ARTICLE Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions Ramhari Kumbhar1,2, Anthony Sanchez1,2, Jullian Perren1,2, Fade Gong4, David Corujo5, Frank Medina1,2, Sravan K. Devanathan1,2, Blerta Xhemalce1,2,3, Andreas Matouschek1,2, Marcus Buschbeck5,6, Bethany A. Buck-Koehntop7, and Kyle M. Miller1,2,3 The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)–binding region unique to KDM5A. Loss of the PAR- binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi’s) blocks KDM5A–PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity. Introduction DNA is recurrently damaged by endogenous processes, in- HR factors breast cancer gene types 1 and 2 (BRCA1 and BRCA2) cluding transcription and replication, as well as by dysregu- found recurrently mutated in breast and ovarian cancer lated proteins and chemical reactions within cells (Chatterjee (Stratton and Rahman, 2008). While mutations in DNA repair and Walker, 2017; Lindahl and Barnes, 2000; Tubbs and genes result in genome instability, these genetic alterations also Nussenzweig, 2017; Xia et al., 2019). Exogenous agents, in- provide vulnerabilities that can be targeted therapeutically in cluding UV light, chemicals, and cancer therapies (i.e., radiation), some genetic backgrounds (Ashworth and Lord, 2018; Jackson also damage DNA (Ciccia and Elledge, 2010; Jackson and Bartek, and Helleday, 2016; O’Connor, 2015; Pilié et al., 2019). This 2009). An inability to constrain and repair DNA damage can result strategy has been successfully employed against BRCA-deficient in mutations and genome instability. Loss of genome integrity cancers, which display synthetic lethality with poly(ADP-ribose; can have dire consequences for cellular and organismal homeo- PAR) polymerase (PARP) inhibitors (PARPi’s), a treatment now stasis, including the development of cancer (Jeggo et al., 2016; used clinically (Bryant et al., 2005; Farmer et al., 2005; Fong Negrini et al., 2010). Cells employ an extensive network of et al., 2009; Lord and Ashworth, 2017). proteins, referred to as the DNA damage response (DDR), that Chromatin is an integral component of the DDR as DNA is counteract DNA damage by sensing its presence in the genome, organized by histones into chromatin, thereby controlling ac- activating the appropriate response, and repairing the lesion cessibility and DDR factor functions at DNA lesions that reside (Ciccia and Elledge, 2010; Jackson and Bartek, 2009). Mutations within the chromatin environment (Agarwal and Miller, 2016; within DDR-related genes are frequent in cancer (Knijnenburg Caron et al., 2019; Chiu et al., 2017; Clouaire and Legube, 2019; et al., 2018), which helps explain the prevalence of genome in- Dabin et al., 2016; Gong and Miller, 2019; Kim et al., 2019c; Tan stability in cancer. For example, DNA double-strand breaks and Huen, 2020). One such chromatin mechanism involved in (DSBs) are repaired mainly by homologous recombination (HR) DDR regulation is histone post-translational modification. Histone and nonhomologous end joining (Chapman et al., 2012), with the marks, including phosphorylation, methylation, acetylation, and ............................................................................................................................................................................. 1Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX; 2Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX; 3Livestrong Cancer Institutes, Dell Medical School, The University of Texas at Austin, Austin, TX; 4Department of Biochemistry & Molecular Biology, Baylor College of Medicine, Houston, TX; 5Cancer and Leukemia Epigenetics and Biology Program, Josep Carreras Leukaemia Cancer Institute, Barcelona, Spain; 6Program for Predictive and Personalized Medicine of Cancer, Germans Trias i Pujol Research Institute, Badalona, Spain; 7Department of Chemistry, University of Utah, Salt Lake City, UT. Correspondence to Kyle M. Miller: kyle.miller@austin.utexas.edu. © 2021 Kumbhar et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1083/jcb.202006149 1 of 20 J. Cell Biol. 2021 Vol. 220 No. 7 e202006149 ubiquitylation, act to regulate chromatin structure and function to provide valuable insights into their potential involvement in coordinate the DDR with DNA-templated processes such as tran- other biological processes dependent on this lysine demethylase, scription, replication, and repair (reviewed in Kim et al., 2019c). including transcription and cancer. Histone variants, including the histone H2A family variants H2AX, H2AZ, and macroH2A, also participate in the regulation of the DDR (Buschbeck and Hake, 2017; Corujo and Buschbeck, 2018; Results Kim et al., 2018; Turinetto and Giachino, 2015). Collectively, KDM5A promotes genome integrity chromatin employs multiple mechanisms to engage DDR factors to We previously demonstrated the involvement of KDM5A in facilitate DNA damage signaling and repair within the chromatin regulating the ZMYND8-NuRD chromatin remodeling complex environment. at DNA damage sites. The KDM5A–ZMYND8–NuRD axis func- Histone methylation plays important roles in the DDR (Gong tions to demethylate H3K4me3 at DNA damage sites, allowing and Miller, 2019; Kim et al., 2019c). For example, lysine-specific ZMYND8-NuRD to localize to DNA lesions within transcrip- demethylase 5 (KDM5) subfamily proteins, consisting of four tionally active chromatin, where this pathway represses tran- jmjC proteins, KDM5A–D, target the transcription-associated scription and promotes HR repair (Gong et al., 2015; Gong et al., marks H3K4me2/3 to regulate transcription and the DDR, in- 2017; Gong and Miller, 2018; Savitsky et al., 2016; Spruijt et al., Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 cluding DSB repair, p53 regulation, and the response to oxidative 2016). To gain further insight into the involvement of KDM5A in stress (Batie et al., 2019; Bayo et al., 2018; Christensen et al., genome integrity pathways, we obtained colon carcinoma cell 2007; Gong et al., 2017; Hu et al., 2018; Klose et al., 2007; Li lines lacking KDM5A (see Materials and methods). Western blot et al., 2014b; Xu et al., 2018). KDM5A (alias JARID1A) is re- (WB) analysis confirmed the loss of KDM5A protein in this cruited to DSBs, where it demethylates H3K4me3 to regulate the HCT116 KDM5A-knockout (KDM5A-KO) cell line (Fig. S1 A). We ZMYND8–nucleosome remodeling and histone deacetylation also observed increased expression of the KDM5A target gene (NuRD) chromatin remodeling complex (Gong et al., 2017). E-cadherin in HCT116 KDM5A-KO cells versus WT parental ZMYND8 binds acetylation marks at DSBs through its acetyl- HCT116 cells, providing additional evidence for the functional binding bromodomain, recruits NuRD to repress transcription, loss of KDM5A in these cells (Fig. S1 B; Dabiri et al., 2019; Feng and promotes HR repair at DSBs (Gong et al., 2015; Gong et al., et al., 2017; Liang et al., 2015; Wang et al., 2013). 2017; Gong and Miller, 2018; Savitsky et al., 2016; Spruijt et al., Consistent with the involvement of KDM5A in HR, cells de- 2016). KDM5A is also found to be dysregulated in several can- ficient for KDM5A were sensitive to the PARPi olaparib (Fig. 1 A; cers. For example, KDM5A is up-regulated in multiple cancers KDM5A protein levels of cells analyzed in Fig. S1 A). PARPi (Cao et al., 2014; Choi et al., 2018; Li et al., 2014a; Teng et al., sensitivity often correlates with HR deficiency because cells 2013; Zeng et al., 2010) and promotes resistance to cancer deficient for HR factors, such as BRCA1 or BRCA2, are sensitive treatments, including to DNA-damaging agents (Hou et al., 2012; to PARP inhibition (Bryant et al., 2005; Farmer et al., 2005; Lord Sharma et al., 2010; Vinogradova et al., 2016; Yang et al., 2019). and Ashworth, 2017; O’Connor, 2015). To validate that the ob- KDM5B is also involved in cancer and radioresistance (Bayo served effects were due to KDM5A loss, we reconstituted et al., 2018; Xhabija and Kidder, 2019). These findings have KDM5A-KO HCT116 cells with GFP-tagged full-length KDM5A highlighted KDM5A and other KDM5s as potential therapeutic (Fig. S1 A). The observed sensitivity of KDM5A-KO cells to targets in cancer. However, the potential connection between PARPi’s was largely rescued by complementation with ectopi- the DNA repair activities of KDM5A and its alterations in cancer cally supplied KDM5A, confirming that these results were due to have not been investigated. Although the function of histone loss of KDM5A in these cells (Fig. 1 A). PARP1 activity is required demethylation by KDM5A in transcription and DNA damage for KDM5A localization to the DNA damage sites to allow effi- repair have been described, the factors that regulate KDM5A in cient DSB repair by HR (Gong et al., 2017). Similar to KDM5A-KO these processes are poorly understood. cells, human osteosarcoma (U2OS) cells treated with the KDM5A Here, we have identified two mechanisms that regulate inhibitor CPI455, which blocks the catalytic activity of this en- KDM5A localization to DNA damage sites. First, we identified a zyme, displayed reduced cell survival after PARPi treatment PAR interaction domain (PID) within KDM5A in its C terminus compared with untreated cells or cells treated with either single that is required to recruit KDM5A to DNA damage sites. This inhibitor (Fig. 1 B). Thus, KDM5A deficiency results in loss of cell domain is unique to this KDM5 lysine demethylase and likely viability because of PARP inhibition. contains a coiled-coil domain, which is required for binding to KDM5A-deficient cells treated with PARPi’s exhibited several PAR chains. KDM5A-PID by itself can localize to damage sites in phenotypes consistent with defective genome integrity path- a PARP-dependent manner, and its presence in KDM5A is re- ways in these cells that were antagonized by PARPi treatment. quired for its genome integrity–promoting functions, including This includes increased formation of micronuclei and persis- DNA repair. Second, the histone H2A variant macroH2A1.2 tence of DSBs as detected by the DNA damage marker histone promotes KDM5A recruitment to DNA damage. Functionally, variant H2AX phosphorylation (γH2AX; Fig. 1, C–F). These loss of macroH2A1.2 results in defective HR and DSB-induced conditions also result in the appearance of persistent DSBs in transcriptional repression, which phenocopies KDM5A defi- KDM5A-KO cells as detected by neutral comet assays (Fig. 1 G). ciency. Thus, we propose that PAR binding and macroH2A1.2 Depletion of PARP1 by siRNA gave similar phenotypes in recognition engage KDM5A to target this enzyme to DNA dam- KDM5A-KO cells, validating that PARP1 deficiency by either age sites. Identification of KDM5A regulators in the DDR may protein loss or inhibition resulted in increased DNA break Kumbhar et al. Journal of Cell Biology 2 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 1. KDM5A deficiency results in genome instability and sensitivity to PARP inhibition. (A) Clonogenic survival assay of HCT116 WT, KDM5A-KO, and KDM5A-KO + GFP-KDM5A complemented cells. Cells were treated with the PARPi olaparib at the indicated doses for 24 h, and colonies were quantified after 2 wk. Error bars represent SD; n = 3. (B) Clonogenic survival assay of U2OS cells treated with DMSO, 25 µM CPI455, and olaparib at the indicated doses for 24 h. Colonies were analyzed as in A. Error bars represent SD; n = 3. (C) KDM5A suppresses micronuclei formation. HCT116 WT and KDM5A-KO cells were treated with 5 µM olaparib for 24 h and immunostained with DAPI to detect micronuclei. Scale bars, 10 µm. White arrowheads mark micronuclei. (D) Quantification of C. n = 2; >100 cells quantified per condition per replicate. (E and F) KDM5A deficiency leads to DNA damage that is exacerbated by PARP inhibition. HCT116 WT or KDM5A-KO cells were treated with or without 5 µM PARPi (olaparib) for 24 h and immunostained for the DNA break marker γH2AX. γH2AX foci are quantified and plotted in F. n = 2; >100 cells were quantified per condition per replicate. Error bars represent SEM. Scale bars, 20 µm. (G) Loss of KDM5A increases DSBs as detected by neutral comet assay. Cells were treated as in C. Scale bars, 100 µm. Right panel: quantification of left panel. Olive tail moment for >100 cells quantified per condition per replicate and plotted as a box and whiskers; n = 2. Error bars represent SEM. P values were calculated by Tukey’s multiple comparison test. P values for A, B, D, and E were calculated with unpaired Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). formation and loss of genome integrity in cells lacking KDM5A of KDM5A did not alter the expression of other JARID1 histone (Fig. S1, C–F; efficiency of siRNA depletion of PARP1 was con- demethylases, KDM5B and KDM5C, suggesting these results are firmed by WB analysis in Fig. S1 G). specifically due to KDM5A deficiency (Fig. S2 H). Collectively, To further support the involvement of KDM5A in genome these results validate our cell models for KDM5A loss and integrity, we performed the same experiments in U2OS cells provide strong evidence for the role of KDM5A in genome lacking KDM5A, which we generated using CRISPR-Cas9 gene integrity pathways, including those impacted by PARPi’s editing (Fig. S2 A). Like HCT116 cells, U2OS cells lacking KDM5A (i.e., DNA repair). derepressed E-cadherin and were sensitive to PARPi treatment (Fig. S2, B and C). U2OS cells lacking KDM5A displayed in- KDM5A interacts with PARP1 and PAR chains in cells creased micronuclei formation after PARPi treatment and an PARP1 recruitment and formation of PAR chains at sites of DNA inability to clear DNA breaks following PARP1 inhibition by ei- damage are one of the earliest responses to DNA damage. PAR- ther olaparib or siRNA depletion of PARP1 (Fig. S2, F and G). Loss ylation promotes the rapid accumulation of DNA damage factors Kumbhar et al. Journal of Cell Biology 3 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 2. KDM5A interacts with PARP1 and PAR chains. (A) Endogenous KDM5A and PARP1 interact. KDM5A was immunoprecipitated from HEK293T cells with or without 10-Gy IR. Samples were evaluated by WB analysis with the indicated antibodies. (B) Reciprocal IP with endogenous PARP1 was performed as in A. (C) KDM5A binds to PAR in cells in a PARP-dependent manner. HEK293T cells were treated with olaparib (5 µM, 1 h) followed by 10-Gy IR or hydrogen peroxide (H2O2; 1 mM, 15 min) and examined by IP-WB analysis with an anti-PAR antibody. (D and E) KDM5A binds PAR noncovalently. U2OS cells were treated with 10-Gy IR for 2 min, and cell lysates were incubated with KDM5A or IgG antibodies with or without 1% SDS. Purified samples were either blotted on nitrocellulose membrane to detect PAR chains or resolved on SDS-PAGE gel and immunoblotted to detect KDM5A. In E, experiments were performed as in D in cells expressing GFP, GFP-KDM5A, or GFP-PARP1. Immunoprecipitated samples with or without SDS were blotted onto nitrocellulose membrane and detected with anti-PAR and anti-GFP for loading. (F) Purified KDM5A from U2OS cells binds PAR chains in vitro. Purified proteins were spotted onto nitrocellulose membrane and probed with purified PAR chains with binding detected with an anti-PAR antibody. to DNA lesions, including DSBs (Ray Chaudhuri and Nussenzweig, and poly(ADP) ribosylation nucleates the localization of many 2017). To further delineate how PARP1 regulates KDM5A in the proteins in the vicinity of DNA damage sites (Gibson and Kraus, DDR, we tested whether KDM5A interacts with PARP1 directly by 2012; Liu et al., 2017; Ray Chaudhuri and Nussenzweig, 2017), we performing coimmunoprecipitation (co-IP) followed by WB next assessed if KDM5A was associated with PAR in cells. IP of analysis. IP of endogenous KDM5A with an anti-KDM5A antibody KDM5A or anti-PAR antibodies revealed an association of in human embryonic kidney (HEK293T) cells detected an inter- PARylated proteins with KDM5A (Fig. 2 C and Fig. S3 A), sug- action between endogenous PARP1 and KDM5A (Fig. 2 A). We gesting that KDM5A or its associated proteins are PARylated obtained similar results in U2OS cells and further controlled for and/or bind PAR chains. The increased binding between KDM5A the specificity of the KDM5A antibody, as the immunoprecipitated and PAR was further enhanced by treatment with hydrogen signal and PARP1 interaction were abolished in samples from peroxide, another DNA damage–inducing agent and potent ac- KDM5A-KO U2OS cells (Fig. S2 A). Reciprocal co-IP WB analysis tivator of PARP1 (Fig. 2 C and Fig. S3 B). Treatment of cells with using PARP1 antibodies in HEK293T cells corroborated the observed the PARPi olaparib inhibited KDM5A–PAR interactions (Fig. 2 interaction between KDM5A and PARP1 (Fig. 2 B). The interaction C), further supporting that these interactions are PARP depen- between PARP1 and KDM5A was still detected and not increased in dent. These results established a connection between KDM5A cells treated with ionizing radiation (IR) compared with cells that and PARylation in cells but do not distinguish between PAR were undamaged (Fig. 2, A and B). Reduced PARP1–KDM5A inter- binding or direct PARylation of KDM5A and/or an interacting actions upon IR treatment could be due to increased binding of protein. To address these various possibilities, KDM5A was PARP1 to DNA or DNA damage–induced autoPARylation of PARP, immunoprecipitated from cells following DNA damage by IR behaviors of PARP1 that are known to disrupt protein–protein in- under denaturing conditions using 1% SDS, and the presence of teractions (Thomas and Tulin, 2013). PAR chains was evaluated by WB analysis. KDM5A–PAR inter- Given that PARP1 catalytic activity promotes the recruitment actions were readily detected under native conditions (i.e., in of KDM5A to laser-induced DNA damage sites (Gong et al., 2017) the absence of SDS) and were abolished by the addition of SDS to Kumbhar et al. Journal of Cell Biology 4 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 denature the samples, which suggested KDM5A was not itself cells. We observed rapid recruitment of GFP-KDM5A-F8 to DNA PARylated (Fig. 2 D). We further confirmed our assay conditions damage sites after microirradiation (Fig. 3 F; quantified in using PARP1, which is known to be directly PARylated upon Fig. 3 G). Retention of KDM5A-F8 was transient, with the DNA damage. Unlike KDM5A, anti-PAR dot-blot analysis of maximum intensity of recruitment occurring within the first immunoprecipitated GFP-PARP1 after IR treatment revealed minute after damage induction followed by a steady decline in signals in both native and denaturing conditions (Fig. 2 E). Thus, localization at damage sites. These recruitment dynamics were these results are consistent with KDM5A binding PAR chains reminiscent of the short lifespan of PAR chains, which are directly and not being itself a direct substrate for PARylation rapidly removed by PAR hydrolase (Alvarez-Gonzalez and Al- under these conditions. These interactions are also likely not thaus, 1989; Kassab et al., 2020; O’Sullivan et al., 2019). Con- mediated by nucleic acids, given that cell lysates were treated sistent with the in vitro binding of PAR chains by KDM5A-F8, with a broad-spectrum DNA/RNA nuclease. Collectively, these recruitment to DNA damage sites of this domain-containing results indicated that KDM5A interacts with PARP1 and PAR fragment was highly reduced in cells treated with PARPi’s chains in cells in response to DNA damage. (Fig. 3 F; quantified in Fig. 3 G). In sum, these results map the PID of KDM5A within aa 1491–1610 and show that this region is KDM5A directly binds PAR chains in vitro sufficient for localization to damage sites in a PARP-dependent Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Our results could be explained by the direct binding of KDM5A manner, supporting the identification of a PID within this region to PAR chains as a mechanism to direct its association to DNA of KDM5A. damage lesions. To test this hypothesis, we determined if KDM5A was capable of binding PAR chains directly using Characterization of the PAR-binding domain within KDM5A in vitro PAR-binding assays (Ahel et al., 2008; Kumbhar et al., We next considered whether other KDM5 proteins interact di- 2018). Purified target proteins, including immunoprecipitated rectly with PAR chains. To address this question, we performed KDM5A, histone H2A (a strong PAR-binding protein), and BSA DNA damage recruitment analyses of GFP-tagged KDMs and (negative control), were blotted onto nitrocellulose membrane PAR-binding assays with purified GFP-KDM5A, GFP-KDM5B, and probed with purified PAR chains. Using purified KDM5A and GFP-KDM5C from U2OS cells. We did not include the male- from U2OS cells, we observed KDM5A binding to PAR chains specific KDM5D protein for this analysis. KDM5 proteins share (Figs. 2 F and S3 C). These results were consistent with KDM5A similar domain structures and are highly homologous (Fig. 4 A). binding directly to PAR chains and prompted us to identify the While GFP-KDM5A is readily detected at laser-induced DNA PAR-binding region within KDM5A. Several PAR-binding do- damage sites, GFP-tagged KDM5B and KDM5C were not re- mains have been reported (Krietsch et al., 2013; Teloni and cruited to these lesions (Fig. 4, B and C). This result is consistent Altmeyer, 2016; Wei and Yu, 2016), but none of these are an- with another report that did not observe KDM5B recruitment to notated within KDM5A in the Pfam database. Thus, to identify DNA damage after laser microirradiation (Li et al., 2014b). Since the PAR-binding region of KDM5A, we dissected full-length KDM5A and KDM5B are both reported to demethylate H3K4me3 KDM5A into eight partially overlapping maltose-binding pro- at DNA damage sites, we also tested if KDM5B could be recruited tein (MBP)-tagged fragments (F1–F8), ensuring that the main to laser-induced DNA damage in cells lacking KDM5A. Even in functional domains of KDM5A were preserved within the frag- cells lacking KDM5A, KDM5B was unable to associate with DNA ments (Fig. 3 A). These fragments were expressed and purified damage sites to the extent detectable by this approach (Fig. S3 from Escherichia coli, then used in in vitro PAR-binding assays E), suggesting different mechanisms are responsible for regu- (Fig. 3 B). These experiments identified three putative PAR- lating these two demethylases in the DDR. KDM5A, but not binding regions, and the C-terminal fragment of KDM5A (F8), KDM5B and KDM5C, displayed PAR-binding activities (Fig. 4 D). which contains aa 1431–1690, exhibited the strongest binding to Taken together, these data suggest that direct binding to PAR PAR chains (Figs. 3 C and S3 D). chains by KDM5A is a unique mechanism that regulates its The KDM5A-F8 fragment contains the third plant homeo- damage localization compared with other KDM5 demethylases. domain (PHD) of KDM5A, which interacts with its substrate To further define the molecular properties of the PID in H3K4me3 and is dispensable for supporting KDM5A localization KDM5A, we performed a multicoil sequence analysis of KDM5A, to DNA damage sites (Gong et al., 2017; Wang et al., 2009). To KDM5B, and KDM5C. The MultiCoil algorithm predicts coiled- further map the PID within KDM5A, we created four additional coil location and oligomerization in protein sequences (Wolf truncation mutations within F8, including one containing the et al., 1997). This analysis revealed that the PAR-binding re- PHD3 domain (Fig. 3 D). F8-2 (aa 1491–1560) and F8-3 (aa gion of KDM5A contains a highly probable coiled-coil domain, 1551–1610) displayed PAR binding, and, as expected, the frag- which is absent in KDM5B and KDM5C (Fig. 4 E). The region ment containing the PHD3 domain did not bind PAR (Fig. 3 E). consists of highly basic and hydrophobic amino acids and is These data show that the PAR-binding domain resides within aa predicted to be intrinsically disordered by IUPred, a prediction 1491–1610 in the C terminus of KDM5A and, importantly, is in- tool designed to identify protein regions with these biophysical dependent from the PHD3 domain. properties that are known to be ideal for PAR binding (Fig. S4, A We next sought to determine if the KDM5A-F8 region was and B; Dosztányi et al., 2005). This putative coiled-coil PAR- sufficient to support DNA damage localization. To this end, we binding domain of KDM5A is not conserved between KDM5B generated GFP-tagged KDM5A-F8 (GFP-KDM5A-F8) and ana- and KDM5C but is highly conserved across KDM5A homologues lyzed its ability to be recruited to laser-induced DNA damage in from higher eukaryotes (Fig. S4, B and C). Intrigued by these Kumbhar et al. Journal of Cell Biology 5 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 3. Mapping of a PAR interaction domain within KDM5A. (A) Domain organization of KDM5A. Schematics for overlapping KDM5A fragments (F1–F8) are indicated. (B) Expression of affinity-purified, MBP-tagged KDM5A fragments. Purified fragments were analyzed by SDS-PAGE and Coomassie blue staining. (C) Purified MBP and MBP-KDM5A fragments F1–F8 were analyzed for PAR binding as in Fig. 2 F. (D) Schematics for KDM5A-F8 and derivatives. Blue box indicates PHD3 domain. (E) PAR binding resides in KDM5A-F8-2 and F8-3. PAR binding was performed as in C with indicated proteins. (F) GFP-KDM5A-F8 sufficient for PARP-dependent recruitment to laser damage. Laser microirradiation was performed in U2OS cells transfected with GFP-KDM5A-F8 with or without the PARPi olaparib (5 µM, 1 h). Damaged region is indicated by dotted white circle. Scale bars, 5 µm. (G) Quantification of F from two representative experiment where n ≥ 20 cells. Error bars represent SEM. P values were calculated with unpaired Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). observations, we set out to further demonstrate that the pu- These analyses, along with our previous binding studies, support tative coiled-coil region containing the PID (residues 1501–1562) the presence of a PID within the C terminus of KDM5A that is is required for PAR binding. To this end, we deleted this re- specific to this KDM5 demethylase family member and that con- gion from MBP-tagged KDM5A-F8 to create KDM5A-F8ΔPID. tains a putative coiled-coil domain. KDM5A-F8 and KDM5A-F8ΔPID were recombinantly purified To gain further insight into whether the KDM5A-F9 region from E. coli and tested for PAR binding using an in vitro PAR- contained characteristics of a coiled-coil domain, we performed binding assay. This analysis confirmed that the KDM5A-PID circular dichroism (CD) spectroscopic analysis on both KDM5A- contained within F8 was required for PAR binding because F9 and F9ΔPID. Consistent with the domain mapping and pre- removal of the proposed PID abolished KDM5A-F8 interactions diction analyses, KDM5A-F9 displayed an apparent α-helical with PAR chains (Fig. 4, F and G; purified proteins shown in Fig. component, as exemplified by the presence of CD spectral S5 A). We had previously identified two overlapping fragments minima at both 222 nm and ∼208 nm (Fig. 4 J). Notably, how- within F8 of KDM5A that interacted with PAR (Fig. 3 E; frag- ever, the spectral region between 200 and 215 nm is more ments F8-2 and F8-3). We purified an untagged KDM5A 1491–1610 negative than would be expected for a predominantly helical fragment (referred to as F9) containing these two domains along protein and is indicative of the F9 region also containing in- with an F9ΔPID (1491–1610Δ1501–1562) mutant of KDM5A and trinsically disordered protein elements, which is also consistent performed PAR-binding assays (schematic of KDM5A-F8 and -F9 with our prediction models based on sequence (Fig. S4 A). constructs shown in Fig. 4 H). The results using these protein Further CD analysis of F9ΔPID showed a significant reduction in products were similar, showing PAR binding to be dependent on helical signatures and resulted in a spectrum consistent with a the PID of KDM5A (Fig. 4 I; purified proteins shown in Fig. S5 B). predominantly disordered protein (Fig. 4 J). Together, these data Kumbhar et al. Journal of Cell Biology 6 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 4. KDM5A contains a coiled-coil domain that facilitates preferential binding to longer PAR chains. (A) Schematic of human KDM5A, KDM5B, and KDM5C. (B) Recruitment of GFP-KDM5A, GFP-KDM5B, and GFP-KDM5C to laser-induced DNA damage sites using live-cell confocal microscopy and laser microirradiation. White dotted lines indicate path of laser damage. Scale bars, 5 µm. (C) Quantification of B from one representative experiment. n > 7 in- dividual cells per condition. Two-way ANOVA with Sidak’s multiple comparison test was used for statistical analysis comparing KDM5A with KDM5B and KDM5C recruitment. Error bars represent SEM (***, P < 0.001). (D) KDM5A uniquely interacts with PAR chains. GFP-tagged KDM5A, KDM5B, and KDM5C were Kumbhar et al. Journal of Cell Biology 7 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 expressed and purified from U2OS cells. PAR binding was performed as in Fig. 2 F. (E) KDM5A contains a predicted coiled-coil region that is absent from KDM5B and KDM5C. Predicted per residue coiled-coil scores for dimers were calculated using MultiCoil (see Materials and methods). (F) KDM5A region predicted as a coiled-coil (residues 1501–1562) is required for PAR binding (PID). PAR-binding assay performed as in D. Diagram of KDM5A-F8 and KDM5A-F8ΔPID with putative coiled-coil (cc) and PHD3 domains indicated. (G) Far-WB analysis of KDM5A PAR binding. The indicated proteins were resolved by SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was incubated with PAR chains, and the indicated proteins were detected as in D. (H) Comparative diagram of KDM5A-F8 with KDM5A-F9 (1491–1610) and KDM5A-F9ΔPID (1491–1610Δ1501-1562). (I) PAR-binding analysis of KDM5A-F9 and KDM5A-F9ΔPID. PAR binding assay was performed as in D. (J) CD spectrum for the F9 region of KDM5A-F9 (purple) is consistent with the presence of α-helical protein el- ements, while the CD spectrum for F9ΔPID (1491–1610Δ1501-1562; red) exhibits an appreciable loss in this helicity, supporting the predictive model of a coiled- coil domain between residues 1501 and 1562. Notably, both KDM5A-F9 and F9ΔPID harbor significant intrinsically disordered protein regions. deg, degree; MRE, mean residue ellipticity. (K) KDM5A-F9, but not F9ΔPID, has preferential nM binding for medium and long PAR chains. (L) EMSA titrations demonstrate that KDM5A-F9 has nM binding for a medium-length 27-mer PAR chain, while PAR recognition by F9ΔPID is severely compromised. suggest that an α-helical structural element resides within the residues 1501–1562 that binds PAR, with a preference for longer region spanning KDM5A residues 1501–1562, which encom- PAR chains. passes the predicted coiled-coil region and domain responsible Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 for PAR binding. KDM5A-PAR binding is essential for KDM5A recruitment and To better quantify the ability of KDM5A-F9 to recognize PAR function at DNA damage sites chains, electrophoretic gel mobility shift assay (EMSA) analysis To test for the functional importance of the identified KDM5A was performed. To date, only a few studies have been directed PAR-binding region, we analyzed the ability of KDM5A protein toward quantitively characterizing the molecular recognition of lacking the PID to bind to sites of DNA damage and act in the PAR-binding proteins with their PAR ligands (Barkauskaite DDR. Interestingly, GFP-KDM5A lacking the PAR-binding region et al., 2013; Fahrer et al., 2007; Fahrer et al., 2010; Fischer (KDM5AΔPID) displayed reduced recruitment to DNA damage et al., 2014; Krüger et al., 2019; Zhang et al., 2015). This has in sites compared with full-length GFP-KDM5A (Fig. 5, A–C). To part been due to limitations in obtaining sufficient quantities of assess if PAR binding by KDM5A is involved in supporting PAR polymers of a defined length, an impediment that has been its DDR activities, we reconstituted KDM5A-KO HCT116 cells overcome (Tan et al., 2012). One intriguing trend that has with WT and ΔPID GFP-KDM5A, allowing us to directly address emerged from these studies is that PAR-binding proteins appear this question. While KDM5A-KO cells were sensitive to IR and to have PAR chain length–dependent binding preferences PARPi’s, expression of WT KDM5A, but not KDM5AΔPID, was (Fahrer et al., 2007; Fahrer et al., 2010; Fischer et al., 2014). able to rescue the inability of KDM5A-KO cells to survive these Consistent with these previous observations, KDM5A-F9 ex- treatments (Fig. 5, D and E; protein expression of KDM5A de- hibits preferential recognition for medium- and long-chained rivatives analyzed in Fig. 5 F). Similarly, KDM5A-KO cells dis- PAR polymers, as evidenced by a significant depletion of the played increased micronuclei formation upon PARPi treatment, free PAR bands, while comparatively at this protein concentra- which was rescued by WT KDM5A but not KDM5AΔPID (Fig. 5 G). tion (250 nM), KDM5A F9ΔPID was significantly compromised Many of these phenotypes in KDM5A-KO cells could be explained in recognizing any of the PAR chain lengths (Fig. 4 K). Notably, by a defect in HR repair, which we previously reported in KDM5A in the EMSA studies, a supershifted band representing the siRNA-depleted U2OS cells (Gong et al., 2017). Using WT, KDM5A- F9–PAR complex was not detectable even after an extensive KO, and KDM5A-KO reconstituted HCT116 cells, we measured HR effort to optimize the assay. The lack of smearing in the gel and efficiency using a CRISPR-Cas9/mClover gene-targeting assay absence of complex being trapped in the well suggests that the (Kim et al., 2019b; Pinder et al., 2015). While KDM5A-KO cells complex was not able to enter the gel for detection. Neverthe- displayed reduced HR compared with WT or KDM5A-KO rescued less, it is unequivocally clear from these studies that the F9 re- cells expressing WT KDM5A, expression of KDM5AΔPID was gion of KDM5A is responsible for PAR chain recognition, while unable to rescue HR deficiencies in KDM5A-KO cells (Fig. 5 H). PAR binding by F9ΔPID is severely compromised. Interestingly, we observed that PARPi treatment in U2OS cells While the lack of an observable F9–PAR complex band pre- resulted in increased expression of the KDM5A repressed gene, vented quantification of binding affinities, it can nevertheless be E-cadherin, suggesting PAR binding by KDM5A may also par- seen from EMSA titration that under our assay conditions, ticipate in regulating the transcriptional repressive activities KDM5A-F9 qualitatively binds a medium-sized 27-mer PAR of this chromatin regulator (Fig. S5 C). Taken together, these chain with a relatively low nM affinity (∼100–175 nM), a range data demonstrate that KDM5A utilizes a domain consisting of a consistent with known PAR-binding domains (Fig. 4 L; Fahrer coiled-coil region in its C terminus to bind PAR chains, which et al., 2007; Teloni and Altmeyer, 2016). A moderate capability is required to localize this enzyme to damage sites, where it for binding the 27-mer PAR chain by KDM5A F9ΔPID can only be functions on chromatin to promote genome integrity and HR seen at low μM concentrations (Fig. 4 L). The fact that KDM5A repair. F9ΔPID can exhibit weak recognition of PAR chains at higher protein concentrations suggests that KDM5A-F9 may use ad- KDM5A interacts with the histone variant macroH2A1.2 ditional protein elements outside of the coiled-coil domain to Given the importance of KDM5A in chromatin-based DNA repair enhance PAR recognition. Taken together, these results are activities, we sought to test if other histone pathways may consistent with KDM5A containing a coiled-coil domain within regulate KDM5A activities. We focused on the histone variant Kumbhar et al. Journal of Cell Biology 8 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 5. The PID containing a coiled-coil region is required for damage localization and DDR functions of KDM5A. (A) Diagram of KDM5A constructs used in B. (B) KDM5A-PID is required for damage localization. Experiments were performed as in Fig. 3 F. Damaged regions are indicated by dotted white outlines. Scale bars, 5 µm. (C) Quantification of B from one representative experiment. Error bars represent SEM; n ≥ 10 cells. Damaged regions are indicated by a dotted white line. (D and E) KDM5A-PID is required for survival in response to IR and PARPi. Clonogenic survival assays were performed as in Fig. 1 A with the indicated ectopically expressed KDM5A genes. Error bars represent SD; n = 3. (F) WB analysis of WT and KDM5A-KO HCT116 cells, including GFP-KDM5A WT and ΔPID reconstituted cells. (G) KDM5A-PID suppresses micronuclei formation. Experiments were performed as in Fig. 1 C with the indicated KDM5A-KO cells and complemented cells as in D. Error bars represent SD; n = 2. P values were calculated by Tukey’s multiple comparison test (**, P < 0.01; ***, P < 0.001). (H) KDM5A-PID is required for efficient homology-directed repair (HDR). HDR efficiency in HCT116 WT and KDM5A-KO cells with or without SFB-KDM5A and SFB-KDM5AΔPID was determined using a CRISPR-mClover HR assay. GFP+ cells represent a repair event. The percentage of GFP+ cells was normalized to WT. Error bars represent SEM; n = 2. P values were calculated for C, D, E, and H with unpaired Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). macroH2A because this variant can bind and be regulated by KDM5A interacted with, we performed endogenous KDM5A IP PARP and is involved in HR (Khurana et al., 2014; Ruiz et al., in HEK293T cells and immunoblotted with antibodies specific to 2019; Timinszky et al., 2009). macroH2A1 has two splice var- macroH2A1.1 and macroH2A1.2. We observed that KDM5A in- iants, macroH2A1.1 and macroH2A1.2, which differ in 32 amino teracted specifically with macroH2A1.2 (Fig. S5 E; antibody acids within the C-terminal macrodomain, leading to the pres- specificity for macroH2A1.1 and macroH2A1.2 is shown in Fig. ence of a PAR-binding domain in macroH2A1.1 but not in S5, F and G). To further validate these results, we performed co- macroH2A1.2 (Kozlowski et al., 2018; Timinszky et al., 2009). IP experiments with KDM5A and macroH2A using an HepG2 cell macroH2A2 is expressed from a second, independent gene and line in which all macroH2A variants are stably knocked down does not bind to PAR but is ∼80% identical with macroH2A1 and complemented with stably expressed Flag-macroH2A1.2 or (Posavec et al., 2013). IP of S protein–Flag-streptavidin–binding Flag-macroH2A2 (Douet et al., 2017). Co-IP WB analysis using peptide (SFB)-tagged KDM5A from HEK293T cells revealed an HepG2 control and macroH2A-depleted derivatives identified an interaction between KDM5A and macroH2A1, including after interaction with macroH2A1.2 but not macroH2A2 (Fig. 6 B; DNA damage induction by IR (Fig. 6 A). Reciprocal IP analysis verification of macroH2A variant expression shown in Fig. S5, H using endogenous macroH2A1 antibodies corroborated these and I). We next considered that the interaction between KDM5A, results (Fig. S5 D). To determine which macroH2A1 variant PARP1, and macroH2A1.2 may be regulated by DNA damage. To Kumbhar et al. Journal of Cell Biology 9 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 6. KDM5A interacts with the histone variant macroH2A1.2. (A) KDM5A interacts with macroH2A1. Inducible SFB-KDM5A–expressing HEK293T cells were treated with or without 10-Gy IR. SFB-KDM5A was immunoprecipitated with streptavidin beads, and interactions were detected by WB analysis. Input shows expression and loading of proteins. (B) Endogenous KDM5A interacts with macroH2A1.2. Co-IPs were performed using HepG2 cells depleted with shRNA control (shCtrl) or shRNA macroH2A (mH2A knock down [KD]) and complemented shRNA-macroH2A with Flag-macroH2A1.2 or Flag- macroH2A2. (C) KDM5A and macroH2A1.2 interactions are PARP dependent. SFB-KDM5A–expressing HEK293T cells were treated with DMSO or olaparib (5 µM, 1 h) with or without subsequent 10-Gy IR treatments. Co-IPs were performed as in A. (D) The PID of KDM5A is required for macroH2A1.2 and PARP1 interactions. HEK293T cells expressing GFP, GFP-KDM5A, or GFP-KDM5AΔPID were analyzed by co-IP WB analysis with the indicated antibodies. (E) Com- parison of domain structure of histone H2A and macroH2A variants with PAR- and KDM5A-binding summary. Dark blue represents a highly conserved region between canonical H2A and macroH2A variants; gold represents a linker region; light blue represents the PAR-binding region within the macrodomain; dark gray represents a region that has lost PAR binding. (F) KDM5A and macroH2A1.2 promote HDR. Measurement of HR efficiency in WT and KDM5A-KO cells with or without siRNA depletion of macroH2A1.2 was performed as in Fig. 5 H (n = 2). Error bars represent SEM. P values were calculated using an unpaired Student’s t test (*, P < 0.05; **, P < 0.01). Kumbhar et al. Journal of Cell Biology 10 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 pursue this question, we performed co-IP experiments in cells recruitment of ZMYND8, a protein that relies on KDM5A to untreated or treated with IR in the presence or absence of support its accumulation at DNA damage sites within tran- PARPi’s. As we previously demonstrated, KDM5A interacted scriptionally active chromatin through H3K4me3 demethylation with PARP1 and macroH2A1.2 in undamaged and damaged (Gong et al., 2017). Indeed, macroH2A also supported ZMYND8 conditions (Fig. 6, A–C). However, specifically in IR-treated damage recruitment (Fig. S5, J and K). Furthermore, similar conditions, these interactions were dependent on PARP activ- and complementary results were obtained in HepG2 macroH2A- ity because the addition of PARPi’s reduced the interaction be- deficient cells, where reconstitution of these cells with tween KDM5A and both PARP1 and macroH2A (Fig. 6 C). macroH2A1.2 specifically led to a rescue of KDM5A localization KDM5A is known to interact with chromatin using its PHDs, at damage sites (Fig. 7, C and D). Finally, we reasoned that if with PHD1 interacting with unmodified histone H3 and PHD3 macroH2A1.2 participated in the KDM5A–ZMYND8 DDR axis, binding to H3K4me3 (Torres et al., 2015). Interestingly, KDM5A this histone variant would also be required to suppress tran- PHD1, but not PHD3, is required for DNA damage localization, scription after DSB induction, a pathway reliant on KDM5A and and H3K4me3 is reduced following DNA damage (Gong et al., ZMYND8. To test this idea, we used a well-established cell-based 2017). Thus, KDM5A engagement with chromatin, including system to analyze transcriptional repression following DSB for- macroH2A1.2, at damaged versus undamaged chromatin regions mation. This system contains a lactose operator (LacO) array that Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 may rely on different binding modules within the protein that is bound by an inducible Lac repressor protein (LacI)-FokI nu- mediate different interactions that are themselves regulated by clease that creates DSBs upstream of a transcriptionally active DNA damage, including PARylation. The requirement for PARP gene (Fig. 8 A). U2OS cells containing this system also express to support macroH2A1.2 and KDM5A interactions is also in- YFP-tagged MS2, which binds to the hairpin structures that oc- triguing, given that macroH2A1.2 does not interact with PAR cur within the mRNA that is driven by the transgene down- chains directly. We reasoned that PAR binding by KDM5A may stream of the LacO array. Upon DSB induction, the downstream mediate these interactions. To test this hypothesis, we evaluated gene is suppressed, which halts production of the mRNA and whether KDM5A-PID is required for KDM5A interactions with abolishes the formation of colocalized mCherry-FokI and YFP- PARP1 and macroH2A1.2. Indeed, KDM5AΔPID failed to associ- MS2 (Fig. 8, B and C). As expected, depletion of KDM5A resulted ate with either macroH2A1.2 and PARP1 as efficiently as WT in defective transcriptional repression, causing an increased KDM5A (Fig. 6 D). These results identify KDM5A PAR binding as frequency of colocalized mCherry-FokI and YFP-MS2 foci. De- an important component of both PARP1 and mH2A1.2 interac- pletion of macroH2A1.2 resulted in a loss of transcriptional re- tions (summarized in Fig. 6 E). pression, which phenocopied the loss of KDM5A (Fig. 8, B and C). These results demonstrate that macroH2A1.2 is required for Histone variant macroH2A1.2 is required for KDM5A break-induced transcriptional repression and suggest that mac- recruitment to DNA damage sites roH2A controls the KDM5A-ZMYND8 DDR pathway at least at Histone variant macroH2A1.2 but not macroH2A1.1 has been the level of facilitating the recruitment of these factors to DNA reported to be required for efficient DSB repair through HR by damage sites. promoting BRCA1 retention at DSBs (Khurana et al., 2014). Since macroH2A1.2 and KDM5A are associated with DSB repair by HR, and because we identified an interaction between these pro- Discussion teins, we set out to determine if macroH2A1.2 and KDM5A col- Here, we report the identification of two molecular mechanisms laborate to promote DNA repair. To begin to address this that govern the DNA damage recruitment and function of question, we measured HR efficiency using a CRISPR-Cas9/ KDM5A in the DDR through the employment of cellular, genetic, mClover assay in cells singly or doubly deficient for KDM5A and and biochemical approaches. Our results reveal that KDM5A macroH2A1.2 (Kim et al., 2019b; Pinder et al., 2015). In agree- directly binds to PAR chains at DNA damage sites through a ment with our previous results and other studies, we observed newly identified PID that resides within the C terminus of that KDM5A or macroH2A1.2 deficiency resulted in decreased KDM5A that is not found in other KDM5 demethylases. We also gene-targeting efficiency, which is a readout for HR, compared find that the histone variant macroH2A1.2 specifically interacts with WT control cells (Fig. 6 F). Depletion of H2A1.2 in KDM5A- with KDM5A and promotes its recruitment to DNA damage sites. KO cells did not further decrease HR efficiency significantly Our work thus identifies a PARP1/macroH2A1.2 pathway that (Fig. 6 F; efficiency of H2A1.2 protein depletion by siRNA is acts upstream of KDM5A to control its interaction and functions shown in Fig. S5 F). at DNA break sites (Fig. 8 D). Considering these results, we speculated that macroH2A may Chromatin structure is known to be dynamically regulated regulate the recruitment of KDM5A to DNA damage sites. We upon DNA damage. Interestingly, PARP1 and macroH2A1.2 have previously generated CRISPR-Cas9 gene-edited U2OS cells that opposing effects on chromatin structure. PARP1 and PARylation lack macroH2A (Leung et al., 2018). Using these cells, we sought decompact chromatin, while macroH2A1 compacts chromatin, to determine if macroH2A is required for KDM5A recruitment including through a mechanism involving PAR binding by the to laser-induced DNA damage sites. Consistent with this idea, macrodomain of macroH2A1.1 (Khurana et al., 2014; Kozlowski we observed that GFP-KDM5A is recruited less efficiently to et al., 2018; Krishnakumar and Kraus, 2010; Timinszky et al., DNA damage sites in cells lacking macroH2A than in WT cells 2009). If sufficiently expressed to high levels, macroH2A1.1 in- (Fig. 7, A and B). We extended these results by analyzing the hibits PARP1 activity in a manner dependent on its PAR-binding Kumbhar et al. Journal of Cell Biology 11 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 7. Histone variant macroH2A1.2 promotes KDM5A recruitment to DNA damage sites. (A) macroH2A1 promotes KDM5A damage localization. Analysis of GFP-KDM5A damage recruitment in macroH2A1-KO U2OS cells was performed as in Fig. 3 F. Scale bars, 5 µm. (B) Quantification of A. n ≥ 10 cells. Error bars represent SEM. (C) macroH2A1.2 promotes KDM5A damage recruitment. GFP-KDM5A damage recruitment was performed as in A. Dotted white circles indicate damaged regions. Scale bars, 5 µm. (D) Quantification of C as in B. n > 10 cells per condition. P values were calculated using an unpaired Student’s t test for B and D (**, P < 0.01; ***, P < 0.001). macrodomain (Kozlowski et al., 2018; Posavec Marjanović et al., sites. In addition, we observed that KDM5A deficiency resulted 2017). MacroH2A1.2 does not bind PAR but is still able to com- in toxicity and genome instability as a result of PARPi treatment. pact chromatin through an activity located in the linker region While this may potentially be at odds with the finding that (Kozlowski et al., 2018). This biphasic behavior of chromatin PARP1 promotes KDM5A recruitment to DNA damage sites, decompaction followed by compaction is observed at DNA PARPi’s broadly impact genome integrity pathways in cells, in- damage sites and has been shown to correlate with HR through a cluding those that are HR proficient, during replication through macroH2A1.2-dependent mechanism (Khurana et al., 2014). Our PARP trapping and DSB formation, replication fork instability data demonstrating PARP1-macroH2A1.2 regulation of KDM5A and Okazaki fragment processing, and other cellular functions, may shed light on how these previous observations relate to including chromatin remodeling and transcription (Azarm and KDM5A. Smith, 2020; Gupte et al., 2017). Indeed, while BRCA-deficient KDM5A is rapidly recruited to damage sites in a PAR- cells are exquisitely sensitive to PARPi’s through a concept binding–dependent manner (Fig. 5 B). While the PAR-binding known as “synthetic lethality,” HR-proficient cells also display region of KDM5A alone was rapidly recruited to damage sites, its cell toxicity to PARPi’s (Ashworth and Lord, 2018; Michelena association with breaks was very transient (Fig. 3, F and G). et al., 2018). Thus, the sensitivity of KDM5A-deficient cells to Transient PAR chains at breaks may be involved in the initial PARP inhibition may not be entirely related to the involvement recruitment of KDM5A, but additional mechanisms may be of PARylation in promoting KDM5A recruitment and involve- needed for retention of this enzyme at DNA lesions. We posit ment in HR repair. that additional, multivalent interactions may act to channel Chromatin compaction at break sites may be related in part to PAR-binding proteins into a specific pathway versus others that our observation that macroH2A1.2 is required for transcriptional are regulated also by PARylation, such as single-strand break repression at DNA breaks. Indeed, KDM5A demethylates the repair, Okazaki fragment sensing, and transcription (Gibson transcription active mark H3K4me3 at DSBs and promotes the and Kraus, 2012; Hanzlikova et al., 2018; Ray Chaudhuri and damage association of the ZMYND8–NuRD complex, which to- Nussenzweig, 2017). Our data may suggest that macroH2A1.2 gether act to repress transcription and promote HR repair of likely serves this purpose for KDM5A at DSBs, given that they DSBs (Gong et al., 2015; Gong et al., 2017; Gong and Miller, 2018; interact with each other and the requirement of this histone Savitsky et al., 2016; Spruijt et al., 2016). Several other tran- variant for KDM5A recruitment and function at DNA damage scriptional repressive complexes, including PRC1 (polycomb Kumbhar et al. Journal of Cell Biology 12 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure 8. Histone variant macroH2A1.2 and KDM5A promote transcriptional repression at DNA breaks. (A) Schematic of FokI-inducible DSB reporter cell system (Tang et al., 2013). Upon 4-OHT and Shield1 treatment, mCherry-FokI endonuclease induces DSBs upstream of a Dox-inducible reporter gene. Nascent transcription is visualized by YFP-MS2 protein binding to stem-loop structures in the mRNA. DSBs are visualized by mCherry-FokI localization to the LacO loci. (B) macroH2A1.2 is required for transcriptional repression following DSBs. Nascent transcription was analyzed at FokI-induced DSBs in siControl-, siKDM5A-, and simacroH2A1.2-treated cells. The presence of MS2 foci indicates loss of DSB-induced transcriptional repression. Arrows indicate the location of the LacO array. Scale bars, 5 µm. (C) Quantification of B; n = 2 with >100 cells analyzed per condition per replicate. Error bars represent SEM. P values were calculated using an unpaired Student’s t test (**, P < 0.01). (D) Model for KDM5A regulation by PARP1 and macroH2A1.2. Following DNA damage, PARP1 promotes PARylation and recruitment of KDM5A to DNA damage sites. KDM5A engages PAR chains through its C-terminal coiled-coil–containing PID. macroH2A1.2 also promotes KDM5A accrual at DSBs to facilitate DSB-induced transcriptional repression and HR repair. repressive complex 1), PRC2, and NELF (negative elongation are features of coiled-coil domains (Truebestein and Leonard, factor), as well as the histone variant H2AZ, are also required for 2016), further strengthen the likelihood that this region is a DNA break–induced transcriptional repression (Caron et al., bona fide coiled-coil domain. Our evidence reveals that this 2019; Puget et al., 2019; Tan and Huen, 2020). Given the iden- coiled-coil domain can facilitate PAR binding, which is necessary tification of the histone variant macroH2A1.2 in this DDR for KDM5A recruitment and activities at DNA damage sites. pathway here, future studies are warranted to decipher other These findings have several important implications. Coiled-coil potential interactions between these DSB-associated transcrip- domains are common in proteins, including several involved in tional regulators. the DDR, and play important roles in protein–protein interac- PARP1 was one of the first factors identified that is required tions, protein multimerization, and conformational changes for break-induced transcriptional repression (Chou et al., 2010), (Truebestein and Leonard, 2016). In the DDR, dimerization and it was previously shown to regulate DNA damage recruit- of CtIP, a BRCA1-interacting protein, requires an N-terminal ment of KDM5A (Gong et al., 2017). The identification of a PID in coiled-coil region (Dubin et al., 2004), while the BRCA1–PALB2 KDM5A was unexpected for several reasons. KDM5B, a related interaction is mediated by coiled-coil regions present in both KDM5 demethylase, is also involved in DSB repair but is proteins (Song et al., 2018). It will be important to test additional PARylated directly, which inhibits its activity at transcription- coiled-coil domains in other proteins for PAR binding, which ally active sites (Bayo et al., 2018; Krishnakumar and Kraus, may reveal additional mechanisms by which PARP1 regulates 2010; Li et al., 2014b). In addition, KDM5A does not share ho- not only the DDR but also other PARP-dependent processes, such mology with any known PAR-binding domains (Krietsch et al., as transcription. Finally, the KDM5A-PID is unique to KDM5A 2013; Teloni and Altmeyer, 2016; Wei and Yu, 2016). Our ex- demethylase, as KDM5B and KDM5C do not contain an identi- tensive mapping, functional, and biochemical analyses identi- fiable coiled-coil domain, nor do they readily bind PAR (Fig. 4). fied a putative coiled-coil domain near the C terminus of KDM5A Given that KDM5A is overexpressed in cancer and is involved in able to bind PAR both in vitro and in cells, which is sufficient to therapy-associated drug resistance (Cao et al., 2014; Choi et al., localize to DNA damage sites in a PARP-dependent manner. Our 2018; Hou et al., 2012; Li et al., 2014a; Sharma et al., 2010; Teng data identifying α-helical properties within this region, which et al., 2013; Vinogradova et al., 2016; Yang et al., 2019; Zeng et al., Kumbhar et al. Journal of Cell Biology 13 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 2010), our data provide the rationale for testing the potential use retroviral particles. 4 × 106 GP2-293 cells were seeded in a 10-cm of PARPi’s in these settings, which may selectively inhibit plate and cultured to 60–70% confluency, then transfected with KDM5A function. 10 µg of pBabe.puro plasmids containing either Flag-tagged full- In sum, this work has identified two regulatory mechanisms, length mouse macroH2A1.2 or macroH2A2 sequences and 3 µg of PAR binding and macroH2A, that control KDM5A interactions pCMV-VSV-G mixed in a 1× Hepes-buffered saline solution (2× and function at DNA damage sites (Fig. 8 D). These findings Hepes-buffered saline: 272 mM NaCl, 2.8 mM Na2HPO4, 55 mM exemplify how chromatin acts as a platform for DNA-templated Hepes, pH 7) containing 125 mM CaCl2. The supernatant con- processes, including DNA repair and transcription, that must be taining viral particles produced by GP2-293 cells was collected at remodeled by chromatin-modifying enzymes to transition from 24 h and 48 h after transfection, filtered using a 0.45-µm filter, one activity to another. In this example, upon DNA damage, supplemented with 8 µg/ml Polybrene (Sigma-Aldrich), and PARP1 and macroH2A1.2 regulate the chromatin environment to added to the target HepG2 cells cultured in 6-well plates at 60–70% trigger KDM5A damage association, resulting in histone de- confluency. Cells were then centrifuged for 45 min at 1,200 rpm methylation and recruitment of the ZMND8–NuRD complex, at 37°C, incubated at 37°C for 45 min, and then cultured over- which collectively act to repress transcription and promote DNA night in fresh media. The same process was repeated 24 h after DSB repair to maintain genome and epigenome integrity. the first infection. The cells were selected with 2 µg/ml puro- Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 mycin. The necessary selection time was determined by using a negative control plasmid without resistance. Materials and methods Cell lines and cell culture Cloning and plasmids U2OS and HEK293T cells were purchased from American Type KDM5A was cloned into the pDONR201 Gateway vector. The Culture Collection. HCT116 WT and HCT116 KDM5A-KO cells pDONR201 clones were transferred into GFP- or SFB-tagged were purchased from Horizon Discovery. To knock out KDM5A, destination vectors using the Gateway LR cloning system (In- a PGK promoter neomycin resistance cassette flanked by LoxP vitrogen). KDM5AΔPID deletion mutant was generated by PCR sites was integrated into both alleles of KDM5A at exon 11, which in pDONR201 vector following a standard cloning method and removes this exon, making both KDM5A alleles nonfunctional in then subcloned into a Gateway destination vector derived from this cell line. All cell lines were maintained in DMEM supple- pcDNA5/FRT/TO plasmids containing the NLS and SFB or GFP mented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, tagged in the N terminus. KDM5A fragments were amplified and 100 µg/ml streptomycin at 37°C and 5% CO2. Inducible cell from KDM5A cDNA using Q5 High Fidelity polymerase (New lines (SFB-tagged KDM5A) were established and maintained in England Biolabs) with primers containing SalI and NotI recog- medium with 0.2 mg/ml hygromycin B (Invitrogen). To estab- nition sites. PCR products were then cloned into SalI and NotI lish the inducible SFB-tagged KDM5A-expressing cell lines, sites of a pMAL-C5X vector (New England Biolabs). All con- pcDNA5/flippase (Flp) recognition target (FRT)/pCMV-tetO2– structs were validated by DNA Sanger sequencing. containing, SFB-tagged constructs were transfected with pOG44 Flp recombinase expression vector into Flp/In T-REx HEK293T cells. Gene editing by CRISPR-Cas9 After 48 h, cells were treated with 0.2 mg/ml hygromycin B KDM5A gene KOs in U2OS cells were generated as previously (Invitrogen) for the selection of cells with integration of the described (Gong et al., 2017). The sequence of the single-guide construct at the FRT locus. All cell lines were routinely checked RNA used to edit the gene for human KDM5A was 59-CGGATG for mycoplasma contamination. CGGCCGATAAAGCT-39. Loss of KDM5A protein expression by The U2OS DSB reporter cell line used for transcriptional gene editing in U2OS clones was validated by WB analysis using analysis was a gift from Roger Greenberg (University of Penn- a mouse anti-KDM5A antibody (ab78322; Abcam). sylvania, Philadelphia, PA). This cell line stably expresses an mCherry-LacI-Fok1 nuclease domain fusion protein that also siRNA depletion and overexpression contains a modified ER receptor and destabilization domain. For knockdown experiments, 2.5 × 105 cells were seeded in 6- Upon Shield1 and 4-hydroxytamoxifen (4-OHT) addition, the well plates. Cells were transfected with siRNA duplexes targeting mCherry LacI-FokI protein is stabilized and induces DSBs within the corresponding protein or scrambled mixes using Lipofectamine the LacO genomic loci, which are located upstream of a doxy- RNAiMAX (Invitrogen) reagents as per the manufacturer’s rec- cycline (Dox)-inducible YFP-MS2 reporter gene (Tang et al., ommendations. Knockdown cells were cultured for 48–72 h before 2013). Cells were treated with 1 µM Shield1 and 1 mM 4-OHT further processing. The siRNAs used were as follows: KDM5A, 59- for 3 h to induce site-specific DSBs and 1 µg/ml Dox for an ad- GCAAAUGAGACAACGGAAA-39; macroH2A1.2, 59-CUGAACCUU ditional 3 h to induce transcription. AUUCACAGUGAA-39. For KDM5A rescue experiments or over- The generation of HepG2 cells (American Type Culture Col- expression, cells were transfected with 2 µg of emGFP-KDM5A or lection; HB-8065) with stable shRNA-mediated knockdown of emGFP-pcDNA 6.2 empty vector using either Lipofectamine 2000 both macroH2A1 and macroH2A2 proteins is described in or Lipofectamine 3000 (Invitrogen) and then allowed to recover for Douet et al. (2017). Cell lines stably expressing Flag-tagged 24 h before proceeding to assay further. In the case of Dox-inducible macroH2A1.2 and macroH2A2 proteins in the double- SFB- or GFP-tagged pcDNA5/FRT/TO plasmids containing KDM5A knockdown background were achieved by retroviral trans- or KDM5AΔPID, 1 µg/ml Dox was used for at least 24 h to induce duction. GP2-293 cells were used as packaging cells to produce expression of the indicated transgene. Kumbhar et al. Journal of Cell Biology 14 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 IP incubated with increasing concentrations of PARPi (olaparib), Cells (U2OS or HEK293T) were lysed in NETN buffer (10 mM and, 24 h later, PARPi-containing media were replaced with Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, protease inhibitor normal DMEM and incubated for 14 d in a tissue culture incu- cocktail) containing TurboNuclease (Accelagen) at 4°C for 1 h. bator (37°C, 5% CO2). DMSO was used as a control. Cells were Cell lysates were centrifuged at 15,000 rpm at 4°C for 10 min. pretreated with KDM5A inhibitor (CPI455, 2573; Axon Med- The lysate was then incubated with appropriate antibodies for chem) alone or in combination with olaparib wherever indi- 12 h and then conjugated with Dynabead Protein A or Protein G cated. Colonies were fixed and stained with crystal violet beads (Invitrogen) for an additional 1 h. SFB- or GFP-tagged solution (0.5% crystal violet in 20% ethanol). Results were proteins were immunoprecipitated with streptavidin Dyna- normalized to WT untreated cells. beads (Invitrogen) or GFP-Trap (Chromotek). After three or four washes with NETN buffer, bead-bound proteins were eluted Neutral comet assay with 2× sample loading buffer (120 mM Tris-HCl, pH 6.8, 4% HCT116 WT or KDM5A-KO cells were treated with 5 µM DMSO SDS, 20% glycerol, 1% bromophenol blue) and resolved on 4–15% or olaparib for 24 h. After treatment, DNA breaks were analyzed gradient SDS-PAGE gel. Proteins were identified by an appro- using the CometAssay Reagent Kit (Trevigen) according to the priate antibody by WB analysis using anti-rabbit or anti-mouse manufacturer’s instructions. After electrophoresis, DNA was Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 IgG HRP-conjugated secondary antibodies (7074S and 7076S; stained with SYBR Green (Invitrogen), and images were ac- Cell Signaling Technology). quired with a FluoView 3000 confocal microscope (Olympus) using a 10× objective with 0.3 NA (Olympus). Comet tail mo- Antibodies ments (Olive moments) were calculated by using CometScore The following antibodies were used in this study: mouse anti- 2.0 software. KDM5A (ab78322; Abcam), rabbit anti-KDM5B (A301-813; Bethyl Laboratories, Inc.), rabbit anti-KDM5C (A301-034; Bethyl Lab- X-ray irradiation oratories, Inc.), rabbit anti–β-tubulin (ab6046; Abcam), mouse Indicated doses of IR were delivered by an x-ray generator anti-γH2AX (05-636, clone JBW301; Sigma-Aldrich), rabbit anti- (Faxitron x-ray system; RX650). GFP (A11122; Invitrogen), rabbit anti-PARP1 (9542; Cell Signaling Technology), mouse anti-PAR (4335-MC-100; Trevigen), rabbit Laser-induced live-cell imaging anti–pan-ADP-ribose (MABE1016; Sigma-Aldrich), rabbit anti- Laser-induced live-cell imaging was performed as previously MBP (Ab9084; Abcam), mouse anti-Flag (F1804; Sigma-Aldrich), described (Kim et al., 2019a). In brief, cells were seeded onto rabbit anti-macroH2A1 (Ab37264; Abcam), rabbit anti-macroH2A1.1 glass-bottomed dishes (Pelco; Ted Pella Inc.) and were pre- (12455S [D5F6N]; Cell Signaling Technology), mouse anti- sensitized by adding 10 µM BrdU (B9285; Sigma-Aldrich) for macroH2A1.2 (MABE61 [14G7]; Sigma-Aldrich), rabbit IgG 24 h before laser-induced damage. For PARPi treatment, cells (NI01; Calbiochem), mouse IgG (NI03; Calbiochem), and rabbit were pretreated with 5 µM PARPi (olaparib) for 1 h before laser- anti-ZMYND8 (A302-089; Bethyl Laboratories, Inc.). induced DNA damage. Laser damage was induced in a defined region of interest (ROI) using a 405-nm, 50-mW laser (150 Immunofluorescence frames at 60% at a scan speed of 20.0 µs/pixel) mounted on a For γH2AX foci and micronuclei analysis, cells were seeded onto FluoView 3000 confocal microscope (Olympus). Cells were in- glass coverslips and 24 h later pretreated with DMSO or 5 µM cubated during laser damage experimentation in a 37°C olaparib (10621; Cayman Chemical). After 24 h, cells were fixed temperature-controlled WSKM series Tokai Hit top-stage incu- with 2% (vol/vol) PFA for 15 min, permeabilized with 0.5% bator chamber with 5% CO2. Cell imaging was performed on Triton X-100 in PBS for 15 min on ice, and blocked with 5% BSA/ the FluoView 3000 confocal microscope (Olympus) using Tru- PBS for 1 h. Coverslips were then incubated with anti-γH2AX Spectral high-efficiency gallium arsenide phosphide spectral antibody (1:1,000) for 1 h at RT. After three washes in PBS, detectors. Cells were incubated in DMEM supplemented with samples were incubated with Alexa Fluor 594 goat anti-mouse 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml IgG (H+L) secondary antibody (A11032; Invitrogen) for 1 h at RT, streptomycin during imaging. The relocalization intensity of and slides were mounted with VECTASHIELD mounting me- GFP fused proteins at the damage sites were analyzed using FV- dium with DAPI (Vector Laboratories). Cells were imaged using 10 ASW3.1 software (Olympus). All images were captured using a FluoView 3000 confocal microscope (Olympus) using Tru- a 60× apochromatic oil objective lens with a 1.42 NA. Recruit- Spectral high-efficiency gallium arsenide phosphide spectral ment intensities of GFP fused proteins were quantified by FV-10 detectors with a 60× plan apochromat oil objective lens with 1.42 ASW3.1 software (Olympus). To calculate the fluorescence in- NA. Image acquisition was performed with FV-10 ASW3.1 soft- tensity at the DNA lesions, the intensity of the damaged ROI ware (Olympus). Quantification of γH2AX foci and micronuclei (ROID) and the undamaged ROI (ROIU) are first normalized to was performed using ImageJ software. the fluorescence intensity at time point 0 (before damage). The relative fluorescence intensity for each time point is calculated Clonogenic cell survival assay as follows: after DNA damage (ROID/ROIU)/before DNA damage Clonogenic cell survival was analyzed using a colony-forming (ROID/ROIu) × 100. GraphPad Prism (version 6.0) software was assay. Briefly, 500 U2OS or HCT116 WT and KDM5A-KO cells used to calculate the SEM for the fluorescence intensity at were plated in 6-well plates 24 h before treatment. Cells were damaged sites. Kumbhar et al. Journal of Cell Biology 15 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 CRISPR-mClover HR repair assay visualized using a FluoView 3000 confocal microscope (Olym- CRISPR-mClover HR assay was performed as previously de- pus) as described above. scribed (Kim et al., 2019b). Briefly, mClover-HR donor plasmid (98970; Addgene) and Cas9-gRNA vector (98971, Addgene; RT quantitative PCR (RT-qPCR) single-guide RNA target sequence: 59-GGTTGGCAGCGCTGCCCG For analysis of E-cadherin mRNA levels upon PARPi treatment, CG-39) were transfected into U2OS WT cells, U2OS KDM5A KO total RNAs from untreated and PARPi-treated (0.25 µM olaparib cells, and U2OS KDM5A KO cells expressing SFB-tagged WT or overnight) U2OS cells were purified using the RNeasy Mini Kit ΔPID KDM5A constructs. Cells were transfected 24 h before (Qiagen) and treated with RNase-free DNase I (Qiagen) follow- mClover-HR donor plasmid and Cas9-gRNA vector transfection. ing the manufacturer’s protocol. 1 µg of total RNA for each After transfection, cells were incubated for 48 h, and GFP- sample was used to synthesize cDNA by the SuperScript III First- positive cells representing repair events by HR were analyzed Strand Synthesis System (Invitrogen). For E-cadherin mRNA using a BD Accuri flow cytometer. Quantification was performed analysis in KDM5A-KO cells, RNA was purified using the RNA/ using FlowJo software. Protein Purification Plus Kit (48200; Norgen) according to the manufacturer’s recommendations. 250 ng of total RNA was re- Recombinant protein purification verse transcribed into cDNA with the qScript cDNA SuperMix Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 U2OS cells expressing full-length GFP-tagged KDM5A, KDM5B, kit (95048; Quantabio). RT-qPCR was performed using the and KDM5C were harvested and lysed in high-salt buffer StepOnePlus Real-Time PCR system (Applied Biosystems) with (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1% Triton X-100, and SYBR Green Master Mix (Applied Biosystems) and the indicated 1 mM DTT). The cell lysate was incubated with GFP-Trap beads gene-specific primers: ALAS1 forward: 59-CCTTGGCCTTAGCAG for 1 h. Beads were extensively washed five times with high-salt TTTTG-39; ALAS1 reverse: 59-CCAAGATGATGGAAGTTGGG-39; buffer, and GFP fusion protein was eluted with 200 mM glycine, B2M forward: 59-AATGTCGGATGGATGAAACC-39; B2M reverse: pH 2.5. Eluted proteins were neutralized with 1 M Tris, pH 10.4. 59-TCTCTCTTTCTGGCCTGGAG-39; GAPDH forward: 59-CAATGA Eluted proteins were used in a PAR-binding assay, as described CCCCTTCATTGACC-39; GAPDH reverse: 59-GATCTCGCTCCT below. MBP fused KDM5A fragments were expressed in BL21 GGAAGATG-39; E-cadherin primer 1 forward: 59-TGCCCAGAA codon plus RIPL competent E. coli cells (230280; Agilent Tech- AATGAAAAAGG-39; E-cadherin primer 1 reverse: 59-GTCACC nologies) purified and eluted from amylose resin with maltose TTCAGCCATCCTGT-39; E-cadherin primer 2 forward: 59-CAG (E8021; New England Biolabs) as described previously (Kumbhar GTCTCCTCTTGGCTCTG-39; E-cadherin primer 2 reverse: 59-ACT et al., 2018). KDM5A 1491–1610 (F9) and KDM5A F9ΔPID were TTGAATCGGGTGTCGAG-39. cleaved from MBP with factor Xa protease (P8010; New England Biolabs) and further purified using a HiTrap SP HP column CD spectroscopy (GE29-0513-24; GE Healthcare) on an ÄKTA Pure fast protein All CD spectra were measured at RT with a JASCO J-815 spec- liquid chromatograph (GE Healthcare). tropolarimeter. Each protein spectrum was collected as an av- erage of four scans using a quartz 0.1-cm cell on 50 µM samples PAR-binding assay in 20 mM Tris (pH 7.2), 100 mM NaCl, and 1 mM tris(2-car- An in vitro PAR-binding assay was performed as previously boxyethyl)phosphine. CD spectral data were acquired using a described (Kumbhar et al., 2018). Briefly, proteins were spotted 1-nm bandwidth scanning from 185 to 250 nm at a speed of 50 nm/ onto nitrocellulose membrane (10600007; GE Healthcare). The min, data pitch of 0.5 nm, and response time of 4 s. Each protein air-dried membrane was then blocked with 5% BSA in PBS and sample was autocorrected with a baseline spectrum of matching incubated membrane with 10 nM PAR chains for 1 h at RT. The sample buffer that was collected under the same conditions. Raw membrane was washed extensively and incubated overnight ellipticities were converted to mean residue ellipticity to normalize with an anti-PAR antibody. Immunoblot analysis was performed the CD spectra between protein samples. to detect the PAR signal. To check for PAR binding of KDM5A fragments, an equal quantity of each fragment was spotted onto Synthesis, purification, and fractionation of PAR chains the nitrocellulose membrane, and the assay was performed as Synthesis, purification, and fractionation of PAR chains were explained above. conducted following previously established protocols (Chen et al., 2018; Tan et al., 2012). Briefly, PAR polymers were en- Nascent transcript detection at DNA damage sites zymatically synthesized in a 10-ml reaction containing 50 mM U2OS reporter cells (from the Greenberg laboratory, described Tris-HCl (pH 8), 4 mM MgCl2, 250 µM DTT, 20 mM NaCl, 5 mM above) with siRNA transfections were seeded onto 12-mm round NAD+, 10 µg/ml 8-mer oligonucleotide (59-GGAATTCC-39), and coverslips in 6-well plates. To induce site-specific DSBs, U2OS 150 nM human PARP1 at 37°C for 60–90 min. PAR chains were reporter cells were treated with 1 µM Shield1 ligand (632189; detached from modified PARP by treating the reaction with Takara) and 1 mM 4-OHT for 4 h for mCherry-FokI expression. DNase I for 1 h and proteinase K for 2 h, respectively, and pre- Transcription was induced from an integrated reporter gene by cipitated with 20% TCA (wt/vol). Further purification and treating cells with 1 µg/ml Dox for an additional 3 h. Cells on fractionation of PAR chains (0.85 μmol) was performed by ion coverslips were fixed with 4% PFA, permeabilized with 0.5% exchange HPLC using a DNAPac PA 100, 4 × 250–mm analytical Triton X-100 in PBS, washed, and mounted on glass slides column preequilibrated with buffer A (25 mM Tris, pH 9.0) and with VECTASHIELD mounting medium with DAPI. Cells were a previously published 152-min variable gradient (0 min [0% B], Kumbhar et al. Journal of Cell Biology 16 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 6 min [0% B], 12 min [30% B], 18 min [40% B], 48 min [47% B], Primers 78 min [49% B], 138 min [55% B], 144 min [63% B], 145 min [100% Primers used for KDM5A molecular cloning (with their re- B], 148 min [100% B], 149 min [0% B], 152 min [0% B]) with striction sites in primers underlined) are as follows: KDM5A F1: buffer B (25 mM Tris, pH 9.0, and 1 M NaCl) at a flow rate of forward: 59-TTGCGGCCGCATGGCGGGCGTGGGGCCGGGGGGC 1 ml/min and a detector setting of 260 nm (Tan et al., 2012). PAR TA-39, reverse: 59-TTGTCGACATCAGTGCTGAGAACCTCAGG chain HPLC fractions were manually collected, dialyzed against CTC-39; KDM5A F2: forward: 59-TTGCGGCCGCGTGCAGATG double-distilled water (ddH2O) for 24 h to desalt, and lyophi- CCTAATTTAGATCTTA-39, reverse: 59-TTGTCGACGCTGCTTAC lized. Dried powders were resuspended in a minimal volume CAGCCGCCAAAATTC-39; KDM5A F3: forward: 59-TTGCGGCCGC (15–20 µl) of 20 mM Tris (pH 7.0), and fractionated PAR chain CCAGTCCATATGGTTCCCACAGAA-39, reverse: 59-TTGTCGAC concentrations were determined by UV analysis at 258 nm using ATTTACACATTGACGTCCAATGGG-39; KDM5A F4: forward: 59- an extinction coefficient of 1,300 cm−1 M−1. The sizes of purified TTGCGGCCGCGCTGAAGCTGTGAACTTCTGTACT-39, reverse: PAR chains in each fraction were identified by mass spectrometry at 59-TTGTCGACCAGCTGAGCCACAGAAGCACA-39; KDM5A F5: the University of Utah Department of Chemistry Mass Spectrom- forward: 59-TTGCGGCCGCGCTGAGACCTGTGCTTCTGTGGCT-39, etry Core Facility. In brief, 10–50 µM fractionated PAR chains in reverse: 59-TTGTCGACTGCTGCTGCTACCTGTGATTCCAC-39; KDM5A ddH2O were submitted for analysis on a Waters ACQUITY I-class F6: forward: 59-TTGCGGCCGCCGCCCTATTCCTGTGCGTCTT Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 ultra-high-pressure liquid chromatograph coupled to a Waters Xevo GAA-39, reverse: 59-TTGTCGACTGTCAAACACTGCAGGGCCTC G2-S quadrupole time-of-flight mass spectrometer operating in TCC-39; KDM5A F7: forward: 59-TTGCGGCCGCGTATCCCTT negative ion mode (2.5-kV capillary voltage, 25-V sampling cone CAGAAGTTGCCCGTA-39, reverse: 59-TTGTCGACCATCATAAG voltage, 150°C source temperature, 500°C desolvation temperature, TTCTTCCAGTTGTGC-39; KDM5A F8: forward: 59-TTGCGGCC nitrogen desolvation at 800 liters/h). An ACQUITY UPLC BEH C18 GCGAACCTCCAGTGCTGGAGTTGTCA-39, reverse: 59-TTGTC VanGuard precolumn (2.1 × 5 mm) preequilibrated in solvent A GACCTAACTGGTCTCTTTAAGATCCTCCAT-39; KDM5A F8-1: ] (1 mM ammonium formate) was used for filtering and further de- forward: 59-TTGCGGCCGCGAACCTCCAGTGCTGGAGTTGTCA- salting samples before analysis using mass spectrometry. The 39, reverse: 59-TTGTCGACGTCCTTTCCTTTCACTTTTAGTGG- samples were eluted from the column using a linear solvent gra- 39; KDM5A F8-2: forward: 59-TTGCGGCCGCGAGAAACCACTA dient: {0–1 min [1% B (methanol)] or 1–5 min [90% B]} at a flow rate AAAGTGAAAGGA-39, reverse: 59-TTGTCGACCTCTTCTTCTTT of 0.3 ml/min. The acquired mass spectra were processed and de- TGCTAGTTTCTT-39; KDM5A F8-3: forward: 59-TTGCGGCCGC convoluted using MassLynx 4.1 software. CTGGCCAAGAAACTAGCAAAAGAA-39, reverse: 59-TTGTCGAC GCACACAGCATTCTCATCATCAGA-39; KDM5A F8-4: forward: EMSA 59-TTGCGGCCGCGAGGAGTCTGATGATGAGAATGCT-39, reverse: For EMSAs, fractionated PAR chains (75 nM) were incubated 59-TTGTCGACCTAACTGGTCTCTTTAAGATCCTCCAT-39; with a single concentration (250 nM) or varying amounts of KDM5A F8ΔPID: forward: 59-AAAAAGAAGGAGAAGGCTGCT either KDM5A-F9 or KDM5A F9ΔPID in 10 µl binding buffer GCAGCC-39, reverse: 59-GTCCTTTCCTTTCACTTTTAGTGGTTT (20 mM Tris, pH 7.2, 100 mM NaCl, 1 mM tris[2-carboxyethyl] CTC-39; KDM5A F9: forward: 59-TTGCGGCCGCGAGAAACCA phosphine, 10% wt/vol sucrose). After incubation at RT for CTAAAAGTGAAAGGA-39, reverse: 59-TTGTCGGACGCACACAGC 30 min, reaction mixtures were separated by electrophoresis in ATTCTCATCATCAGA-39. 8% (wt/vol) polyacrylamide gels using 1× Tris-borate-EDTA (pH 8.3) for 50 min at 100 V. Gels were soaked in ddH2O supple- Online supplemental material mented with 1× SYBR Gold Nucleic Acid Gel Stain (Thermo Fig. S1 is related to Fig. 1 and shows validation of KDM5A KO in Fisher Scientific) and visualized on an Amersham Typhoon HCT116 cells and the defects in genome integrity pathways re- biomolecular imager (GE Healthcare). sulting from KDM5A deficiency in these cells. Fig. S2 is related to Fig. 1 and analyzes KDM5A loss in human U2OS cells and reports Bioinformatics the associated deficiencies in genome integrity in these cells. Fig. The MultiCoil algorithm hosted at http://cb.csail.mit.edu/cb/ S2 also shows that PARPi interacts with KDM5A and that other multicoil/cgi-bin/multicoil.cgi was used for prediction of the KDMs are expressed normally in KDM5A U2OS KO cells com- coiled-coil region in KDM5A–C (Wolf et al., 1997). A coiled-coil pared with WT U2OS cells. Fig. S3 is related to Fig. 2 and Fig. 4 probability cutoff score of 0.5 and window size 28 were used to and depicts the biochemical analysis of KDM5A interactions obtain dimeric or trimeric coiled-coil predictions. KDM5A pre- with PAR and PARylated proteins. Fig. S3 also shows that dicted intrinsic disorder was determined using IUPred (Dosztányi KDM5B is not recruited to laser damage in either WT or KDM5A- et al., 2005). Multiple sequence alignments were performed using KO HCT116 cells. Fig. S4 is related to Fig. 4 and shows the Clustal Omega. intrinsically disordered regions of KDM5A, including in the predicted coiled-coil region, and compares sequence homology Quantification and statistical analyses of this putative coiled-coil region of human KDM5A with human GraphPad Prism (version 6.0) software was used for statistical KDM5B and KDM5C, as well as KDM5A proteins from other analysis. All results are presented as mean ± SEM unless oth- species. Fig. S5 is related to Figs. 4, 5, 6, and 7 and depicts the erwise specified. P values were calculated using unpaired t tests purified protein fragments of KDM5A (Fig. 4) and the induction unless otherwise indicated. P values <0.05 were considered as of E-cadherin expression by PARPi treatment (Fig. 5). Fig. S5 statistically significant. also shows that KDM5A interacts with macroH2A1.2, validates Kumbhar et al. Journal of Cell Biology 17 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 macroH2A variant expression and depletion by siRNAs (Fig. 6), Alvarez-Gonzalez, R., and F.R. Althaus. 1989. Poly(ADP-ribose) catabolism in mammalian cells exposed to DNA-damaging agents. Mutat. Res. 218: and demonstrates that ZMYND8 recruitment is deficient in 67–74. https://doi.org/10.1016/0921-8777(89)90012-8 macroH2A1-KO U2OS cells (Fig. 7). Ashworth, A., and C.J. Lord. 2018. Synthetic lethal therapies for cancer: what’s next after PARP inhibitors? Nat. Rev. Clin. Oncol. 15:564–576. https://doi.org/10.1038/s41571-018-0055-6 Azarm, K., and S. Smith. 2020. Nuclear PARPs and genome integrity. Genes Acknowledgments Dev. 34:285–301. https://doi.org/10.1101/gad.334730.119 We thank Roger Greenberg (University of Pennsylvania) for Barkauskaite, E., A. Brassington, E.S. Tan, J. Warwicker, M.S. Dunstan, B. Banos, P. Lafite, M. Ahel, T.J. Mitchison, I. Ahel, et al. 2013. Visualiza- providing the FokI DSB cell line and Hung-wen Liu (University tion of poly(ADP-ribose) bound to PARG reveals inherent balance be- of Texas at Austin, Austin, TX) for providing PAR chains and tween exo- and endo-glycohydrolase activities. Nat. Commun. 4:2164. recombinant PARP1. We thank Dr. A.M. Fleming (University of https://doi.org/10.1038/ncomms3164 Batie, M., J. Frost, M. Frost, J.W. Wilson, P. Schofield, and S. Rocha. 2019. Utah, Salt Lake City, UT) for his ion exchange HPLC expertise. Hypoxia induces rapid changes to histone methylation and repro- We thank members of the Miller laboratory for helpful dis- grams chromatin. Science. 363:1222–1226. https://doi.org/10.1126/ cussions and comments on the manuscript. science.aau5870 B. Xhemalce acknowledges support from the National In- Bayo, J., T.A. Tran, L. Wang, S. Peña-Llopis, A.K. Das, and E.D. Martinez. 2018. Jumonji inhibitors overcome radioresistance in cancer through changes stitutes of Health (R01 GM127802). Funding in A. Matouschek’s in H3K4 methylation at double-strand breaks. Cell Rep. 25:1040–1050.e5. Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 laboratory was provided by the National Institute of General https://doi.org/10.1016/j.celrep.2018.09.081 Medical Sciences (R01GM124501) and the Welch Foundation (F- Bryant, H.E., N. Schultz, H.D. Thomas, K.M. Parker, D. Flower, E. Lopez, S. Kyle, M. Meuth, N.J. Curtin, and T. Helleday. 2005. Specific killing of 1817). Research support for work performed in M. Buschbeck’s BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) poly- laboratory was provided through the Fondo Europeo de De- merase. Nature. 434:913–917. https://doi.org/10.1038/nature03443 sarrollo Regional/Ministerio de Ciencia e Innovación—Agencia Buschbeck, M., and S.B. Hake. 2017. Variants of core histones and their roles in cell fate decisions, development and cancer. Nat. Rev. Mol. Cell Biol. Estatal de Investigación (RTI2018-094005-B-I00 and ISCIII 18:299–314. https://doi.org/10.1038/nrm.2016.166 PIE16/00011). The work performed by B.A. Buck-Koehntop was Cao, J., Z. Liu, W.K. Cheung, M. Zhao, S.Y. Chen, S.W. Chan, C.J. Booth, D.X. supported by the National Science Foundation (MCB-1715379). Nguyen, and Q. Yan. 2014. Histone demethylase RBP2 is critical for breast cancer progression and metastasis. Cell Rep. 6:868–877. https:// Funding for this project from K.M. 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Cell Rep. 13:678–689. .org/10.1042/EBC20190094 https://doi.org/10.1016/j.celrep.2015.09.040 Kumbhar et al. Journal of Cell Biology 20 of 20 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Supplemental material Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure S1. KDM5A loss results in reduced genome integrity in siPARP1-depleted HCT116 cells. Related to Fig. 1. (A) WB analysis of cell lines from Fig. 1 A with the indicated antibodies. (B) Gene expression analysis of E-cadherin in WT and KDM5A-KO HCT116 cells. Total RNA was isolated for each cell type and analyzed using gene-specific primers. Data are normalized to B2M, ALAS1, and WT samples. Shown is mean ± SEM (n = 3). P values were calculated by unpaired Student’s t test (***, P < 0.001). (C) Micronuclei formation analysis in HCT116 WT and KDM5A-KO cells with or without siPARP1. PARP1 was depleted by siRNA for 48 h followed by DAPI staining to detect micronuclei. Scale bars, 10 µm. White arrowheads mark micronuclei. (D) Quantification of C. n = 2 with >100 cells quantified per condition per replicate. P values were calculated by unpaired Student’s t test (**, P < 0.01; ***, P < 0.001). (E) KDM5A loss results in increased DSBs as detected by neutral comet assay in cells deficient for PARP1. Cells were treated as in C and analyzed by comet assay as in Fig. 1 G. (F) Quantification of E. Olive tail moment for >100 cells quantified per condition per replicate and plotted as a box and whiskers; n = 2. P values were calculated by Tukey’s multiple comparison test (**, P < 0.01; ***, P < 0.001). (G) Validation of PARP1 protein depletion by siRNAs. WT and KDM5A-KO HCT116 cells were treated with siCtrl or siPARP1, and samples were evaluated by WB analysis with the indicated antibodies 48 h after transfection of the siRNAs. Error bars in B, D, and F represent SEM. Kumbhar et al. Journal of Cell Biology S1 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure S2. KDM5A loss results in reduced genome integrity in siPARP1-depleted U2OS cells. Related to Fig. 1. (A) KDM5A co-IP and WB analysis in U2OS cells. KDM5A was immunoprecipitated from WT and KDM5A-KO cells, and precipitates were evaluated by WB analysis with the indicated antibodies. Analysis confirms loss of KDM5A protein in KDM5A-KO cells. (B) Gene expression analysis of E-cadherin in WT and KDM5A-KO U2OS cells. Experiments were performed as in Fig. S1 B. n = 3. Error bars represent SEM. P values were calculated by unpaired Student’s t test (*, P < 0.05). (C) U2OS cells lacking KDM5A are sensitive to PARPi. Experiments were performed as in Fig. 1 A. Error bars represent SD; n = 3. (D) Loss of KDM5A increases micronuclei in U2OS cells treated with PARPi. Experiments were performed as in Fig. 1 C. Scale bars, 10 µm. White arrowheads mark micronuclei. (E) Quantification of D. >100 cells were quantified per condition per replicate; n = 2. (F and G) KDM5A loss in U2OS cells results in persistent DSBs as detected by neutral comet assay in cells deficient for PARP1 by either inhibition (F) or siRNA depletion (G). Cells were treated and analyzed as in Fig. 1 G and Fig. S1 E. Olive tail moment for >100 cells quantified per condition per replicate; n = 2. Error bars represent SEM. P values were calculated by Tukey’s multiple comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (H) WB analysis of KDM5 proteins in WT and KDM5A-KO HCT116 cells. P values were calculated by unpaired Student’s t test for B, C, and E (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Kumbhar et al. Journal of Cell Biology S2 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure S3. KDM5A interacts with PAR chains. Related to Figs. 2 and 4. (A) Endogenous KDM5A interacts with PAR chains. Untreated or IR-treated U2OS cells were immunoprecipitated with KDM5A antibody and analyzed with the indicated antibodies. (B) PAR IP identifies increased KDM5A interactions following DNA damage. Experiment was performed as in Fig. 2 C. (C) Immunoprecipitated KDM5A from U2OS cells binds PAR. Experiment was performed as in Fig. 2 E. (D) Analysis of MBP-KDM5A fragments binding to PAR. 10 pM of H2A, BSA, MBP, and MBP-KDM5A fragments F1–F8 were spotted onto nitrocellulose membrane, and PAR-binding analysis was performed as in C. (E) GFP-KDM5B is not recruited to laser-induced DNA damage in either HCT116 WT or KDM5A-KO cells. Scale bars, 5 µm. Dotted white circles specify damaged region. Kumbhar et al. Journal of Cell Biology S3 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure S4. Identification of a conserved putative coiled-coil domain unique to KDM5A. Related to Fig. 4. (A) KDM5A PID is intrinsically disordered. Diagram of KDM5A aligned with a disorder tendency plot (red graph). The disorder tendency of KDM5A was calculated using IUPred. Globular, structured domains in KDM5A are shown in light blue boxes. Magenta box indicates PID. (B) Alignment of human KDM5A aa 1408–1623 and the corresponding regions of human KDM5B and KDM5C. Amino acids are color coordinated as indicated in the legend. PAR-binding–predicted coiled-coil region is indicated by the magenta dotted line. Multiple sequence alignment was performed using Clustal Omega. (C) A multiple sequence alignment of Homo sapiens KDM5A aa 1496–1615 with Mus musculus, Rattus norvegicus, Pan troglodytes, Canis familiaris, Bos taurus, Gallus gallus, Equus caballus, Sus scrofa, Ovis aries, Felis catus, and Xenopus tropicalis. KDM5A exhibits high conservation in this C-terminal region. Colored amino acids are as in B. For B and C, asterisks denote fully conserved residues; colon indicates conservation between strongly similar residues; period indicates conservation between weakly similar residues. Kumbhar et al. Journal of Cell Biology S4 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149 Downloaded from http://rupress.org/jcb/article-pdf/220/7/e202006149/1415959/jcb_202006149.pdf by guest on 24 January 2023 Figure S5. DNA damage recruitment dynamics, dependencies, and interactions. Related to Figs. 4, 5, 6, and 7. (A and B) Purified proteins from Fig. 4, G and I, respectively, were analyzed by Coomassie blue staining after SDS-PAGE. (C) PARPi treatment increases E-cadherin expression. U2OS cells were treated with 25 µM PARPi for 16 h, and samples were analyzed by RT-qPCR. E-cadherin expression was normalized to untreated and GAPDH. Error bars are SEM; n = 3. P values were calculated by unpaired Student’s t test (****, P < 0.0001). (D) Co-IP with endogenous macroH2A1 was performed as in Fig. 2 A. (E) KDM5A interacts with macroH2A1.2 but not macroH2A1.1. Co-IP with endogenous KDM5A from HEK293T cells was performed as in Fig. 2 A. Asterisks mark bands that are not full-length/unmodified macroH2A1. (F) Validation of macroH2A1.2 antibody. KDM5A WT and KDM5A-KO HCT116 cells were treated with siCtrl or simacroH2A1.2 and evaluated by WB analysis with the indicated antibodies 48 h after transfection. (G) Validation of macroH2A1.1 antibody. HEK293T cells were treated with siCtrl, simacroH2A1.1, and simacroH2A1.2 and analyzed as in F. (H and I) WB analysis of HEPG2 macroH2A variant–expressing cells. Samples from Fig. 6 B were probed with macroH2A1.2 (H) and macroH2A2 (I) antibodies to confirm expression of these variants in these cells. (J) Damage recruitment of GFP-ZMYND8 is abolished in macroH2A1-KO U2OS cells. Dotted white circles indicate damaged region. Scale bars, 5 µm. (K) Quantification of J. n > 10 cells. Error bars represent SEM. P values were calculated by unpaired Student’s t test (**, P < 0.01). Kumbhar et al. Journal of Cell Biology S5 DNA damage regulation of KDM5A by PAR and macroH2A https://doi.org/10.1083/jcb.202006149