Peer review of: BRD4 prevents the accumulation of R-loops and protects against transcription–replication (2020)


Reviewers' comments: 

Reviewer #2 (Remarks to the Author): 

The authors have done an excellent job in addressing my previous concerns and the new data greatly 
improve the manuscript. However, the authors should explain how BET bromodomain protein loss 
leads to increased phosphorylation of RPA2 Ser33 (Figure 8B and S7B), while impairing the ATR-
TopBP1-Chk1 pathway (Figure 10). 

Reviewer #3 (Remarks to the Author): 

The manuscript by Lam et al,Yaffe and Floyd has been revised with extensive new data, 
experimentation and validation. Many of these new experiments are in response reviewers 
suggestions (isoforms, cell cycle defects) and I appreciate the authors careful attempts to address our 
comments. One problem with this new data is that the manuscript is now dramatically extended in its 
scope which clouds the molecular mechanism and model that the authors are really proposing. While I 
appreciate the tremendous amount of work, the authors may be better to reframe this story in simpler 
terms. Below I will outline my persistent concerns. 

The major issue that remains is that the article does not present a clear and cohesive model for how 
BRD4 prevents transcription-replication conflicts. The authors present models in Figure 7 and Figure 
10 which incorporate their new observations that gene expression of key R-loop regulators is altered, 
leading to R-loops, TRCs and DNA damage. This is a perfectly reasonable model that I believe is likely 
to be true, but statements throughout the manuscript and inconsistencies in timing of their 
experiments undermine this clear message. 

The paper should be simplified to focus on the mechanism of TRCs in BRD4 depleted cells and whether 
or not this is due to changes in gene expression alone, or whether there are direct physical effects of 
BRD4 on R-loops or the replisome. 

- On Page 23, line 567, the authors still invoke RNAPII stalling as a short term mechanism of TRCs 
after BRD4 loss. If this is true and part of their model then they should experimentally support it or 
describe the literature in more detail. 

- Loss of expression of SETX, DHX9 and SRSF1 is a great explanation for their observations, except 
that DNA damage and R-loops are seen after 6 hours of BRD4 depletion (Fig. 2 and 3) while SETX is 
depleted after 24 hours and DHX9 and SRSF1 are depleted after 48 hours (Figure 7). So this leaves a 
major gap in our understanding of the mechanism. If the early effects of failure to elongate 
transcription lead to R-loops and TRCs in cis to BRD4 sites then the article should focus on that rather 
than the late effects of gene expression. If there is a direct effect on all BRD4 target genes then the 
downregulation of SETX 18 hours later just confuses the issue. The same problem exists for the 
ATRIP/ATR/TOPBP1/CHK1 qPCR data which are done at 48 hours. 

- The confusion around the proposed model is enforced by the wording in places. For example, on 
page 22 line 525-526 the authors state “… supports our model that a normal function of BRD4 in these 
cells is to suppress collisions between the transcription and replication machinery.” I feel this invokes 
a very direct model not clearly articulated or supported by the authors. Based on the model where loss 
of R-loop interactor expression is the key driver, the function of BRD4 is just to regulate transcription 
not suppress collisions as indicated. 

- The bottom of Page 15 also begins to invoke a kind of two-step model that BRD4 regulated genes 
form R-loops when BRD4 is lost, and that these genes themselves are R-loop regulators. Which of 
these R-loop promoting activities of BRD4-loss are important for the authors observations is not clear. 

Other issues: 
- I am not convinced by the data in 6B that only a subset of BRD4-loss-induced R-loops cause DNA 
damage. I doubt very much that the foci represent single R-loops that can be classified as with or 
without H2AX signal – there are 1000s of R-loop sites in the genome. It would be necessary to show 
BRD4-loss-induced R-loops that are damaging and BRD4-loss-induced R-loops that are not damaging 
to understand that a specific subset are damaged. I felt that this data was unnecessary. 

- I did not understand the DNA combing data as presented. The authors inhibit BRD4 with JQ1 or 
ARV825 for 16 or 6 hours respectively, then they add IdU, then CldU for equivalent times. The fiber 
images show that IdU is incorporated normally, but that absolutely no CldU is incorporated. If 
replication is stalled after BRD4 inhibition, then neither label should be incorporated effectively. Or if 
there were more induced replisome stalling events, then the ratios of green to red track lengths 
should not equal 1. Given the representative images and the bar graph presentation, what the data 
actually shows is unclear and if the fibers shown are representative then there seems to be a technical 
problem.