MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks
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Abstract
Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino 1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.
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Referee #4
Evidence, reproducibility and clarity
In this manuscript by Torres Esteban et. al., the authors investigate how Pellino1/2 are recruited to chromatin surrounding DSBs. These two proteins contain the well-established FHA phospho-peptide binding domain that are present in other DDR chromatin binding such as RNF8. These two proteins were recently implicated as being involved in the DNA damage response, however, the exact phosphorylation-dependent recruitment method Pellino1/2 is not well defined.
The authors first attempt to identify proteins that bind to the multiple MDC1 TQXF motif, specifically when phosphorylated on the Threonine residue. In two independent experiments …
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Referee #4
Evidence, reproducibility and clarity
In this manuscript by Torres Esteban et. al., the authors investigate how Pellino1/2 are recruited to chromatin surrounding DSBs. These two proteins contain the well-established FHA phospho-peptide binding domain that are present in other DDR chromatin binding such as RNF8. These two proteins were recently implicated as being involved in the DNA damage response, however, the exact phosphorylation-dependent recruitment method Pellino1/2 is not well defined.
The authors first attempt to identify proteins that bind to the multiple MDC1 TQXF motif, specifically when phosphorylated on the Threonine residue. In two independent experiments both Pellino1 and Pellino2 were pulled down with a phosphorylated MDC1 pTQXF peptide along with RNF8, as expected. They further show that this interaction between Pellino1/2 and the MDC1 is not specific to just a single MDC1 TQXF motif and that it requires a phosphorylated Threonine residue both from nuclear extracts and an in-vitro binding assay. The authors also show that Pellino1/2 cannot be pulled down from nuclear extracts using a pS139 H2AX peptide, as had been suggested by immunoprecipitation methods previously. Next, in Figure 2 the authors investigate if any of the 4 MDC1 TQXF motifs are preferentially bound by Pellino1/2 in comparison to RNF8. The authors further provide evidence for this phosphor-dependent interaction with a crystal structure of Pellino2-FHA along with a pTQXF MDC1 peptide. They then show that while this Pel1/2-MDC1 interaction can be observed using immunoprecipitation of HA/Flag-MDC1, it appears to be IR-induced DNA damage independent with this method. Using PLA as a second approach they validate that Pellino1 and MDC1 can be found in close proximity within the nucleus in cells and that this interaction is enhanced upon IR-induced DNA damage. As a control they see no PLA foci in MDC1 KO U2OS cells. The authors further this notion that MDC1 is important for Pellino1/2 recruitment to DSBs with laser micro-irradiation and see that GFP-Pellino1 is recruited to DNA damage in an MDC1-dependent manner. They further show that this MDC1-dependent recruitment of Pellino1 requires the TQXF motif of MDC1 as mutation of all 4 TQXF residues to AQXF abolished Pellino1 recruitment to laser induced DNA damage. The authors then show that this same AQXF mutant is also devoid of Pellino1 recruitment ability using IR.
Overall, the manuscript is very clear and concise and presents a reasonable model that MDC1 pTQXF motifs are required for recruitment of Pellino1/2 to DSBs. The explanations of data and figures, including the rationale behind experiments are easily understood and well presented. There are a few points that should be addressed.
Major Comments:
What were the other proteins that came down with the phospho-MDC1 pTQXF peptide pulldown, and what were the statistics of the pulldowns from the mass spectrometry experiments (i.e. number of distinct peptides, coverage, etc, for all of the interacting proteins that were identified. I was not able find a table showing all of the other interactors (Table S1? Not included in the files). These data are critical and need to be presented in the manuscript.
Given that the FHA domain structure primarily recognizes the three amino acids C-terminal to the pThr residue, why do the authors think that the measured KD for the pT765 site (pTQPF) is 5-fold lower than the measured KD for the pT752 site (pTQPF)? Please provide some rationale for this in the text. (See point 4 below as well)
In Figure 2, the pT699 peptide binds strongest to RNF8 and PELI1/2. Is this a consequence of other factors (conformational or allosteric changes, or other modifications on these proteins from the nuclear extracts) in the full length proteins compared to the isolated FHA domains? If the authors repeat this pulldown experiment using only the isolated FLAG- or GST-tagged FHA domains, do they also get the same result? The data in Table T1 suggests this will not be the case. Please confirm this, and if the data and discordant, please comment on the difference in full-length RNF8 and PELI1/2 binding from the HeLa nuclear extracts versus the isolated FHA domains in the text.
In Figure 3, I am confused by the reference to Asp+5. The sequence around pT765 is pTQPFDT, so isn't the critical Asp in the +4 position? Is this interaction sufficient to account for the 5-fold tighter binding of this site than to the pT752 site? Or is this higher affinity a consequence of the pT-1 Glu residue, or both? Perhaps showing a full side chain interaction map would help here.
It would be helpful if the authors looked at RNF8 levels in the FLAG Ips shown in Figure 4A. It looks like DNA damage slightly reduced PELI1/2 binding which might be accounted for by RNF8 competition.
Do the authors have any data, or can they speculate, on whether the entire MDC1 699-770 region can simultaneously bind to both RNF8 and PELI1/2 though different sites at the same time on a single MDC1 molecule or is the RNF8-PELI1/2 co-localization from adjacent MDC1 molecules? While clearly not required, it would be interesting to ask whether transfecting MDC1-knockout cells with variations of MDC1 containing 1, 2, or 3 Thr-Ala mutations at these sites compromises co-localization and DNA damage responses? Can co-transfection of two constructs containing distinct single TQXF sites - one optimized for RNF8 binding and one optimized for PELI1/2 binding, can rescue the co-localization phenotype.
What effect does the combined knock-down of PELI1/2 have on some aspect of DNA damage signaling or cell cycle progression? Is the repair/loss of gH2AX or 53BP1 foci delayed if PELI1/2 are absent?
Figure 4A shows that the interaction between MDC1 and Pellino1/2 is independent of IR-induced DNA damage as observed using immunoprecipitation. 4B shows that PLA foci between MDC1 and Strep-HA-GFP-Pellino1 can be observed in undamaged cells and PLA foci are increased when observed 3h post 3Gy IR damage. Is this discrepancy explainable by differences between PELI1/2 recruitment given that the antibody in 4A used recognizes both isoforms but the PLA in 4B is specific to Pellino1? It would be nice to see if there are any differences between Pellino1/2 by performing IP +/- DNA damage using exogenously expressed Pelliono1 or 2 variants. Additionally, repeating PLA in 4B with a Pellino2 construct would help address this question.
Similar to the comment above, a further investigation into if there are any differences in recruitment between Pellino1/2 using the experimental systems used in Figure 5 would greatly support the model presented, particularly since the crystal structure presented in Figure 3 is with Pellino2. Is there any rationale for this based upon amino acid sequence differences between Pellino1/2?
Using the experimental approaches in Figures 4 & 5 and the insight from the crystal structure in Figure 3, making point mutations in Pellino1/2 that would be predicted to disrupt the phospho-specific interaction would provide further confidence in the model proposed.
Minor Comments:
Could the authors please comment on if Pellino3a/b came down in the initial IP experiment and if not, is there potentially an explanation based upon sequence similarities/differences as inferred by the crystal structure of Pellino2?
Is there a rationale for why only single TQXF motifs were tested in isolation for binding in 2A? Given the proximity of the TQXF sites one could imagine that 2 or 3 TQXF sites being simultaneously phosphorylated could provide an even better peptide for Pellino1/2 FHA binding.
In Figure 2B, how is the data "Adjusted"?
Table T2 is provided twice, but the Table of co-precipitating proteins from the mass spectrometry experiments is not shown.
REFEREE CROSS-COMMENTS
It seems that most of us hit on the same set of core issues - whether the MDC stretch of 4 pTQxF motifs simutaneously recruits RNF8 and Pellino1/2 to the same MDC1 molecules or not, a few more mutational studies, and whether the Pellino recruitment is important in events downstream of MDC1 binding. This latter issue could entail significantly more work, though its inclusion would likely influence which of the journals that use Review Commons was interested in pursuing the paper. A purely biochemical study is fine, at least in my opinion, depending on the type of journal, but adding some functional DNA damage context and relevant cell biology would bring the work to a deeper level. Just my thoughts, of course....
Significance
The general significance of the data presented by Torres Esteban et. al., provide a mechanistic understanding of how Pellino1/2 are recruited to DNA damage, a role that has previously not been characterized. While this manuscript does not delve into the role of Pellino1/2 downstream of its recruitment, much of this has been investigated by a paper by Ha et. al., 2019 as cited in this manuscript. In fact, the Ha et. al., propose that Pellino1/2 directly interacts with pS139 H2AX. Torres Esteban et. al., convincingly show that this is likely not the case and provide robust biochemical and cellular evidence to support their mechanistic model, very nicely contributing to the body of knowledge regarding Pellino1/2 in the DDR. The audience that this paper would be important for would be those in the DNA damage field or structural biologists interested in phospho-peptide binding domains.
Our expertise is in DNA damage signaling, FHA and other protein modular domains, and determination of protein structure by X-ray crystallography.
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Referee #3
Evidence, reproducibility and clarity
The authors in this work provide compelling evidence to support a critical role of MDC1 as a molecular scaffold that dock Pellino1/2 at DSBs. Using an unbiased approach Pellino1/2, alongside RNF8, were identified as FHA-containing DNA damage responsive factors that bear affinity for an MDC1 TQXF phospho-peptide. Results from a series of interaction studies suggest that Pellino1/2 highly likely interact with MDC1 in a phosphorylation-dependent manner. Consistently, Pellino1/2 assembly at DSBs required MDC1 and its TQXF phosphorylation. Overall the work is of high quality and observations that highlight the MDC1-Pellino1/2 interaction …
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Referee #3
Evidence, reproducibility and clarity
The authors in this work provide compelling evidence to support a critical role of MDC1 as a molecular scaffold that dock Pellino1/2 at DSBs. Using an unbiased approach Pellino1/2, alongside RNF8, were identified as FHA-containing DNA damage responsive factors that bear affinity for an MDC1 TQXF phospho-peptide. Results from a series of interaction studies suggest that Pellino1/2 highly likely interact with MDC1 in a phosphorylation-dependent manner. Consistently, Pellino1/2 assembly at DSBs required MDC1 and its TQXF phosphorylation. Overall the work is of high quality and observations that highlight the MDC1-Pellino1/2 interaction are solid.
Significance
The work offers an alternative perspective that details MDC1 as an important molecular scaffold that allows DSB targeting of Pellino1/2. I have only two minor comments -
As opposed to examining interaction between Pellino1/2 and the MDC1 fragment (Figure 4A), to unequivocally demonstrate that Pellino1/2 interacts with MDC1 in response to genotoxic stress the authors should show that Pellino1/2 interacts with full-length MDC1 but not its AQXF mutant. In this setting IR should further enhance the Pellino1/2-MDC1 interaction.
For visualisation purpose the authors should provide density map that shows the MDC1-pTQXF and the key interacting residues on the PELI2 FHA.
OPTIONAL:
Given the plausibility that Pellino1/2 may co-occupy MDC1 with NBS1 and RNF8 it would be of interest to examine if that is indeed the case. & Does Pellino1/2 expression affect RNF8-dependent ubiquitylation?
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Referee #2
Evidence, reproducibility and clarity
The authors present a series of experiments demonstrating a physical interaction between the phosphorylated TQXF motifs of MDC1 and Pellino1 / Pellino 2 proteins (PELI1/PELI2). Both proteins are enriched in a peptide pull-down mass spectrometry experiment using HeLa nuclear extracts, and shown to bind preferentially to MDC1-pThr699 and -pThr765. These data are nicely supported by iso-thermal titration calorimetry experiments with a dissociation constant of 230nM determined for interaction with a synthetic pT765 phospho-peptide. The authors go on to determine an X-ray crystal structure of the FHA domain from PELI2 in complex with …
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Referee #2
Evidence, reproducibility and clarity
The authors present a series of experiments demonstrating a physical interaction between the phosphorylated TQXF motifs of MDC1 and Pellino1 / Pellino 2 proteins (PELI1/PELI2). Both proteins are enriched in a peptide pull-down mass spectrometry experiment using HeLa nuclear extracts, and shown to bind preferentially to MDC1-pThr699 and -pThr765. These data are nicely supported by iso-thermal titration calorimetry experiments with a dissociation constant of 230nM determined for interaction with a synthetic pT765 phospho-peptide. The authors go on to determine an X-ray crystal structure of the FHA domain from PELI2 in complex with the MDC1-pThr765, to reveal the molecular determinants underpinning the interaction. Additional experiments, including immunoprecipitation, proximity ligation assay, and laser micro-irradiation support a phospho-specific interaction, driven by DNA damage, between MDC1 and PELI1/2 - and discount a direct interaction of PELI1/2 with the phosphorylated tail of histone H2AX as proposed previously in Ha et al (2019).
Major comments:
a) In all cases, RNF8 binds more tightly to the MDC1-pTQXF motifs, with the notable exception of pThr765; however, the affinity of RNF8 for the MDC1-pThr765 peptide has not been determined by ITC, which would be a useful comparator. Esp., given the question of how the authors envisage the interchange between binding to RNF8 vs PELI1/2?
b) Does Wing-II of PELI1/2 provide a secondary interaction pocket, capable of interacting with an extended bis-phosphorylated peptide? Is it highly conserved in terms of amino acid sequence?
c) Do the authors findings support the idea of PELI1 acting as part of a positive feedback mechanism that functions via PELI1-dependent ubiquitylation of NBS1?
d) The manuscript appears a little truncated, and does not explore the downstream (cellular) effects of disrupting the PELI1/2 - MDC1 interaction.
Significance
Overall, the manuscript contains a robust and well-controlled set of experiments that confirm a direct interaction between the PELI1/2 protein and MDC1, dependent on prior phosphorylation of MDC1 (at its TQXF ) sites in response to DNA damage. With that said, it is somewhat limited in scope, and does not explore the cellular consequences of breaking/interfering with the ability of PELI1/2 to interact with MDC1 and how this might be integrated into our understanding of the mammalian DNA damage response.
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Referee #1
Evidence, reproducibility and clarity
Summary:
The manuscript by Esteban et al. provides compelling evidence that MDC1, in addition to its well-known interaction with the E3 ligase RNF8, also directly binds to the E3 ubiquitin ligases Pellino1 and Pellino2, both of which have recently emerged as important players in the DNA damage response. The authors convincingly show that MDC1 mediates the recruitment of Pellino1/2 to sites of DNA double-strand breaks (DSBs) via a phosphorylation-dependent interaction.
Major Comments:
Competitive Binding Dynamics: The manuscript should address whether RNF8 and Pellino1/2 compete for binding to phosphorylated MDC1. Understanding …
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Referee #1
Evidence, reproducibility and clarity
Summary:
The manuscript by Esteban et al. provides compelling evidence that MDC1, in addition to its well-known interaction with the E3 ligase RNF8, also directly binds to the E3 ubiquitin ligases Pellino1 and Pellino2, both of which have recently emerged as important players in the DNA damage response. The authors convincingly show that MDC1 mediates the recruitment of Pellino1/2 to sites of DNA double-strand breaks (DSBs) via a phosphorylation-dependent interaction.
Major Comments:
Competitive Binding Dynamics: The manuscript should address whether RNF8 and Pellino1/2 compete for binding to phosphorylated MDC1. Understanding whether these interactions are competitive, cooperative, or mutually exclusive is crucial for deciphering the functional implications of MDC1's ability to interact with multiple E3 ligases.
DNA Damage-Independent Interaction: The observation that MDC1 and Pellino1/2 interact in the absence of DNA damage (as depicted in Figure 4A) is unexpected. While the authors note that MDC1 is phosphorylated to some degree in non-irradiated cells, this explanation does not fully clarify the mechanism or significance of this interaction in the absence of DNA damage. Since DNA damage typically enhances MDC1 phosphorylation, one would anticipate a corresponding increase in the interaction between MDC1 and Pellino1/2. Further investigation into this aspect is needed to better understand the context in which these interactions occur.
Minor Comments:
Figure 1E Labeling: There is a labeling error in Figure 1E; the labels should be corrected to "H2AX" and "pS139-H2AX."
Significance
While the discovery of this interaction is intriguing, the manuscript would benefit from a more thorough exploration of its physiological significance.
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