BRD4 Phosphorylation Regulates the Structure of Chromatin Nanodomains

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

I thanks the authors for their work in addressing the outstanding comments in the manuscript and the supplementary information, and as such and I am now happy to recommend publication of this manuscript.

Author Response

We would like to thank the reviewer again for the insightful suggestion and the rigorous examination. 

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

In this manuscript, Seitz et al studied the relationship between BRD4 and chromatin architecture, using live-cell imaging and molecular dynamics simulation. The authors concluded that both chromatin binding and phase separation of BRD4 might co-regulate the nanoscale chromatin architecture and its microenvironment. However, more evidence is necessary to support these conclusions. 

Major concerns:

1. Regarding the impact of the various BRD4 mutants on the colocalization of BRD4 with nucleosomes, the difference between the mutants and the wildtype is too subtle (Fig 1C and 1D).
2. The authors claimed that a constitutively phosphorylated mutant of BRD4 (7D) condenses nucleosome clutches, while inhibiting chromatin binding of BRD4 by treatment with (+)-JQ1 increases the diffusion dynamics of single nucleosomes and decondenses nucleosome clutches. However, there are two concerns: (i) as shown in Fig 2C, all the three mutants, 7A, 7D, and BD seems lead to  reduced nucleosome dynamics; (ii) The BD mutant that disrupted the chromatin binding of BRD4 showed opposite effect on nucleosome dynamics to the inhibition of chromatin binding of BRD4 by  (+)-JQ1.
3. As shown in Fig 3e and 3f, the BD mutant and the (+)-JQ1 treatment showed opposite effect on the compacting of nucleosomes too.

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

In the revised manuscript is acceptable.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Many problems in the initial manuscript have been solved in this version.

Author Response

Comment: Many problems in the initial manuscript have been solved in this version.

Responses: We would like to thank the reviewer for the rigorous and insightful comments on the manuscript. 

Reviewer 2 Report

Comments and Suggestions for Authors

Unfortunately I am still unable to recommend publication of this manuscript as the authors have not addressed my concerns.

The authors have still not reported how many independent experiments were performed throughout the manuscript. For example, I think the 10 cells analyzed per condition in Figure 1 is not sufficient and I wonder how many independent experiments these 10 cells derive from? Statistical testing should be performed between independent experiments. 

Q1 is still not addressed adequately in my opinion. There is not sufficient detail in the new density distribution analysis to determine how this data shows equivalent expression levels between the wt and mutant proteins. Firstly, the actual data is not presented, so the reader/reviewer cannot appreciate what kind of analysis is performed or the variability in the data. What do they mean by a density distribution? The authors write that they ‘count the number of puncta in a box of width 10um centered on the nucleus’. Thus, it sounds like they are just comparing number of puncta between groups, but this is not clear?  Was the counting performed manually or automatically? If done manually was blinding performed to avoid subconscious bias? If done manually how was segmentation performed? Does the intensity or size of puncta differ between conditions? Simply counting number of puncta is not sufficient to demonstrate comparable expression levels.

Q7 is now addressed.

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for now adding reproducibility information to the manuscript. It is now more convincing to me from the authors response that the various Brd4 mutants are expressed at similar levels, although this is still not apparent within the manuscript itself. The only demonstration in the manuscript that the mutants are expressed at similar levels is the western blot in Figure 1b, which still seems to come from 1 experiment. In the methods section regarding immunoblotting it is not described how many independent experiments were performed. In the legend to Figure 1 it is stated that 3 biological replicates were performed for each group. However this is inconsistent with the authors response that 'we collected 10 cells in WT and mutant groups from one biological replicate to construct the colocalization dataset'. I disagree with the authors assessment that this is sufficient due to the large number of H2B puncta collected from each cell. This data is not independent and thus would not account for numerous potential uncontrolled factors occurring during the experimental process that would influence the results effecting every puncta at the same time and/or every cell in that experiment at the same time. Thus the assumption of the Mann-Whitney test that the observations are independent of each other is violated. Independent experiments are necessary in scientific research to ensure results are reliable and robust. I do not see this in this Figure.