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

Version Changes

Revised. 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.

Abstract

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.

Introduction

PC4 and SF2 interacting protein (PSIP1) also known as Lens epithelium derived growth factor (LEDGF) is a multifunctional chromatin protein that has been implicated in regulation of homeotic genes, cell survival, cancers and autoimmune diseases. PSIP1 gene encodes two splice variants – a shorter isoform called p52 and a longer isoform called p75. Both these isoforms possess common domains in their N-terminal regions, namely, PWWP domain and adenine-thymine (AT) hook-like DNA binding domain ( Figure 1A). The PWWP domain binds specifically to Histone H3 trimethylated at lysine 36 (H3K36me3); both PSIP1 isoforms and H3K36me3 co-occur at expressed gene bodies ( Pradeepa et al., 2012; Pradeepa et al., 2014; van Nuland et al., 2013a). The C-terminal integrase binding domain (IBD) is unique to p75, and is shown to interact with HIV integrase ( Cherepanov et al., 2003; Ciuffi et al., 2005); the same domain binds to the mixed lineage leukaemia proteins (MLL1, MLL2 and Menin) ( Pradeepa et al., 2014; van Nuland et al., 2013b; Yokoyama & Cleary, 2008). Apart from other functions, p75 has also been shown to promote homology directed repair (HDR) by recruiting C-terminal binding protein interacting protein (CtIP) to double stranded breaks (DSB) ( Basu et al., 2012; Ciuffi et al., 2005; Daniels et al., 2005; Desfarges & Ciuffi, 2010; Pradeepa et al., 2014; Sutherland et al., 2006).

Figure 1. PSIP/p75 domains and its functional interactors.

( A) Cartoon of PSIP1 p52 and p75 isoforms showing methylated histone binding PWWP domain, DNA binding AT hook domain at the N-terminus and also the integrase binding domain (IBD) at the C-terminus of p75 that interacts with MLL1, Menin and JPO2. ( B) Illustration showing crosslinked chromatin immunoprecipitation (xChIP) followed by mass spectrometry (MS) to identify proteins associated with histone modifications.

PSIP/p75 is a multifunctional protein interacts with JPO2 (also known as R1, RAM2 and CDCA7L) ( Bartholomeeusen et al., 2007), pogo transposable element with zinc finger domain (PogZ) ( Bartholomeeusen et al., 2009), the activator of the S-phase kinase complex (CDC7-ASK) ( Hughes et al., 2010), methyl CpG binding protein 2 (MeCP2) ( Bartholomeeusen et al., 2009), CtIP ( Daugaard et al., 2012), SSRP1 ( Lopez et al., 2016), and Spt6 (IWS1) ( Tesina et al., 2015), in different genomic location or cellular contexts.

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; Daugaard 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.

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.

Results

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).

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.

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 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).

Figure 3. PSIP/p75 role in facilitating DNA repair.

( A) Illustration showing schematics of SILAC immunoprecipitation for using rabbit IgG, anti-MOF and anti-PSIP/p75 specific antibodies in cells labelled with light (R0K0), medium (R6K4) and heavy (R10K8) SILAC media. ( B) Western blotting with antibodies recognising MLL1, phosphorylated H2A.X (γH2A.X), H2A and H3K36me3, for HA-tag pulldowns from nuclear extracts of Psip1 ^–/– MEFs rescued with HA-p52 and HA-p75. ( C) Immunoprecipitation using anti- PSIP1/p75 and normal IgG followed by western blotting with PSIP/p75, PARP1, hnRNPU and SSRP antibodies, PCNA antibodies served as a negative control.

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).

Table 1. List of proteins that associate with PSIP/p75 in mouse embryonic fibroblasts and stem cells.

*proteins also found associated with H3K36me3 chromatin ( Dataset 1); ND, proteins not detected.

Proteins with higher SILAC ratio	Known function	P75/IgG (MEFs)	P75/IgG (mESCs)
PC4 and SFRS1-interacting protein *	Transcription, alternative splicing Promotes DNA repair	93.5	22
Cell division cycle-associated 7-like	Ser/Thr kinase protein	17.7	6
Histone H2A.X *	DNA damage response	ND	23
Isoform Alpha of DNA ligase 3	NHEJ, BER, SSBR	10.9	ND
PARP1 *	DNA repair	8.7	4
XRCC1	single-strand DNA breaks repair	7.3	ND
FACT complex subunit (SSRP1)	Nucleosome exchange, DNA repair	5.9	ND
FACT complex subunit (SPT16) *	Nucleosome exchange, DNA repair	ND	6.3
Heterogeneous nuclear ribonucleoprotein U Top2 alpha *	DNA Double-Strand Break Signaling and Repair DNA replication & transcription	5.0 9.2	ND ND
Top2 beta *	DNA replication & transcription	ND	2.9
Tyrosine-protein kinase BAZ1B *	H2A.X kinase	3.6	ND

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.

Figure 4.

( A) Microscopic images of WT and Psip1 ^–/– MEFs after Comet assay, a representative image from 16 microscopic fields are shown. (n=2 biological replicates). ( B) Working model showing various DNA repair proteins that are recruited to H3K36me3 chromatin to modulate repair choice or enhance DNA repair at the site of transcription.

SILAC proteomic analysis of MOF/KAT8 complex

Acetylation of histone H4 at lysine 16 (H4K16ac) and enzymes responsible for H4K16ac – TIP60 (KAT5) and MOF (KAT8) have been implicated with DNA repair ( Kumar et al., 2013; Sharma et al., 2010; Tang et al., 2013). Recent work suggests a link between H4K16ac and H3K36me3 in DNA damage response ( Li & Wang, 2017). 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 ( LeRoy et al., 2019). These evidences suggests FACT and PSIP function together or in same manner to facilitate efficient DNA repair.

Several pieces of evidence have emerged in recent years for the role of histone modifications especially H3K36me3 in DNA repair. Mammalian SETD2 (homolog of Set2) catalyses H3K36me3 at expressed gene bodies in a transcription dependent manner. H3K36me3 in turn recruits PSIP1 ( Daugaard et al., 2012; Pradeepa et al., 2012; Pradeepa et al., 2014), MRG15 ( Bleuyard et al., 2017; Luco et al., 2010), BS69 ( Guo et al., 2014), DNMT3a ( Dhayalan et al., 2010) and MSH6 ( Li et al., 2013), which modulate transcription, DNA methylation, alternative splicing, and DNA repair choice. A clear association of H3K36me3 with PSIP1 at expressed gene bodies and their association with several DNA repair proteins involved in NHEJ (this work) and HDR ( Aymard et al., 2014; Daugaard et al., 2012), suggests wider role of H3K36me3/PSIP1 axis in DNA damage response and genome stability.

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.

Methods

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.

ChIP mass-spectrometry

mESCs were cultured in GMEM as described previously ( Pradeepa et al., 2016). Cells were harvested by trypsinization and fixed immediately with 1% formaldehyde (Thermo Fisher, Cat. 28906) (25°C, 10 min) in PBS, and stopped with 0.125M Glycine. Cross linked cells were re-suspended in Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, Complete Mini EDTA-free protease inhibitor; Roche) for 30 minutes and centrifuged at 228 g for 5 minutes at 4°C. Nuclei were resuspended in RIPA buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (filtered 0.2 -0.45 micron filter unit) + Complete Mini EDTA-free protease inhibitor; Roche) and sonicated using a Bioruptor ^® Plus sonication device (Diagenode) at full power for 50 minutes (30 seconds on, 30 seconds off) to produce fragments of 200–500 bp. 10 µg of each antibody was incubated with Protein A Dynabeads (ThermoFisher Scientific, 10001D) in 5 mg/ml bovine serum albumin (BSA) in PBS on a rotating platform at 4°C for two hours. An arbitrary concentration of 200 µg chromatin was incubated with antibody bound Dynabeads on a rotating platform at 4°C for 16 hours. Beads were washed 5 times (5 minutes each) on a rotating platform with cold LiCl wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% Sodium deoxycholate) and one time with RT TE buffer.

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.

HA-pulldown of p52 and p75

PSIP1 knockout MEFs stably rescued with HA-PSIP/p52 and HA-PSIP/p75 were immunoprecipitated with anti-HA antibodies. 5% input lysate and IPed proteins were separated on Novex 4–20% gels and transferred to PVDF membranes. Western blotting was performed by immunoblotting with MLL1 (Active Motif, 61295), γH2A.X (Millipore, 05-636), H2A (Abcam, ab18255) and H3K36me3 (Abcam, ab9050) 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 data for this study are available from OSF http://doi.org/10.17605/OSF.IO/UAX7G ( Pradeepa, 2017). Dataset 1: ChIP MS data in mESCs, Dataset 2: PSIP/p75 SILAC results in MEFs; Dataset 3: PSIP/p75 SILAC results in mESCs, WT MEFs Comet assay data, PSIP1 KO Comet assay data, and uncropped blots for Figure 3B.

Funding Statement

This work was supported by the Wellcome Trust [085767]; and the Medical Research Council and centenary award to MP and a H2020 Marie-Curie Individual Fellowship (896079, PSIPCancer) awarded to JS. MP is supported by the UKRI/MRC grant (MR/T000783/1) and Barts charity (MGU0475) grants.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.