRF1WT causes increased DNA damage, 
RAD51 foci and growth arrest in BIBR-treated HCT75 PD245 cells. A major concern in this manuscript 
is about the effect of overexpressed TRF1WT on various phenotypes including DNA damage and 
growth arrest (see the point #2 above). Therefore, it is critical that the authors address this issue to 

substantiate their findings.  
 
We did not decline to address the effect of TRF1.WT in BIBR-treated HTC75 PD245 cells. We pointed 
out that the purpose of the BIBR-HTC75 experiment was to artificially generate short telomeres and 
ask if that (in itself) would induce persistent telomere cohesion (in a telomerase positive cancer cell 
line that did not normally exhibit persistent telomere cohesion) and support subtelomere copying upon 
TRF1.WT (but not TRF1.AA) overexpression, which it did. We did not see the value in using this 
artificial system (where telomerase activity is reduced but not eliminated and where cells have been 
extensively passaged in culture) to perform additional analyses, since we had already shown the 
consequences of TRF1.WT (versus TRF1.AA) on RAD51, DNA damage, and growth in cells that 
(naturally) completely lack telomerase (normal aged cells and ALT cancer cells).  
 
Nonetheless, as requested we now provide new data in new Fig. S5F-S5K to address the issue. We 
infected BIBR-treated HTC75 PD245 cells with Vec, TRF1.WT, and TRF1.AA lentiviruses and performed 
immunoblot analysis, immunofluorescence analysis for RAD51 and for DNA damage markers H2AX 
and 53BP1, and growth curve analysis. Similar to our results in normal aged cells and ALT cancer cells 
TRF1.WT, but not TRF1.AA, led to an increase in RAD51 foci (Fig. S5F-S5H) and DNA damage foci (Fig. 
S5I-S5J). We did not observe a growth arrest (Fig. S5K) perhaps due in part to residual telomerase 
activity in BIBR-treated HTC75 cells. We now state these results at the end of the first paragraph on 
page 11.