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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This manuscript provides new insights into how DSBs are induced by inhibition of BET family proteins 
including BRD4. Previously, these authors demonstrated that a variant of BRD4 protected chromatin 
from DNA damage response by insulating chromatin from DNA damage signaling. The source of this 
damage upon inhibition of BET family was not clear. Here, this manuscript tries to address this 
question by showning that BRD4 inhibition induces transcription-replication conflicts as a result of R-
loop formation. This suggests that the BET family proteins are important for the correct loading of 
transcription co-activator complexes such as pTEFb to regulate transcription and avoid R-loop 
formation. The observation that R-loop formation by BRD4 inhibition is novel and builds upon this 
groups previous work showing that inhibition of BRD4 induced DNA damage responses. Overall this 
work is interesting but there are still several issues that need to be addressed before publication. 

Major comment: 

1. Inhibition of BRD4 induced transcription-replication conflicts in S phase. In this state, BRD4 knock 
down cells are likely to be arrested in S and G2 phase. However, in these groups previous studies, 
BRD4 knockdown cells do not display any cell cycle defects (Scott R. Floyd et al., Nature, 2013). In 
addition, JQ1 treatment also did not induce phospho-H2AX (Figure 4e of this paper). One possibility is 
that this phenomenon occurs in a sub-set of cancer cells and its mechanisms are not universal. These 
data seem at odds and need to be explained. 

2. Given that the authors claim that these effects occur in S-phase, cell cycle analyses for all key 
experiments need to be performed. This includes Triptolide and RNase H1 o/e analyses. In addition, 
growth curves -/+ JQ1 with and without RNase H1 should be performed to see how these treatments 
effect cell proliferation and not just the phenotypic readouts of DNA damage. 

3. The data in figure 5B, in the quantification graph show that BRD4 knock down cells only increased 
phospho-H2AX level but in WB image, BRD3 knockdown cells also increased phospho-H2AX even if the 
actin level was less than control and BRD4 knockdown cells. The graph and WB image seem 
inconsistent. Given the poor quality of these western blots, new data should be provided and for these 
experiments unmodified H2AX should be used and total PARP staining should be analyzed. 

4. There are three isoform of BRD4 in the cells (Scott R. Floyd et al., Nature, 2013). Which BRD4 
isoform affects R-loop formation? 

5. In this paper, BRD4 inhibition induces DNA damage and it would be interesting to know if this 
relates with deregulated transcription. Is it dependent on dysfunction of pTEFb on transcription? One 
might think that BRD4 inhibition should reduce transcription. Similarly, Triptolide also inhibits 
transcription but has opposite effects. This should be explained. 

6. Based on these data, it is curious that BRD4 isoform A knock down in cells does not increase 
phospho-H2AX levels in undamaged cells ((Scott R. Floyd et al., Nature, 2013)? BRD4 isoform A 
contain the CTM domain, which is critical for interaction with pTEFb. Does BRD4 CTM mutant show the 
same phenotype as BRD4 knock down? 

7. If R-loop formation induces DNA damage, why do the foci of S9.6 not co-localize with DNA damage 
markers. This is a major issue that must be fully addressed. One possibility is that the phospho-H2AX 
seen is a result from apoptosis and not R-loops. All the data shown shows that phospho-H2AX and 
apoptosis are occurring. Perhaps altering treatment conditions could provide a scenario where DNA 

damage is induced but not enough to provoke apoptosis. One could also block apoptosis and show 
that DNA damage still occurs. 

Minor comment: 

1. In the figure1, panel C, phospho-H2AX should be white bar not black. 

2. In page 6, 2nd low, DBS formation should be DSB formation. 

Reviewer #2 (Remarks to the Author): 

In this manuscript, the authors uncover a previously unappreciated function of BET bromodomain 
proteins, particularly BRD4, in the regulation transcription-replication conflicts by preventing R-loop 
formation. They show that inhibition of BET bromodomain proteins leads to increased DSB formation 
and apoptosis in different cancer cell lines. The authors use a specific transcription initiation inhibitor 
(triptolide) to show that the DNA damage and apoptotic phenotype observed with BET bromodomain 
inhibitors requires active transcription. Next, they propose that this damage originates primarily as a 
consequence of replication-transcription conflicts because it occurs primarily within the S-phase. 
Finally, they conclude that DNA damage is due to the accumulation of R-loops by combining 
experiments with the S9.6 antibody that specifically detects R-loops and RNase H1 that suppresses R-
loop formation. Lastly, they selectively knockdown distinct BET bromodomain proteins by siRNA and 
suggest that BRD4 is primarily responsible for the observed phenotype. Collectively, this is an 
interesting study that points to a novel function of BET bromodomain proteins in regulating R-loop 
formation and replication-transcription conflicts. The studies are however too preliminary and have not 
been developed to the point that it should be considered for publication in a high-profile journal. 

Major concerns: 

1. The authors’ statement that BET bromodomain proteins are important to prevent R-loop formation 
is not supported by the data. The authors cannot distinguish whether loss/inhibition of BET 
bromodomain proteins leads to increased R-loop accumulation because these proteins are needed to 
prevent R-loop formation, as the authors suggest, or to favor R-loop resolution. The authors should 
include further experiments to distinguish between these two alternative possibilities. 

2. The authors should include more experiments to provide mechanistic insights into the actual role of 
BRD4 in R-loop regulation. 

3. The authors should explore the role of the previously reported interactions of BRD4 with the 
positive elongation factor (P-TEFb) and the transcriptional co-activators JMJD6 and CHD4 in regulating 
transcription-replication conflicts and R-loop formation. 

4. The authors should include additional experiments to strengthen their conclusion that the DNA 
damage observed with BET bromodomain inhibitors originates from transcription-replication conflicts. 
For example, they could use single-molecule DNA fiber assays to directly monitor replication 
perturbations and demonstrate that replication is impaired when cells are treated with the with BET 
bromodomain inhibitors. 

5. The authors should include a quantification for the EdU incorporation experiments shown in Figure 
2A. 

6. Figure 3. The authors should measure the percentage of S-phase cells. On the basis of the authors’ 
model that R-loop accumulation perturbs replication progression, they might find an increased 
percentage of S-phase cells following treatment with BET bromodomain inhibitors, due to S-phase 
arrest. Along the same line, they might find a decreased EdU incorporation because of the increased 
fork stalling associated with R-loop accumulation. 

Reviewer #3 (Remarks to the Author): 

This article describes the discovery that BRD4 inhibition leads to transcription-coupled DNA damage. 
This damage is at least partly driven by the accumulation of R-loops which the authors speculate is 
due to transcriptional dysregulation. Overall the story presented is very straightforward and the 
authors have been rigorous in applying multiple reagents to inhibit or deplete BRD proteins and show 
their results are consistent in different cell lines. The flip side of this is that the story can be viewed as 
quite superficial from a mechanistic perspective, and the authors themselves point out that the 
mechanism of R-loop induced DNA damage in BRD4-depleted cells remains unknown, even concluding 
the discussion with a suggested experiment: DRIP-sequencing of BRD4-depleted cells. 

The work is solid and supports the conclusions, but is at an observational stage, rather than a 
mechanistic stage. The observations are interesting and below I make suggestions for improvements. 

1. The S9.6 antibody has now been used repeatedly for precipitation of R-loops. I suggest that DRIP 
experiments, followed by qPCR at known BRD4-target loci, (or by deep sequencing if feasible), would 
enhance the work. It would also allow some direct tests of mechanisms. For example, to show that 
BRD4 ChIPs to the same locus, then upon depletion, R-loops accumulate at that locus. This would at 
least implicate a direct model of R-loop induction rather than other indirect effects on gene expression 
networks. 

2. The article uses comet assays and total g-H2AX staining intensity throughout to monitor DNA 
damage. While they do correlate the damage with S-phase, exploration of the damage response is 
quite limited. The authors might consider native BrdU immunofluorescence, or monitoring RPA2-S33 
phosphorylation, and probing the ATR signaling pathway to really establish that DNA replication stress 
is arising from the BRD protein depletions. 

3. I found it unfortunate that chemical inhibitors with multiple targets were used for all experiments 
except those in Figure 5 where siRNA was used to specifically implicate BRD4. It would be nice to at 
least repeat the triptolide experiments in the BRD4-siRNA setting, or probe the mechanism more 
deeply with the BRD4-siRNA reagents which would appear to be more specific than the 
inhibitors/degraders. 

4. While the model of R-loop associated replication stress rests on considerable literature, the idea 
that ‘deregulated transcription’ leads to these events in BRD4-depleted cells is totally unclear. In the 
discussion the idea is raised that transcriptional pausing in the absence of BRD4 is increasing the 
opportunity of a nascent RNA to re-anneal with the template strand to create an R-loop. However, the 
authors do not provide much data to support this view. As above, additional experiments targeting a 
specific locus that accumulates R-loops in BRD4 depleted cells for co-accumulation of paused RNA 
polymerase could be important. 

5. In general a clearer statement of the proposed mechanism (i.e. what does transcriptional 
deregulation mean as a predisposing factor for R-loop induced DNA damage) and supporting data 

would significantly increase my enthusiasm for this study. There are many possible experiments that 
would be helpful and I don't mean to suggest that all avenues need to be explored, but testing some 
specific models is required. 
For example, if RNA-seq data on transcriptional dysregulation after BRD protein depletion was 
available, then correlating these data with published R-loop maps might be instructive (i.e. if you 
predict that promoter-proximal pausing is driving R-loops then perhaps BRD4 targets would be R-loop 
prone). Alternatively, many groups have now had success using proximity ligation to show R-loop 
association in cells, or an increase in replication-transcription conflicts in situ. 

Minor points: 
The authors state that “Despite potent suppression of MYC, treatment with triptolide alone did not 
result in increased DNA damage signaling, DSBs, or apoptosis in cells…”. Surely triptolide would 
eventually kill cells. I think the time frame of the experiment is what allows the observations with 
triptolide to exert the effects seen and that this should be acknowledged. 

- Why is the time frame of ARV-825 treatment in Figure 3 (6 hours), different than in Figures 1 and 2 
(3 hours)? It would seem that consistency between experiments or an explanation would be preferred. 

- The authors state in the discussion that “… it cannot be excluded that the RNA:DNA hybrid within the 
transcription bubble can remain annealed and be extended as RNAP moves forward.” How likely do the 
authors think this is? RNAPII extrudes nascent RNA through an exit tunnel that is physically displaced 
from the DNA template. This just seemed unlikely to be correct based on what we know about RNAPII.