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


Reviewers' comments:
Reviewer #1 (Remarks to the Author):
In the revised manuscript, the authors have made a good effort to answer my previous comments
On the other hand, I still believe that the differences in the length of S phase (around 10% faster
in siE2F6) are too small and such modest differences can be the result of experimental variation.
Also, the difference of fork speed after siE2F6, around 10%, is too small to make any solid
conclusions along the lines the authors put forward.
Below, I specifically comment on the issues that still remain to be convincingly addressed, in the
interest of authors' own reputation and providing solid and well supported evidence. Hence, before
suggesting the acceptance of their manuscript, the authors should show convincingly that E2F6-
depletion indeed does accelerate fork speed during mid/late S phase. This is an important part of
the mechanism to explain how low-level E2F6 reduces the length of S phase without altering origin
activity. At this stage, I am not satisfied with their answers to the following related questions
raised in the original submission.
Specifically,
11. (original concern): 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.
Authors response: We agree with the reviewer that the effect on S phase length, seen in the single
cell time lapse analysis, is not obvious in a synchronized RPE1 cell population. We believe that this
is due to the lack of a robust synchronous release with RPE1 cells, which probably masks the
decrease in S phase length in this setup. RPE1 cells are hard to arrest and synchronously release.
Whilst the enrichment in the S phase population does allow us to observe an increase in DNA fibre
length in middle/late S phase the extent of the synchrony is insufficient to evaluate S phase
length. It is therefore, unfortunately, not possible to draw any conclusions about S phase length
from this data.
My new comment on the rebuttal in this point:
Therefore it is not possible to draw any conclusion about different stages of S phase and correlate
those with increased fork speed in mid/late S phase due to the lack of robust synchrony that the
authors are experiencing with this model.
Authors could evaluate fork speed using synchronised T98G cells +/-siE2F6 to test fork speed at
mid/late S phase.
Alternatively, following the logic of the results presented in Fig 3, authors could test the length of
S phase in RPE1 cells that have been manipulated to overexpress E2F6.
12. Original concern: 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
 Authors' response: Unfortunately, as mentioned above the lack of synchrony, from a release form
a contact inhibition arrest, masks synchronous progression through S phase of a RPE1 cell
 population. We have therefore established EdU incorporation in asynchronous cells, Fig 2a.
 My new commenton the rebuttal in this point:
 This just further supports my previous serious concern and the fact that the data is still
 inconclusive.
 13. Original concern: Figure 3a. The authors should confirm their findings by investigating the
 level of PCNA by immunoblotting in the same experimental settings.
 Authors' response: We have added this analysis in Supplementary Figs 2d (asynchronous cells)
 and 3a (synchronised cells).
 My new related comment on the rebuttal in this point:
Other comments were addressed adequately.
Reviewer #2 (Remarks to the Author):
 Authors could analyse and plot the PCNA mean intensity vs DNA content using high-content image
 analysis. This will show directly that cells in mid/late S phase contain higher level of PCNA. In
 Cimprich et al, (Science 2018) there are examples of this type of analysis using EdU vs DNA
 content. In Pennycook’s manuscript, authors should pre-extract cells to quantify PCNA associated
 to chromatin and correlate the level with the DNA content +/-siE2F6.
   I am utterly confused by the response to my request to analyze the status of Mcm2-7 binding in
 the second G1 phase (Fig. 4). The authors basically argue that the cells are no longer
 synchronized. However, they don't say this in the results section of their manuscript, and claim
 that the second G1 phase is longer (Fig. 4c). They can't have it both ways. I don't understand why
 they didn't attempt to assess Mcm2-7 loading dynamics in the second G1. If cells are labeled in
 parallel by Edu and DAPI, then the portion of cells in G1 can be clearly quantified and Mcm2-7
 loading can be determined. The authors claim in their response that "Therefore, if there is a
 diminished origin
 licensing in the second cycle we should expect a decrease in Mcm binding,
 which we don’t observe." Where is the data that shows that Mcm2-7 binding is not decreased?
 That's the very piece of data I was asking for. The fact that p21 expression is increased and G1 is
 longer in the granddaughters clearly points to a potential licensing defect in the second G1 phase.
 As I said in my original review, this part of the study needs to be further corroborated.
 Understanding why DNA damage occurs in the 2nd cell cycle is critical to understanding the
 consequences of changes in S phase dynamics.
 Reviewer #3 (Remarks to the Author):
 While authors show that replication dynamics change upon alteration of E2F transcription, in order
 to claim that E2F determines “replication capacity” to regulate S-phase length authors should
 demonstrate that E2F transcription actually responds to changes in replication dynamics (e.g.
 origin firing frequency). In the reviewed manuscript authors have not addressed this major
 concern, nor they have investigated whether regulation of dNTP levels is a key driver for the
 observed changes of replication dynamics upon manipulation of E2F transcription (as opposed to a
 general increase in replication factors postulated to be limiting for replication speed). Hence, the
 
data presented fail to provide strong support to the manuscript’s central conclusion. In order for
 the study to be suitable for publication in Nature Communications authors should provide strong
 evidence that E2F transcription actually dynamically responds to replication changes (origin firing
 or fork speed) to maintain “replication capacity” constant or strictly limit their conclusions to the
 impact of E2F transcription on replication fork speed throughout the manuscript.