PHD1-dependent hydroxylation of RepoMan (CDCA2) on P604 modulates the control of mitotic progression

Reviewer #1 (Public review):

Summary:

The study by Druker et al. shows that siRNA depletion of PHD1, but not PHD2, increases H3T3 phosphorylation in cells arrested in prometaphase. Additionally, the expression of wild-type RepoMan, but not the RepoMan P604A mutant, restored normal H3T3 phosphorylation localization in cells arrested in prometaphase. Furthermore, the study demonstrates that expression of the RepoMan P604A mutant leads to defects in chromosome alignment and segregation, resulting in increased cell death. These data support a role for PHD1-mediated prolyl hydroxylation in controlling progression through mitosis. This occurs, at least in part, by hydroxylating RepoMan at P604, which regulates its interaction with PP2A during chromosome alignment.

Strengths:

The data support most of the conclusions made. However, some issues need to be addressed.

Weaknesses:

(1) Although ectopically expressed PHD1 interacts with ectopically expressed RepoMan, there is no evidence that endogenous PHD1 binds to endogenous RepoMan or that PHD1 directly binds to RepoMan.

(2) There is no genetic evidence indicating that PHD1 controls progression through mitosis by catalyzing the hydroxylation of RepoMan.

(3) Data demonstrating the correlation between dynamic changes in RepoMan hydroxylation and H3T3 phosphorylation throughout the cell cycle are needed.

(4) The authors should provide biochemical evidence of the difference in binding ability between RepoMan WT/PP2A and RepoMan P604A/PP2A.

(5) PHD2 is the primary proline hydroxylase in cells. Why does PHD1, but not PHD2, affect RepoMan hydroxylation and subsequent control of mitotic progression? The authors should discuss this issue further.

Reviewer #2 (Public review):

Summary:

This is a concise and interesting article on the role of PHD1-mediated proline hydroxylation of proline residue 604 on RepoMan and its impact on RepoMan-PP1 interactions with phosphatase PP2A-B56 complex leading to dephosphorylation of H3T3 on chromosomes during mitosis. Through biochemical and imaging tools, the authors delineate a key mechanism in the regulation of the progression of the cell cycle. The experiments performed are conclusive with well-designed controls.

Strengths:

The authors have utilized cutting-edge imaging and colocalization detection technologies to infer the conclusions in the manuscript.

Weaknesses:

Lack of in vitro reconstitution and binding data.

Reviewer #3 (Public review):

Summary:

The manuscript is a comprehensive molecular and cell biological characterisation of the effects of P604 hydroxylation by PHD1 on RepoMan, a regulatory subunit of the PPIgamma complex. The identification and molecular characterisation of the hydroxylation site have been written up and deposited in BioRxiv in a separate manuscript. I reviewed the data and came to the conclusion that the hydroxylation site has been identified and characterised to a very high standard by LC-MS, in cells and in vitro reactions. I conclude that we should have no question about the validity of the PHD1-mediated hydroxylation.

In the context of the presented manuscript, the authors postulate that hydroxylation on P604 by PHD1 leads to the inactivation of the complex, resulting in the retention of pThr3 in H3.

Strengths:

Compelling data, characterisation of how P604 hydroxylation is likely to induce the interaction between RepoMan and a phosphatase complex, resulting in loading of RepoMan on Chromatin. Loss of the regulation of the hydroxylation site by PHD1 results in mitotic defects.

Weaknesses:

Reliance on a Proline-Alanine mutation in RepoMan to mimic an unhydroxylatable protein. The mutation will introduce structural alterations, and inhibition or knockdown of PHD1 would be necessary to strengthen the data on how hydroxylates regulate chromatin loading and interactions with B56/PP2A.

Author response:

Public Reviews:

Reviewer #1 (Public review):

We appreciate the reviewer’s agreement that our data, "support most of the conclusions made”.

With respect to Concerns raised by reviewer 1:

(1) Although ectopically expressed PHD1 interacts with ectopically expressed RepoMan, there is no evidence that endogenous PHD1 binds to endogenous RepoMan or that PHD1 directly binds to RepoMan.

We do not fully agree that this comment is accurate - the implication is that we only show interaction between two exogenously expressed proteins, i.e. both exogenous PHD1 and RepoMan, when in fact we show that tagged PHD1 interacts with endogenous RepoMan. The major technical challenge here is the well known difficulty of detetcing endogenous PHD1 in such cell lines. We agree that co-IP studies do not prove that this interaction is direct and never claim to have shown this, though we do feel that a direct interaction is most likely, albeit not proven.

(2) There is no genetic evidence indicating that PHD1 controls progression through mitosis by catalyzing the hydroxylation of RepoMan.

We agree that our current study is primarily a biochemical and cell biological study, rather than a genetic study. Nonetheless, similar biochemical and cellular approaches have been widely used and validated in previous studies in mechanisms regulating cell cycle progression and we are confident in the conclusions drawn based on the data obtained so far.

(3) Data demonstrating the correlation between dynamic changes in RepoMan hydroxylation and H3T3 phosphorylation throughout the cell cycle are needed.

We agree that it will be very interesting to analyse in more detail the cell cycle dynamics of RepoMan hydroxylation and H3T3 phosphorylation - along with other cell cycle parameters. We view this as outside the scope of our present study and are actively engaged in raising the additional funding needed to pursue such future experiments.

(4) The authors should provide biochemical evidence of the difference in binding ability between RepoMan WT/PP2A and RepoMan P604A/PP2A.

Here again we agree that it will be very interesting to analyse in future the detailed binding interactions between wt and mutant RepoMan and other interacting proteins, including PP2A. We view this as outside the scope of our present study and are actively engaged in raising the additional funding needed to pursue such future experiments.

(5) PHD2 is the primary proline hydroxylase in cells. Why does PHD1, but not PHD2, affect RepoMan hydroxylation and subsequent control of mitotic progression? The authors should discuss this issue further.

We agree with the main point underlining this comment, i.e., that there are still many things to be learned concerning the specific roles and mechanisms of the different PHD enzymes in vivo. We look forward to addressing these questions in future studies.

Reviewer #2 (Public review):

We appreciate the reviewer’s comments that our manuscript uses biochemical and imaging tools to delineate a key mechanism in the regulation of the progression of the cell cycle and their appreciation that our experiments performed are, 'conclusive with well-designed controls.'

With respect to the specific Concern raised by reviewer 2:

Lack of in vitro reconstitution and binding data.

We agree that it will be very interesting to pursue in vitro reconstitution studies and detailed binding data. We view this as outside the scope of our present study and are actively engaged in raising the additional funding needed to pursue such future experiments.

Reviewer #3 (Public review):

We appreciate the reviewer’s comments that our study, “is a comprehensive molecular and cell biological characterisation of the effects of P604 hydroxylation by PHD1 on RepoMan, a regulatory subunit of the PPIgamma complex” and their conclusion that, “we should have no question about the validity of the PHD1-mediated hydroxylation”.

With respect to the specific Concern raised by reviewer 3:

Reliance on a Proline-Alanine mutation in RepoMan to mimic an unhydroxylatable protein. The mutation will introduce structural alterations, and inhibition or knockdown of PHD1 would be necessary to strengthen the data on how hydroxylates regulate chromatin loading and interactions with B56/PP2A.

We do not agree that we rely solely on analysis of the single site pro-ala mutatin in RepoMan for our conclusions, since we also present a raft of additional experimental evidence, including knock-down data and experiments using both fumarate and FG. We would also reference the data we present on RepoMan in the parallel study by Jiang et al, which has also been reviewed by eLife and is currently available on biorxiv (doi: https://doi.org/10.1101/2025.05.06.652400). Of course we agree with the reviewer that even although the muatnt RepoMan features only a single amino acid change, this could still result in undetermined structural effects on the RepoMan protein that could conceivably contribute, at least in part, to some of the phenotypic effects observed. Hopefully future studies will help to clarify this.