The overarching goal of this manuscript by Pradeepa et.al is to identify if PSIP1 isoforms provide a binding platform for DNA repair proteins, which would explain how H3K36me3 functions in DNA repair. They use proteomics to identify, in a somewhat unbiased manner, the interacting partners of H3K36me3 chromatin, and PSIP1/p75. They found a substantial overlap between the binding partners of H3K36me3 and p75. Furthermore, they perform co-IP with Y-H2A.X to validate their Ip-mass spectrometry data. Finally, they show that Psip1-/- cells have higher level of unrepaired DNA damage, thereby alluding to a role for Psip1 -/- in DNA repair.

Specific Comments: As far as I can tell, all of the mass spectrometry experiments have been performed without the induction of any DNA damage. This implies that one is looking at some combination of intrinsic DNA damage associated binding partners, which would be heavily influenced by transcriptional biases (related to gene length, transcriptional frequency and exons), and cell cycle phases. To directly make a conclusive statement regarding if PSIP1 acts as a scaffold for recruiting DNA repair proteins, it would be beneficial to monitor the interaction(s) with and without DNA damage. Given the extensive rewiring of cellular signaling after DNA damage, having a more direct readout of differential interacting partners would be more beneficial to refine the various models presented in figure 3d.

Figure 3 can be improved by testing some other key interacting partners from table 1. Specifically, components of FACT complex and PARP1 should be tested in co-IP experiments since both of them are key regulators of DNA repair as well.

Fig. 3: Additional evidence for unrepaired DNA damage/abrogated DNA damage signaling in Psip1-/- should be provided. E.g. Y-H2A.X retention kinetics after DNA damage in Psip1-/- cells. Datasets1 and 2 should be labeled in the same manner as the OSF files.

The authors state “Interestingly, with the exception of….” and cite Fig.2 for this. They should also refer to table1, alongside Fig.2 to clarify the overlaps.

Can the authors phenocopy fig.3c with a PWWP mutant of p75 in order to directly test if H3K36me3- p75 are part of the same mechanism in DNA repair? Is the work clearly and accurately presented and does it cite the current literature?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests No competing interests were disclosed.

Reviewer Expertise Epigenetics, Genome Stability, Cell Cycle, Cancer Epigenetics, Hematopoiesis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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