Peer review of: E2F-dependent transcription determines replication capacity and S phase length (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
In this manuscript, Pennycook et al. intend to propose a new concept, called ‘replication capacity’, 
which the authors define as the maximum amount of DNA a cell is able to synthesize per unit of 
time throughout S phase. The authors argue that E2F-dependent transcription determines the 
replication capacity of a cell. The manuscript is well written, however, the conclusions are 
overstated and the experimental evidence is rather preliminary. Some of the results are 
inconsistent between experiments and some statistical differences are arguable. In general, the 
authors test the effect of knocking-down E2F6 during S phase. With this, the authors claim in the 
title that E2F activity determines replication capacity, which is an overstatement. The comments 
below indicate the multiple problems with this manuscript, in terms of experimental design, 
controls and interpretation. Overall, the present results do not support the author’s model and 
conclusions. I suggest addressing at least some of the specific comments listed below before 
submitting the manuscript elsewhere. 
 
Major comments: 
 
1. The authors modulate E2F activity only via E2F6, but claim that general E2F activity is the driver 
for replication capacity (the maximal amount of DNA a cell is able to synthesise per unit time 
throughout S phase). Proof for their hypothesis falls short as no general E2F activity readout is 
provided and a positive E2F transcritption factor (e.g. E2F1) is not modulated whatsoever. The 
authors should therefore focus on claims for E2F6-dependent mechanisms on the title and their 
conclusions or include more comprehensive E2F readouts. 
2. Related to 1 above, the authors should also evaluate the effect of knocking-down E2F4 and 
E2F3 (Giangrande et al., Genes and Dev, 2004) in S phase using RPE1 as a cellular model. 
(currently only E2F6 has been looked at). 
3. Many controls are missing in the experimental settings of the cell cycle profile analysis. Cells 
were synchronized by serum starvation and then treated with nocodazole. The authors should 
therefore include siControl and siE2F6 cell cycle profiles without synchronization, only synchronized 
without nocodazole treatment and follow them up to 48 h. 
 
4. The assessment of DNA damage (Fig. 4) is not sufficient. No proper readouts of replication 
stress, such as RPA foci detection or 53BP1 body formation in the following G1 were used. For 
RPA2 foci detection, cells have to be pre-extracted before fixation and analysis, as free 
nucleoplasmic RPA would largely abolish foci detection. Alternatively, WB analysis for 
phosphorylated ATR or Chk1 could have been used. The mere fact that higher levels of p21 exist in 
E2F6-depleted cells in the second cell cycle after G1 release shows that replication stress must be 
present in the first cell cycle. The provided analysis is not sufficient and comprehensive enough to 
draw any conclusions. 
 
 
 
5. Figure 1b. The authors should show western blots of some representative examples of cyclins, 
licensing factors, E2F6a, DNA synthesis and repair proteins and repressors of G1/S, by 
synchronizing cells as in C and blotting different proteins after the release. 
 
6. Figure 1c. These cell cycle profiles of T98G cells should be replaced by an equivalent experiment 
using RPE1 cells. T98G cells are p53 mutant and clearly progress differently through the cell cycle 
after arrest than RPE1 cells (see S3b). Especially that this is the only one experiment where the 
authors used T98G. Perhaps it is due to some historical reasons that this cell line is included, as 
the authors have published other papers using T98G (Bertoli et al., Curr Biol, 2013). 
 
 

 
7. Figure 2a. A color code should be presented in the scattering plots for siControl and siE2F6, 
including gating strategy. The differences in EdU intensity are not convincing and the results from 
triplicates should be presented as a bar plot with S.D. 
 
8. Figure 2b. The authors must measure the distance between origins using DNA fibre/combing 
techniques. PCNA intensity is not a direct measure of origin activity. PCNA is also needed to repair 
DNA and it can be engaged in replication factories for longer time in the presence of arrested 
forks. In any case, the authors should present the scattering plots from flow cytometry from 
siControl and siE2F6 cells. In the current figure, it is not clear, which cells were used as the 
example. 
 
9. Figure 2c. Approximately 200 forks were counted for each condition. Perhaps the number of 
analyzed forks is not sufficient, as there is a discrepancy in the fork length with other similar 
experiments. There is plenty of room in the supplementary figures to show individual fibre 
experiments to judge the difference between experiments. 
 
10. ‘E2F-dependant transcription in S phase results in an increase in replication fork speed, thus 
increasing the overall DNA synthesis rate without  any detectable change in origin licensing or 
firing’ – again this sentence is overstated. The authors should confirm their findings using classical 
biochemistry experiments, like tritium-labelled thymidine incorporation and measurement of the 
ori-to-ori distance by immunofluorescence. 
 
11. Why do siE2F6a-treated cells show no faster S phase after release from contact inhibition 
(Suppl. Fig 3), but faster replication forks? This contradicts the main message of the paper. It 
rather seems that contact inhibition and serum starvation have different impact on S phase length 
after E2F6-depletion despite increased fork speed, which is not addressed by the authors (Fig. 1 
and Suppl Fig. 1 in contrast to Suppl. Fig. 3). 
 
This study is indicative: 
Yang HW, Chung M, Kudo T, Meyer T. Competing memories of mitogen and p53 
signalling control cell-cycle entry. Nature. 2017 Sep 21;549(7672):404-408.  doi: 
10.1038/nature23880.  Epub 2017 Sep 6. PubMed PMID: 28869970. 
 
 
12. Also, the cell cycle profiles in the Figure S3b at 24 h look similar between siControl and siE2F6. 
The authors should quantify the amount of cells in early-mid-late S by EdU incorporation after the 
release 
 
13. Figure 3a. The authors should confirm their findings by investigating the level of PCNA by 
immunoblotting in the same experimental settings. 
 
14. Figure 3b. Interestingly, the authors found that the fork length was similar in siControl cells in 
early and mid/late S phase, (the average length in these cells was ~16 um). In the Figure 2c, for 
siControl the average length in non-synchronized cells was 17.8. Is this difference due to technical 
problems of the DNA fibres or due to synchronization? Fibre results from equivalent experiments 
should be presented and the number of scored fibres for each experiment should be increased. 
 
15. Figure 3c. Is the fork length significantly different between early vs mid/late S, 17.2 vs 18.6? 
Any explanation why are these numbers different from the ones presented in the Figure 3b for 
siControl untreated cells? 
 
16. Figure 4. gH2AX and RPA intensities can be correlated with DNA content. The cell cycle profiles 
from generations 0, 1, 2 should be shown. I speculate that the cells start to arrest in G1 due to 
DNA damage. 

 
 
Minor comments: 
 
• It probably is just semantic but the conclusion from results presented in the Figure 1 are 
overstated (Page 4, the end of paragraph 1). A more precise conclusion should be – kd-E2F6 cells 
spend less time in S phase. 
 
 
• In line 106, incomplete sentence: “This allowed us to monitor.” 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
The study by Pennycook et al. analyzes the role of E2F in controlling S phase length by 
determining what the authors term “replication capacity”. Replication capacity is a novel and useful 
parameter of a cell’s DNA synthesis kinetics. The authors essentially demonstrate that E2F 
regulates the expression of S phase factors and actively adjusts those expression levels to ensure 
completion of genome duplication with a given number of active replication origins. Importantly, 
the study modulates E2F6, which represses E2F-driven transcription of S phase proteins in late S 
phase. The authors show convincingly that E2F6 knockdown accelerates S phase progression in 2 
different (a transformed and nontransformed) cell lines by increasing fork speed in late S phase. 
The data is well-controlled, statistics are solid and the conclusions are well justified. Previous 
studies have shown that an increase in fork speed is associated with DNA damage. The authors 
show here that this is not necessarily a direct effect. This is an important observation. The finding 
that low level damage can be observed in the second cell-cycle after E2F6 knockdown is 
theoretically a consequence of diminished origin licensing, which would explain why p21 
expression is increased and G1 is extended in the KD cells. This has not been explored rigorously 
but could be easily done by analyzing Mcm2 and Mcm7 chromatin association at the end of G1 of 
the second cycle. Examining this point would make this particular section of the paper clearer. 
 
Overall, this is a rigorous and important study. 
 
 
Reviewer #3 (Remarks to the Author): 
 
In this manuscript Pennycook et al. explore the impact of E2F transcriptional activity de-repression 
in S-phase dynamics of human cells. Authors observe that increasing E2F transcription by 
interference of the repressor E2F6 results in a faster S-phase completion. A slight increase in EdU 
incorporation is observed in E2F6 interfered cells, which correlates with longer replication tracks in 
DNA fibers, suggesting that enhanced E2F transcription increases fork speed. Conversely, 
overexpression of E2F6 in S-phase reduces replicated fiber length and EdU incorporation. Lastly, a 
slight increase is observed in gH2AX phosphorylation and RPA2 intensity, proxies of DNA damage 
signaling and replication stress, respectively, in E2F6 interfered cells, which correlate with an 
increase in p21 foci and slight lengthening in G1 duration. Based on these results authors propose 
that E2F controls the replication capacity of the cell (defined by fork speed and number of active 
forks), as part of a mechanism that would respond to fluctuations in replication initiation events to 
maintain absolute synthesis rates and S-phase duration. 
 
The data presented are in general of good quality and the conclusions are original and of potential 
interest for researchers in the replication and cell cycle fields. However, there are some issues 
about experimental design and data interpretation raising concerns that should be addressed to 
support the main conclusions and for the model to be convincing. 
 

 
Major points: 
 
- Authors define the concept of a replication capacity, which should be kept somewhat constant to 
promote optimal S-phase length and replication speed, and propose that E2F transcription works 
within a mechanism controlling replication capacity. Replication rates and fork speed seem 
increased upon E2F6 interference. However, to conclude that replication capacity is enhanced by 
E2F transcription in this context authors should prove that the frequency of initiation events is 
unchanged. This should be straight forward, as origin firing frequencies could be measured in the 
double label fiber experiments shown. MCM7 data on figure 2b seem to be an estimate of nuclear 
MCM levels rather than a measure of origin licensing/firing. 
 
- Authors should provide evidence that E2F transcription responds to changes in replication 
initiation event numbers to support the notion that it acts within a mechanism regulating 
replication capacity. 
 
- A key point is which E2F targets promote an enhancement of replication fork speed. A 
straightforward mechanism would be upregulating dNTP pools via induction of ribonucleotide 
reductase expression. Authors should measure dNTP levels in control and E2F6 interfered cells to 
rule out this possibility. 
 
Minor point: 
 
- DNA damage signaling and replication stress are addressed 21 hours after cell cycle block release 
when a small proportion of cells (5-7%) is in S-phase. These experiments would be more 
informative if performed at later time points when more cells have ongoing replication.