H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.

H3K36me3 peptide pulldown followed by SILAC-MS identified PWWP as a putative H3K36me3 reader domain (Vermeulen et al., 2010). Several pieces of evidence support the role of H3K36me3 in the DNA damage response. SETD2 mediated H3K36me3 is shown to recruit PSIP/p75 through PWWP domain to expressed exons (Pradeepa et al., 2012;Pradeepa et al., 2014). Upon DNA damage, PSIP1 recruits the repair factors CtIP and RAD51 to facilitate HDR (Aymard et al., 2014;

Amendments from Version 3
We have now added new data showing the presence of PARP1 in the PSIP/p75 complex. Our mass spec data together with the immunoblotting and comet assays show a clear link to PSIP role in DNA repair. Future studies will be needed to understand the clear mechanism through which H3K36 methylated chromatin, PSIP, and its interacting partners contributing to DNA repair.
Any further responses from the reviewers can be found at the end of the article  et al., 2012;Pfister et al., 2014). All this evidence supports a model in which PSIP1 is anchored to H3K36me3 chromatin at expressed gene bodies through its PWWP domain. Upon DNA damage, chromatin bound PSIP/p75 recruits CtIP and RAD51, which promotes HR repair by efficient resection, and protects these vulnerable regions of the genome from DNA damage. In the absence of SETD2 or H3K36me3, the chromatin association of PSIP1 is reduced, and DNA damage induced recruitment of repair proteins is impaired, leading to reduced resection and HDR. Similarly, another H3K36me3 reader -MRG15 -has been shown to recruit the partner and localiser of BRCA2 (PALB2) complex to undamaged chromatin (Bleuyard et al., 2017). Constitutive association of PALB2 to H3K36me3 chromatin at expressed gene bodies facilitates immediate availability of PALB2 upon DNA damage during active transcription and DNA replication. H3K36me3 is also shown to promote DNA mismatch repair by recruiting the mismatch recognition protein MutSα through its PWWP domain (Li et al., 2013). In contrast to mammalian studies, in budding and fission yeasts, H3K36me3 promotes non homologous end joining (NHEJ) and inhibits HDR (reviewed in (Jha et al., 2014). Similar to yeast studies, H3K36me2, catalysed by SETMAR/Metnase also promotes NHEJ in human cells (Fnu et al., 2011). This suggests a wider and complex role of H3K36 methylation in DNA repair choice and genome stability. Intriguingly, the PWWP domain of PSIP/p75 is also shown to bind H3K36me2 (Zhu et al., 2016) and detected near transcriptional start sites of Hox genes suggesting the possibility of binding of PSIP/p75 to H3K36me2 at TSS and to H3K36me3 at the gene bodies (Pradeepa et al., 2014) SETD2, the only enzyme responsible for H3K36 trimethylation is mutated in cancers and is proposed to function as tumor suppressor (Li et al., 2016;Zhu et al., 2014). The methylated H3K36 reader -PSIP1 -is implicated in a variety of cancers (Basu et al., 2016;Daniels et al., 2005;French et al., 2016;Yokoyama & Cleary, 2008) and also implicated in resistance to chemotherapy induced cell death in prostate cancer (Mediavilla-Varela et al., 2009). This suggests that H3K36me3 and PSIP1 play an important role in DNA repair and that dysregulation of this pathway could cause or promote human cancer.

Daugaard
We hypothesised that PSIP/p75 isoform function as an adaptor protein to recruit various proteins involved in DNA repair to H3K36me3 chromatin. We performed formaldehyde crosslinked chromatin immunoprecipitation (ChIP) followed by label-free quantitative mass spectrometry (xChIP-qMS) to identify proteins associated with H3K36me3 chromatin. We find several proteins implicated in transcriptional elongation, RNA processing and DNA repair associated with H3K36me3. Furthermore, SILAC proteomics analysis of endogenous PSIP/p75 complex shows interaction of several DNA repair proteins with PSIP/p75, many of them overlap with proteins enriched in H3K36me3 ChIP. We also detect a higher level of DNA damage in mouse embryonic fibroblasts (MEFs) derived from a Psip1 knockout mouse (Psip1 -/-) compared to wild type MEFs. We propose a wider role of H3K36me3/PSIP1 axis in maintaining genome integrity and efficient DNA repair at the site of transcription.

xChIP-qMS identifies H3K36me3 associated proteins
H3K36me3 is associated with actively transcribed gene bodies, preferentially at the exons of the expressed genes, suggesting its role in splicing and transcriptional elongation. In order to capture the proteins that transiently and stably associate with H3K36me3, formaldehyde cross linked mouse embryonic stem cells (mESCs) were treated with hypotonic buffer to prepare nuclei, sonicated to obtain soluble chromatin, immuno-precipitated using H3K36me3 and pan H3 antibodies ( Figure 1B). This is a useful method to study the proteins that are associated with particular histone modifications. However, since the chromatin is crosslinked and fragmented by sonication to get 100-500bp DNA fragments, many proteins that do not directly bind to H3K36me3 but are bound directly to DNA or to other histone modifications, are also likely to be enriched. Hence, we performed ChIP with the same chromatin using pan-H3 antibodies as control.
Label-free quantitative mass spectrometry analysis of two replicate ChIPs show enrichment of several proteins, implicated in replication, transcription, RNA processing and DNA repair, after anti-H3K36me3 ChIP normalised to anti-pan H3 ChIP ( Figure 2 and Dataset 1). Association of RNA processing proteins with H3K36me3 is consistent with the role of H3K36me3 in RNA processing. (Guo et al., 2014;Luco et al., 2010;Pradeepa et al., 2012). Since H3K36me3 is located at expressed gene bodies, it is not surprising that we find several proteins implicated in transcription and transcriptional elongation (Dataset 1). Interestingly, we found 26 proteins that are implicated in DNA repair and are associated with H3K36me3, with >1.5 ratio of H3K36me3 ChIP/H3 ChIP ( Figure 2 and Dataset 1). These include known interactors of H3K36me3 -PSIP1, SPT16, SSRP1 and MSH6 (Carvalho et al., 2013;Li et al., 2013;Pradeepa et al., 2012;Pradeepa et al., 2014). We detected many PSIP1 peptides mapping to the N-terminal domain that is common to both PSIP1 isoforms. Consistent with our previous work, we also found peptides mapping to the p75 specific C-terminal domain, suggesting the association of both p52 and p75 isoforms of PSIP1 in H3K36me3 chromatin (Pradeepa et al., 2012;Pradeepa et al., 2014). Nuck1 (Paralog of RAD51 AP1) protein had the highest H3K36me3/pan-H3 ratio, which was recently shown to promote homologous recombination DNA repair (Maranon et al., 2020).

SILAC immunoprecipitations followed by Mass
Spectrometry (SILAC-IP-MS) of PSIP/p75 complex PSIP1 is a H3K36me3 reader protein, binds to H3K36me3 and localises to expressed gene bodies. The p52 isoform of PSIP1 binds to H3K36me3 and recruits splicing factors to exons of expressed genes (Pradeepa et al., 2012). Similarly, the p75 isoform binds to H3K36me3 and recruits MLL proteins to expressed HOX genes (Pradeepa et al., 2014;van Nuland et al., 2013a;van Nuland et al., 2013b). p75 is also shown to promote HDR by recruiting CtIP and RAD51 to DSBs in a H3K36me3 dependent manner (Aymard et al., 2014;Daugaard et al., 2012;Pfister et al., 2014). In order to comprehensively identify both stable and transient interacting partners of p75, we Figure 2. H3K36me3 associated proteins that are implicated in DNA repair. The label-free mass spectrometry quantitative output values assigned to each protein following immunoprecipitation from the mouse embryonic stem cells (mESCs). The list of proteins associated with DNA repair function with the H3K36me3 vs H3 ratio of more than 1.5 (y-axis) are plotted (full list of proteins in Dataset 1). Horizontal scatter was added only to aid visibility of each protein and has no data correlate. The position of PSIP1 protein is highlighted in red.
performed immunoprecipitation (IP) of endogenous p75 protein in cells grown in SILAC media using previously characterised antibodies that specifically pull-down the p75 isoform of PSIP1 (Pradeepa et al., 2012;Pradeepa et al., 2014). IP with anti-MOF (KAT8) served as an irrelevant control and rabbit immunoglobulin (IgG) served as a negative control. mESCs and MEFs were first labelled for two weeks in light, medium and heavy SILAC cell culture media, followed by IP-MS with rabbit IgG anti-MOF and anti-PSIP/p75 antibodies, respectively ( Figure 3A, Dataset 2 and Dataset 3). The protein enrichment ratio was then calculated to identify proteins that are quantitatively enriched in PSIP/p75 and MOF IP compared to normal rabbit IgG. Similarly, proteins enriched in PSIP/p75 IP normalised to MOF IP was also calculated (Dataset 2 and Dataset 3).

PSIP/p75 interacts with DNA repair proteins
As expected, SILAC-IP-MS with PSIP/p75 and MOF antibodies showed PSIP1 and MOF proteins with the highest SILAC ratio over negative control in respective IPs in both MEFs and mESCs (Table 1; Dataset 2 and Dataset 3). Proteins identified in PSIP/p75 IPs are specific to this isoform of PSIP1, as the antibody used for IP is specific to the c-terminal domain of PSIP/p75, which is absent in the p52 isoform ( Figure 1A). Cell division cycle-associated 7 like (CDC7L), one of the known interacting partners of p75, had the second highest ratio in MEFs (Hughes et al., 2010). SILAC ratio for γH2A.X was similar to PSIP1 in mESCs, suggesting the co-occurrence of PSIP1 along with γH2A.X at the nucleosomal level. Interestingly, with the exception of XRCC1 all the other DNA repair proteins found in the p75 complex were also enriched in H3K36me3 ChIP-MS (Table 1 and Figure 2).
FACT (facilitates chromatin transcription) complex composed two subunits SPT16 (Suppressor of Ty 16) and SSRP1 (Structure-specific recognition protein-1), both were detected in the PSIP/p75 IP. SSRP1 interacts with PWWP domain of PSIP1 (Lopez et al., 2016), which suggesting the functional interplay between PSIP1 and the FACT complex in transcriptional elongation and DNA repair.
Heterogeneous nuclear ribonucleoprotein U (hnRNPU) is detected in MEFs but not mESCs. Although hnRNPU is involved in RNA metabolism, it is known to be involved in promoting DNA Double-Strand Break Signaling and Repair, hnRNPU proteins promote DNA-end resection and promote ATR dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents (Polo et al., 2012). Immunoprecipitation done in mESCs detected fewer proteins and also lower SILAC ratio for p75 (Table 1; Dataset 3). Intriguingly, other PSIP1 interacting proteins -MLL1, MLL2, Menin, and/or CtIP -were not detected.

Validation of PSIP/p75 SILAC-MS data
We validated the hits from SILAC-IP-MS for PSIP/p75 by performing immunoprecipitation using PSIP/p75 specific antibody followed by western blotting ( Figure 3C), which confirmed the PSIP/p75 interaction with PARP1 and hnRNPU, while SSRP1 was not detected in the p75 IP.
We further validated the interaction of PSIP/p75 with S139 phosphorylated histone H2AX (γH2A.X), MLL1 and H3K36me3, by performing IP with αHA-tag antibodies in Psip1 knockout MEFs (Psip1 -/-), which are stably transduced with HA-Psip1/p75 and HA-Psip/p52 ( Figure 3B) (Pradeepa et al., 2014;Shun et al., 2007). Western blotting of HA IPs with anti-H3K36me3 confirmed the interaction of both p52 and p75 with H3K36me3, which is mediated by a PWWP domain common to both PSIP1 isoforms. Interestingly, we found specific association of a DNA damage marker γH2A.X, with PSIP/ p75, but not the p52 isoform. This is consistent with the previously known function of p75 in DNA damage response (Aymard et al., 2014;Daugaard et al., 2012). PSIP/p75 is known to interact with MLL1, but it was not detected in our endogenous p75 IP-MS analysis. However, western blotting with anti-MLL1 shows specific interaction of HA-p75, but not HA-p52 with MLL1 ( Figure 3B). These results confirm that although both PSIP1 isoforms are localised to H3K36me3 chromatin, only p75 associates with MLL1, γH2A.X and other DNA repair proteins -consistent with previous reports showing both isoforms of PSIP1 have different protein partners and cellular function (Daugaard et al., 2012;Ge et al., 1998;Pradeepa et al., 2012;Pradeepa et al., 2014;Pradeepa et al., 2017).
Higher DNA damage in PSIP1 knockout cells To examine whether absence of PSIP1 lead to accumulation of unrepaired DNA, we performed comet assay in WT and Psip1 -/-MEFs, which is a sensitive method to measure DNA damage in individual cells (Olive & Banáth, 2006). In comet assay, cells are mixed with agarose gel matrix, layered on to a slide, subjected to in-gel lysis after solidification, and followed by electrophoresis. When these slides are stained with DNA binding dye and visualized in the microscope, the cells will look like comets. Depending on the extent of DNA damage in a cell, the mobility of the DNA will be more into the tail part of the comet (tail DNA). The Visual scoring of comets showed a significantly higher number of comets with a higher concentration of tail DNA in Psip1 -/-MEFs compared to WT ( Figure 4A) suggesting the presence of unrepaired residual DNA damage in Psip knock-out cells. This data supports our previous observation that human cells depleted of p75 show higher levels of unrepaired DNA compared to control, confirming the higher level of unrepaired DNA in cells lacking PSIP1. This strengthens the evidence for a role for PSIP/p75 in maintaining genomic integrity. . We labelled cells with light, medium and heavy isotope and performed SILAC IP for three different antibodies and were thus able to include anti-MOF IP along with p75 IP and IgG, allowing us to identify the protein partners of MOF along with PSIP/p75 in mESCs and MEFs. MOF IP also acted as an unrelated chromatin protein control that is known to associate with active genes at regulatory elements (Li et al., 2012;Taylor et al., 2013). MOF protein has been shown to be associated with both male-specific lethal (MSL) and non-specific lethal (NSL) complexes (Cai et al., 2010;Li et al., 2009;Mendjan et al., 2006). Like H3K36me3, MOF/MSL complex mediated H4K16ac is enriched at expressed gene bodies. Intriguingly, canonical MSL complex proteins but not NSL or DNA repair proteins were associated in the MOF IP (Dataset 2). There was also no overlap between PSIP and MOF complex proteins which shows the specificity of the PSIP and MOF IP. Although there have been efforts to study protein partners of PSIP and MOF, to our knowledge, this is the first study exploiting the utility of SILAC proteomics to investigate the cellular interactome of these two proteins without using overexpression or epitope tagging approaches. The SILAC immunoprecipitation strategy used here is a very sensitive and powerful means of detecting both transient and stable protein partners of chromatin associated proteins.

Discussion
Immunoprecipitation of nuclear extracts with specific modified peptides followed by SILAC MS led to identification of PWWP domain containing proteins as putative readers of H3K36me3 (Vermeulen et al., 2010). In vitro pulldown of recombinant PSIP-PWWP domain, modified peptide arrays together with ChIPon chip assays confirmed specific interaction of PSIP1 PWWP domain with H3K36me3 (Pradeepa et al., 2012). We now used x-ChIP-qMS, a useful method to identify proteins that interact transiently to histone modifications or chromatin proteins. A similar method was successfully used in Drosophila cells to identify MSL associated chromatin proteins and histone modifications (Wang et al., 2013). In this study, we identified proteins associated with H3K36me3 using x-ChIP-qMS. Further studies upon targeted mutations to histone modifying proteins (writers) or domains that recognises these histone modifications (readers) using gene editing methods will aid in validating the specific interaction of H3K36me3 reader proteins.
SETD2 is the only enzyme responsible for majority of H3K36 trimethylation in mammals, and its depletion reduces H3K36me3 levels, which results in a lower density of FACT subunits SPT16 and SSRP1 (Carvalho et al., 2013). Our data shows that FACT subunits are associated with both H3K36me3 and p75, suggesting the possibility of functional interplay between PSIP/p75 and FACT complex at transcribing gene bodies and in DNA damage signalling response. PSIP/p75 and the SSRP1 complex are suggested to be important for the life cycle of HIV (Lopez et al., 2016). The FACT complex has been shown to facilitate the exchange between H2A.X and H2A (Heo et al., 2008). FACT also promote new H2A.X deposition coupled to repair synthesis and contribute to DNA damage signalling and repair of DNA damage (Piquet et al., 2018). FACT complex promotes incorporation of H2A.X to DNA damage sites, and shown to associate with PARP1 and DNA-PK (Heo et al., 2008). SSRP1 cooperates with PARP1 and XRCC1 to promote single strand DNA break repair. (Gao et al., 2017). Interestingly, PSIP has been shown to function like FACT by allowing RNAPII to overcome the nucleosome-induced barrier to transcription elongation in differentiated cells that no longer express FACT ( Although we have previously shown that both isoforms of PSIP1 bind to H3K36me3 through the common PWWP domain, it is only the p75 isoform of PSIP1 that associates with γH2A.X ( Figure 3B). Moreover, most of the previously known p75 interacting proteins are shown to bind to IBD in the C-terminus of p75 ( Figure 1A). These data suggest the possibility of other known p75 interacting proteins like PogZ, JPO2, IWS1, MLL and ASK that binds to IBD (Tesina et al., 2015) in DNA repair pathways. It is also possible that PSIP1 binding partners might be interacting with DNA repair proteins leading to enrichment of DNA repair proteins in the H3K36me3 and PSIP1 IP-MS. Future studies involving in vitro reconstitution assays using recombinant proteins will be crucial to demonstrate the direct role of H3K36me/PSIP axis in DNA damage response.
Identification of several DNA repair proteins that interact with PSIP/p75 that are also associated with H3K36me3 suggests that H3K36me3 and PSIP1 have a wider role in DNA repair pathways than previously appreciated. We propose a wide spectrum of roles for SETD2 dependent H3K36me3 and its reader proteins in DNA repair and genome stability than previously suggested ( Figure 4B). PSIP/p75 is a stress survival protein, also implicated in various cancers including breast, ovarian, prostate and leukaemia, promote resistance to chemotherapy induced cell death in prostate cancer. Further research is needed for a better understanding of the importance of PSIP1 in promoting DNA repair during stress response, in chemo or radiotherapy induced cell death in cancers.

Cell lines
Psip1 -/and its corresponding WT MEFs (Pradeepa et al., 2014;Shun et al., 2007) were a kind gift of Prof. Alan Engelman (Dana-Farber Cancer Institute, USA), and were cultured for two weeks in SILAC DMEM media (Dundee Cell Products), containing 10% fetal bovine serum (HyClone, GE Healthcare) and 1% Pen/Strep (Sigma Aldrich). mESCs (OS25, IGMM biostore) were adapted to grow in DMEM media before they were cultured in SILAC DMEM media. MEFs and mESCs to be used as control (rabbit IgG) in the pulldown were grown under R0K0 media; cells used for MOF IP were cultured in R6K4 media; and cells used for P75 IP were cultured in R10K8 media.
Antibodies used: 5 µg of rabbit IgG (Santa Cruz sc-2025), Histone H3 (rabbit polyclonal, Abcam, Ab 1791), H3K36me3 (rabbit polyclonal, Abcam, Ab 9050) were used per IP. For analysis by mass spectrometry, beads were washed 3 times with Tris-saline buffer, and excess buffer removed. ChIPed complexes were digested on beads, desalted and analysed on a Q-Exactive plus mass spectrometer, as previously described (Turriziani et al., 2014). Proteins were identified and quantified by MaxLFQ 17 by searching with the MaxQuant version 1.5 against the mouse proteome data base (Uniprot). Modifications included C Carbamylation (fixed) and M oxidation (variable). Bioinformatic analysis was performed with the Perseus software suite.

Immunoprecipitation
10×14-cm dishes of cells were trypsinized and pelleted, resuspended in 5 ml of ice-cold swelling buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2, 10 mM KCl, 0.5 mM DTT and protease inhibitors (Complete, Roche) for 5 min, and cells were broken open to release nuclei using a pre-chilled Dounce homogenizer (20 strokes with a tight pestle). Dounced cells were centrifuged at 228 g (2,000 rpm) for 5 min at 4°C to pellet nuclei and other fragments. The supernatant was discarded. The resulting nuclear pellet was resuspended in 5 ml of RIPA buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, and protease inhibitors + Benzonase (Novagen, 10 µl/ml), incubated for 30 min on ice, and sonicated briefly on ice (10 × 30 s at full power in bioruptor). Extracts were cleared by centrifugation at 13000 RPM for 10 min at 4°C. Nuclear protein concentrations were measured using a Bradford assay.
Protein A Dynabeads (Life Technologies) were incubated with 5 µg of rabbit IgG (Santa Cruz sc-2025), anti-PSIP/p75 (rabbit polyclonal, Bethyl Laboratories, A300-848A) and anti-MOF (rabbit polyclonal, Bethyl Laboratories A300-992A) in 5% BSA in phosphate buffer saline for two hours, equivalent total protein amounts of extracts were incubated separately with antibodies bound to beads in a rotating platform at 4°C for 30 min. Beads were washed once with RIPA buffer and combined carefully after first wash step. After a further 4 washes, bound proteins were eluted in 4X SDS loading buffer (Life Technologies) with freshly added DTT at 95°C for 5 min. Samples were centrifuged at 11,000 RPM speed for 1 min and supernatant was collect in low binding tube. LC-MS/MS and quantification were carried out by Dundee Cell Products. Briefly, for SDS-PAGE, gel slices per fraction were cut and digested in-gel with trypsin. The purified peptides were then separated (Ultimate U3000, trap-enriched nanoflow LC-system, Dionex), and identified (LTQ Orbitrap XL, Thermoscientific, via nano ES ion source, Proxeon Biosystems). Quantification (MaxQuant, based on 2D centroid of isotope clusters within each SILAC pair) can distinguish between the samples, to give a ratio of protein of interest to IgG. Background proteins would be expected to have a ratio of 1:1 and are therefore disregarded. 10% input nuclear lysate and IPed proteins were separated on Novex 4-20% gels and transferred to PVDF membranes. Western blotting was performed by immunoblotting with PSIP/ p75(Bethyl laboratories A300-848A), PARP1(abcam ab191217), hnRNPU (abcam ab10297-50), SSRP1 (Biolegend 609701) and PCNA (Santa Cruz Biotechnology sc7907) antibodies.

Comet assay
Comet assays for WT and Psip1 knockout MEFs were performed using Comet Assay kit (OxiSelect™), according to the manufacturer's instructions.

Data availability
Raw  Figure 3B.

Are the conclusions drawn adequately supported by the results? Partly
Partly 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.
2. Could the authors rescue the DNA repair defect in -/-cells by a mutant LEDGF/p75 protein not able to interact with IBM proteins?
The datasets in the supplemental material are not clear to me. Dataset 1 and 2 are mentioned but not clearly linked to the supplements. I could not find dataset 1.

We have added the datasets now clearly labeled
Although DNAse is used to overcome DNA bridging in the IP experiments, the xChip experiments are inevitably prone to DNA bridging. One can only claim that these proteins were in the neighborhood of H3K36me3 marks. This should be clearly indicated in the discussion.

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We completely agree with the reviewer's point, hence say these proteins are associated with H3K36me3.
In the text, it is claimed that there is a strong overlay between both datasets in DNA repair proteins. The authors refer to figure 2 to support this statement. However, this figure only mentions H3K36me3 hits. The general overlay between both sets could be presented in a better way.

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We have now referred to Table 1 and Figure 2 and dataset 1 LEDGF is a major hit in the H3K36me3 xChIP. Looking at the sequenced peptides, can you speculate whether this was p52 or p75?

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We have now looked into this and found p75 peptides in the mass spec data. So we cannot rule out the presence of shorter p52 isoform along with the p75 isoform. We didn't find a short peptide that distinguishes between two isoforms. So our data shows both isoforms are likely to present in the H3K36me3 domain. Confirmation of some of the important hits by co-immunoprecipitation experiments as presented in figure 3b would strengthen the credibility of the manuscript.

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We completely agree with the suggested confirmatory experiments by both reviewers will strengthen the credibility of the manuscript. We will be performing these experiments when we have resources, and we will update the manuscript. The fact that LEDGF/p75 has an effect on DNA damage response (figure 3c) is not new. However, neither the present manuscript nor previous manuscripts from other groups could provide a direct link. The authors claim that LEDGF/p75 and not p52 affects DNA repair. As such, there is a good chance that a protein binding to the IBD domain is responsible for this effect. Proteins binding the IBD are characterized by an IBM motif (Tesina et al, 2015).

It is an interesting point; we have now discussed this in the manuscript
Could the authors rescue the DNA repair defect in -/-cells by a mutant LEDGF/p75 protein not able to interact with IBM proteins? ○ Thanks for suggesting this experiment, we would love to do this experiment in the future, but not for this manuscript CDC7/ASK is the only protein in table 1 with an IBM motif. Can HA-LEDGF/p75 rescue DNA repair upon knockdown ofCDC7/ASK? ○ These data could provide a direct link between LEDGF/p75 and DNA repair.
We appreciate the suggestions to improve the manuscript by both the reviewers. Due to limited resources, we cannot perform the suggested experiments at this time. However, we will perform the suggested experiments in future. We wished to share our data with this publication to wider readers at the earliest time point.
Competing Interests: No competing interests were disclosed.