RESEARCH ARTICLE

Inhibition of KDM6 activity during murine ESC differentiationinduces DNA damageChristine Hofstetter1, Justyna M. Kampka1, Sascha Huppertz1, Heike Weber2, Andreas Schlosser3,Albrecht M. Muller1,* and Matthias Becker1,*

ABSTRACTPluripotent embryonic stem cells (ESCs) are characterised by theircapacity to self-renew indefinitely while maintaining the potential todifferentiate into all cell types of an adult organism. Both theundifferentiated and differentiated states are defined by specificgene expression programs that are regulated at the chromatin level.Here, we have analysed the contribution of the H3K27me2- andH3K27me23-specific demethylases KDM6A and KDM6B to murineESC differentiation by employing the GSK-J4 inhibitor, which isspecific for KDM6 proteins, and by targeted gene knockout (KO) andknockdown. We observe that inhibition of the H3K27 demethylaseactivity induces DNA damage along with activation of the DNAdamage response (DDR) and cell death in differentiating but not inundifferentiated ESCs. Laser microirradiation experiments revealedthat the H3K27me3 mark, but not the KDM6B protein, colocalise withγH2AX-positive sites of DNA damage in differentiating ESCs. Lack ofH3K27me3 attenuates the GSK-J4-induced DDR in differentiatingEed-KO ESCs. Collectively, our findings indicate that differentiatingESCs depend on KDM6 and that the H3K27me3 demethylase activityis crucially involved in DDR and survival of differentiating ESCs.

KEYWORDS: Histonemethylation, Polycomb, Histone demethylase,DNA damage, Embryonic stem cells, Differentiation

INTRODUCTIONEmbryonic development with coordinated lineage specificationof stem and progenitor cells requires dynamic packing andunfolding of chromatin to initiate and manifest tissue-specificgene expression patterns (Teif et al., 2012). This process includesthe reversible modification of histone tails. In ESCs, distincthistone tail methylation patterns are associated with pluripotent anddifferentiated states. Among these modifications, tri-methylation(me3) of histone 3 residue K4 (H3K4), di-methylation (me2)of H3K79 and mono-methylation (me1) of H4K20 showdevelopmental stage-specific dynamics that correlate with geneactivation, whereas the H3K27me2 and H3K27me3 modificationsare associated with gene silencing (Zhou et al., 2011). AlthoughH3K4me3 and H3K27me3 are associated with opposingtranscriptional states, they are at times found on the same targetsites (Azuara et al., 2006; Bernstein et al., 2006; Roh et al., 2006).

This bivalent modification state primes developmental genes forrapid transcriptional activation or repression (Bernstein et al., 2006).

The formation of the H3K27me3 mark is catalysed by thePolycomb Repressive Complex (PRC)2. H3K27me3 is notrandomly distributed along the genome but decorates largedomains exceeding 100 kb that comprise high gene and shortinterspersed nuclear element (SINE) densities (Pauler et al., 2009).Chromatin immunoprecipitation sequencing (ChIP-seq) analyseshave revealed that a continous H3K27me3 marking across thebody of genes corresponds to transcriptional inhibition, whereas aH3K27me3 peak around the transcriptional start site marks bivalentdomains and, in contrast to the classic concept of H3K27me3function, a peak in the promoter region is associated with activetranscription (Young et al., 2011). H3K27me3 is also enriched insubtelomeric regions and in long-terminal-repeat retrotransposons(Leeb et al., 2010; Rosenfeld et al., 2009). Demethylation of H3K27by specific demethylases represents a means to regulate thecoordinated transcriptional activation of lineage-affiliated genes inresponse to developmental signals, whereas unused programsbecome packed into heterochromatin (Pedersen and Helin, 2010).Recently, ‘ubiquitously transcribed tetratricopeptide repeat’ onchromosome X (UTX; also known as KDM6A) and jumonji D3(JMJD3; also known as KDM6B) have been identified as histoneH3K27-specific demethylases (Agger et al., 2007). KDM6A andKDM6B catalyse the transition of H3K27me3 and, in vitro, alsoH3K27me2 to H3K27me1 (Agger et al., 2007; De Santa et al.,2007; Lan et al., 2007; Lee et al., 2007). Both KDM6 proteins arecomponents of the mixed lineage leukemia (MLL)–COMPASS-likecomplex (Cho et al., 2007; De Santa et al., 2007; Issaeva et al.,2007). A paralog of the X-chromosome-located KDM6A, termedUTY (also known as KDM6C), is located on the Y-chromosome. Itis so far unknown whether UTY possesses in vivo demethylaseactivity (Hong et al., 2007; Walport et al., 2014). KDM6A andKDM6B take part in the regulation of gene expression programsduring development (Burgold et al., 2008, 2012; Lee et al., 2012;Seenundun et al., 2010). Embryos in which KDM6B has beenknocked out die perinatally owing to respiratory failure. Acatalytically inactive KDM6B mutant fails to rescue thisphenotype, supporting the notion that KDM6B enzymatic activityis crucial for the establishment of respiratory function (Burgoldet al., 2012). Knockout (KO) embryos of KDM6A fail to activateexpression of the early mesodermal marker brachyury (gene symbolT ), and embryos show severe defects in the development of thenotochord, hematopoietic and cardiac tissues (Lee et al., 2012). TheKDM6A-KO phenotype is partially rescued by UTY in maleembryos or by expression of a catalytically inactive KDM6Aprotein, suggesting a demethylase-independent function ofKDM6A (Lee et al., 2012; Morales Torres et al., 2013; Thiemeet al., 2013; Wang et al., 2012). So far it is unknown to what extentthe demethylase activities of KDM6A and KDM6B can compensateReceived 2 June 2015; Accepted 5 January 2016

1Institute for Medical Radiation and Cell Research (MSZ) in the Center ofExperimental Molecular Medicine (ZEMM), University of Wurzburg, Wurzburg97078, Germany. 2Microarray Core Unit, Interdisciplinary Center for ClinicalScience, University of Wurzburg, Wurzburg 97078, Germany. 3Rudolf VirchowCenter for Experimental Biomedicine, University of Wurzburg, Wurzburg 97078,Germany.

*Authors for correspondence (albrecht.mueller@uni-wuerzburg.de;matthias.becker@uni-wuerzburg.de)

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for each other. A recent report suggests that KDM6B and UTY canrescue the loss of KDM6A in a demethylase-dependent and-independent manner, respectively (Morales Torres et al., 2013).To analyse the functional consequences of combined KDM6A

and KDM6B inhibition in ESCs and during ESC differentiation, weused the recently described specific small-molecule inhibitor GSK-J4 (Kruidenier et al., 2012) and employed KDM6A-KO ESCs andknockdown (KD) of KDM6B. We report that differentiating but notundifferentiated ESCs are crucially dependent on KDM6A andKDM6B enzymatic activities. In differentiating ESCs, combinedKDM6A and KDM6B inhibition induces DNA damage.

RESULTSInhibition of KDM6 enzymatic activity leads to cell cyclearrest and cell death in differentiating ESCsTo analyse the requirement for the combined enzymatic activities ofKDM6A and KDM6B in undifferentiated and differentiating wild-type (WT) ESC embryoid bodies, we employed the specific cell-permeable small-molecule inhibitor GSK-J4 (Kruidenier et al.,2012). As assessed by western blot analyses using an H3K27me3-specific antibody, a two- to three-fold increase in H3K27me3 levelswas observed in undifferentiated and differentiating ESC culturesfollowing treatment with GSK-J4. In contrast, no changes in H3K27methylation were observed when cells were treated with DMSO orwith GSK-J5, an inactive regio-isomer of GSK-J4 (Fig. 1A; seeFig. S1A for densitometry analyses). The effect of GSK-J4 wasrestricted to the H3K27me3 mark as H3K27me2, H3K4me3 andHK4me2 levels remained unaltered (Fig. 1A; see Fig. S1A fordensitometry analyses). The elevated H3K27me3 levels cannot beattributed to the loss of KDM6A or KDM6B expression as KDM6Aand KDM6Bwere expressed at constant or higher levels in GSK-J4-treated ESCs and embryoid bodies (Fig. S1B). UTY was expressedat constant levels in GSK-J4-treated ESCs and embryoid bodies(Fig. S1B). To analyse the effect of combined KDM6A andKDM6B inhibition on ESC proliferation, we monitored cumulativepopulation doublings in the presence or absence of GSK-J4. Weobserved that in comparison to GSK-J5- or DMSO-treated ESCs,GSK-J4 treatment had no effect on the cumulative populationdoublings (Fig. 1B, left). Similarly, ESC pluripotency as assessedby alkaline-phosphatase-positive colony formation, RNAexpression of Oct4, Sox2 and Nanog, and SSEA-1 surfacestaining remained unaffected by GSK-J4 treatment (Fig. S1C–E).In contrast, GSK-J4 affected ESCs that had been cultured underdifferentiation conditions as the cellularity of embryoid bodiesrapidly declined under GSK-J4 treatment (Fig. 1B, right). Althoughcontinuous GSK-J4 treatment of undifferentiated ESCs did notyield increased apoptotic cell numbers (Fig. 1C, left), we noticed atwo- to three-fold increase in apoptotic cell numbers at day 2 indifferentiating cultures when treated with GSK-J4 (Fig. 1C, right).In agreement with previously published work (Qi et al., 2015),caspase-3 activation can be observed during embryoid bodyformation as early as 2 days after leukemia inhibitory factor (LIF)withdrawal, consistent with an increased apoptotic rate. We,however, noticed higher cleaved caspase-3 levels in GSKJ-4-treated day 2 embryoid bodies than in untreated cells (Fig. 1D).Furthermore, the cell cycle distributions of undifferentiated ESCscultured in the presence of GSK-J4 were comparable to thedistribution of untreated control cultures (Fig. 1E, left). In contrastand consistent with the reduced cell numbers, we observed anaccumulation of cells in G1 along with a reduced fraction of S-phasecells in GSK-J4-treated embryoid body cultures (Fig. 1E, right). Inconclusion, inhibition of KDM6 protein activity affected viability

and the cell cycle progression of differentiating ESCs but not ofundifferentiated ESCs.

Inhibition of KDM6 protein activity during ESC differentiationincreases DNA damageNext we sought to elucidate the underlying cause of the GSK-J4-induced effects during ESC differentiation. To this end, we firstperformed whole transcriptome analyses of ESC differentiationcultures that had either been treated with GSK-J4, GSK-J5 orDMSO (Fig. 2A).We chose to analyse an early time point (day 0.75)of differentiation to avoid apoptotic cells. In total, 303 genes weredifferentially expressed [false discovery rate (FDR)<0.05] in thepresence of GSK-J4 (Fig. 2A; Table S1). Fewer genes were down-(75) than upregulated (228). Gene ontology analysis (Fig. 2B)revealed that a fraction of the differentially regulated genes werecategorised as being associated with developmental processes (77).Additional germ-layer-specific differentiation markers that had notbeen identified in the global expression analyses showed nodifferential expression under GSK-J4 treatment (Fig. S2A). Asubstantial fraction of responsive genes was categorised as beingassociated with cell death (44) or cell proliferation (33). Consistentwith the observed accumulation of GSK-J4-treated differentiatingESCs in the G1 phase, Ccnd2 (cyclin D2) was downregulated,whereas the cyclin-dependent kinase inhibitor Cdkn1a (p21) andthe retinoblastoma protein RB1 were upregulated (Table S1). Wefurther noticed that several of the cell-death- and/or cell-proliferation-associated upregulated genes (Mdm4, Polk, Btg2,p21CIP, Trp53inp1, Ddit3, Ddit4, cdc42bpg, RB1, Rbl2) were alsoassociated with DNA damage. Quantitative real-time (qRT)-PCRanalyses for a limited gene set confirmed the results obtained fromglobal transcriptome analyses (Table S1, Fig. S2B).

DNA damage signalling is a potent inducer of cell cycleinhibition and apoptosis (Horn and Vousden, 2007). Therefore, weasked whether DNA damage response (DDR) signalling wasactivated in ESCs differentiating in the presence of GSK-J4. Ataxiatelangiectasia mutated kinase (ATM) and ataxia telangiectasia andRad3-related kinase (ATR) are central components of DNA damagesignalling, and they phosphorylate a multitude of substrates,including H2AX at serine residue 139 (S139) or p53 at serineresidue 15 (S15) in response to DNA damage (Matsuoka et al.,2007). Phosphorylated H2AX (S139) (γH2AX) marks DNAdouble-strand breaks (DSB), whereas phosphorylated p53 (at S15)is involved in the regulation of cell cycle progression and survivalupon DNA damage (Horn and Vousden, 2007; Paull et al., 2000).We therefore analysed γH2AX and phosphorylated p53 (S15) levelsin response to GSK-J4 treatment, and separated protein extracts ofESCs or embryoid bodies that had been either cultured in thepresence of DMSO, GSK-J5 or GSK-J4 by using SDS-PAGE,which was followed by western blotting (Fig. 2C). Probing of themembranes with antibodies specific for γH2AX or phosphorylatedp53 (S15) revealed that in agreement with a previous report, basalγH2AX levels were higher in ESCs than in embryoid bodies(Banáth et al., 2009). Similarly, phosphorylated p53 (S15) levelswere also elevated in ESCs. No increase in γH2AX orphosphorylated p53 (S15) levels was observed as a consequenceof GSK-J4 treatment in undifferentiated ESCs. In contrast, elevatedγH2AX and phosphorylated p53 (S15) levels were detected inGSK-J4-treated embryoid bodies. Phosphorylation of p53 at serine15 leads to reduced binding of MDM2 and as a consequence tostabilisation of the p53 protein (Canman et al., 1998; Banin et al.,1998). In agreement with this, we noticed increased p53 proteinlevels in GSK-J4-treated embryoid bodies, whereas p53 levels

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Fig. 1. See next page for legend.

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remained unaltered in GSK-J4-treated ESCs (Fig. 2C). Additionalexperiments employing an antibody specific for substratesphosphorylated by ATM and ATR revealed at least sevendifferentially phosphorylated ATM and ATR substrates inembryoid bodies that had been treated with GSK-J4 (Fig. S2C).Next we asked whether the increased DNA damage signalling in

the presence of GSK-J4 is paralleled by an increased incidence ofDNA breaks. We therefore performed alkaline comet assays withcells isolated from embryoid bodies that had been differentiated inthe presence of GSK-J4, GSK-J5 or DMSO (Fig. 2D). To avoidcomets originating from apoptotic cells that could perturb the results(Choucroun et al., 2001), we analysed cells from 1-day-oldembryoid bodies because apoptosis was not detectable at this timepoint (Fig. 1C,D). As a positive control for DNA damage induction,we used cells that were isolated from day-1 embryoid bodies afterexposure to 6 Gy of ionizing radiation (IR). GSK-J4-treatedembryoid bodies showed a strong increase in tail moment valuesas compared to those formed from GSK-J5- or DMSO-treated cells.The tail moment value of GSK-J4-treated cells was comparable tothe tail moment value of 6-Gy-irradiated cells. Analyses of cometformation in embryoid bodies that had been cultured in the presenceof DMSO, GSK-J5 or GSK-J4 for 12 or 18 h showed a time-dependent increase in tail moment values in the GSK-J4-treatedcells (Fig. S2D). The underlying cause for the increase in DNAdamage might be an impaired capacity of the GSK-J4-treated cellsto repair DNA damage. To start addressing this, we tested whetherone of the main pathways of DSB repair, homologousrecombination, is impaired in the presence of GSK-J4. To thisend, we employed a previously described system withchromosomally integrated SceI-inducible DSBs (Pierce et al.,2001). The results show that homologous recombination isutilised in differentiating ESCs and that it is unaltered in thepresence of GSK-J4 (Fig. 2E). The MRE11 inhibitor Mirin reduced

homologous recombination frequencies, as expected. We concludethat the increase in DNA damage signalling in differentiating ESCsin the presence of GSK-J4 is a consequence of DNA damage andthat impaired homologous recombination is not the underlyingcause for increased DNA damage.

Combined genetic ablation of KDM6A and KDM6B increasesDNA damage in differentiating ESCsTo analyse whether the effect of GSK-J4 on viability, cell cycleprogression and DNA damage signalling is a direct consequence ofcombined inhibition of KDM6A and KDM6B, or a consequence ofoff-target effects, we knocked down KDM6B in KDM6A-KOESCs. The knockout of KDM6A led to a complete loss of KDM6Aexpression on RNA and protein levels, and the knockdown ofKDM6B was evident in RNA and protein levels (Fig. 3A,B;Fig. S3A). Consistent with the results obtained with GSK-J4-treatedWT ESCs, proliferation and cell cycle distribution as well as SSEA1surface marker expression of KDM6A-KOKDM6B-KD ESCs wereidentical to those of control ESCs (Fig. 3A; Fig. S3B,C). In contrast,embryoid body differentiation of KDM6A-KO KDM6B-KD ESCsshowed reduced cell numbers, along with an increased frequency ofcells in G1/G0 (Fig. 3A; Fig. S3B). KDM6B KD in WT embryoidbodies did not lead to reduced cell numbers or to increased G1frequencies (Fig. S3D,E). Taken together, these data indicate thatthe combined abrogation of KDM6A and KDM6B causes defectivecell cycle progression. In addition, we observed elevated γH2AX,phosphorylated p53 (S15) and H3K27me3 levels in KDM6A-KOKDM6B-KD embryoid bodies, whereas H3K27me2 andH3K27me1 levels were constant (Fig. 3B; Fig. S3F). Inagreement with increased global γH2AX levels in KDM6A-KOKDM6B-KD embryoid bodies, the frequency of γH2AX-positivefoci in KDM6A-KO KDM6B-KD embryoid bodies was increased(Fig. S3G). Next, we analysed comet formation and found thatdifferentiating KDM6A-KO KDM6B-KD ESCs showed increasedtail moment values (Fig. 3C). Next, we asked whether the globalincrease in H3K27me3 levels in the knockout and knockdown cellsleads to increased H3K27me1, H3K27me2 or H3K27me3 levels inpromoter regions of putative KDM6 target genes (Fig. 3D). Weincluded Sox2 in the analyses as it becomes tri-methylated atH3K27 during later time points of differentiation (Obier et al.,2015). HoxB1 and Pou2f3 were analysed because both wereupregulated following GSK-J4 treatment (Table S1). ChIP analysesshowed that H3K27 methylation levels were similar in wild-typeand KDM6A-KO KDM6B-KD cells. Thus, these limited ChIPanalyses revealed no increase in H3K27me3 in promoter regions.Finally, as previous work on Drosophila UTX indicates thatknockdown of UTX leads to increased irradiation sensitivity (Zhanget al., 2013), we assessed the radiation sensitivity in wild-type andKDM6A-KO KDM6B-KD cells. As shown in Fig. 3E, exposure to2.5 Gy increased apoptotic cell fractions in wild-type and KDM6A-KO KDM6B-KD cells. The increase in late apoptotic cells(annexinV and propidium iodide double positive) is moderatelyelevated in KDM6A-KO and KDM6A-KO KDM6B-KD cells.

Collectively, these data indicate that the combined abrogation ofKDM6A and KDM6B leads to DNA damage, and to DNA damagesignalling in differentiating ESCs.

No colocalisation of H3K27me3 or KDM6B with γH2AX+ fociafter GSK-J4 treatmentTo ask whether KDM6B or H3K27me3 associate with sites of DNAdamage, we analysed the sub-nuclear distribution of H3K27me3and KDM6B in relation to γH2AX damage foci by

Fig. 1. Inhibition of KDM6 demethylase activity with GSK-J4 isincompatible with embryoid body formation, and induces apoptosis andcell cycle arrest in differentiating but not in undifferentiated ESC cultures.(A) (top) Western blot analyses of undifferentiated ESCs (ESC, left) andembryoid bodies (EB, right) cultured in the presence of DMSO (vehicle), GSK-J5 (J5, control compound) or GSK-J4 (J4, KDM6 inhibitor) for 1 or 2 days.Membranes were probed with antibodies specific for H3K27me3, H3K27me2or histone 3 (H3, loading control). Molecular masses (kDa) are indicated on theleft. (B) Cumulative population doublings (CPD) of ESCs (left) cultured for threepassages in the presence of DMSO, GSK-J5 or GSK-J4, and micrographs ofESCs grown on MEFs for two days with DMSO, GSK-J5 or GSK-J4.Population doublings (PD) of embryoid bodies (right) in suspension cultures(2×106 cells seeded at day 0) and cultured for up to 3 days in the presence ofDMSO, GSK-J5 or GSK-J4, and micrographs of embryoid body cultures at day3. Scale bars: 100 µm (left), 500 µm (right). (C) Frequency of apoptotic cells inresponse to GSK-J4 treatment. ESCs and embryoid bodies were cultured inthe presence of DMSO, GSK-J5 or GSK-J4 for 2 or 4 days (ESCs) or for 1 or2 days (embryoid bodies) before single-cell suspensions were analysed bycombined 7-aminoactinomycin D (7AAD) and annexinV staining, and flowcytometry. (D) Cleaved-caspase-3-specific western blot analysis of ESCs andembryoid bodies cultured in the presence of DMSO, GSK-J5 (J5) or GSK-J4(J4) for the indicated periods. Staurosporine (stauro)-treated NIH3T3 cellextracts and extracts from embryoid bodies that had been exposed to 6 Gy areshown as controls. Membranes were probed with antibodies specific forcleaved caspase 3 and GAPDH (loading control). Shown is a representativeexample of two independent experiments. (E) Relative cell cycle distribution ofESCs or embryoid bodies. After incubation with DMSO, GSK-J5 or GSK-J4cells for the indicated time points, cells were subjected to propidium-iodide-specific flow cytometry analysis for quantification of the percentages of sub-G1, G0/G1, S or G2/M cell frequencies. (B,C,E) Data represent means±s.e.m.,*P≤0.05; **P≤0.01; ***P≤0.001 (two way Student’s t-test). n=3 independentexperiments.

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immunofluorescence staining (Fig. 4). Consistent with western blotanalyses, we observed elevated H3K27me3 levels in GSK-J4-treated ESCs and embryoid bodies (Fig. 4A, lower panels). Also wenoticed a higher frequency of γH2AX-positive nuclei in GSK-J5-treated ESCs compared to GSK-J5-treated embryoid bodies whereγH2AX+ foci were infrequent. Visual inspection of theimmunofluorescence-stained specimens revealed no colocalisationof γH2AX-positive nuclear foci and H3K27me3 signals in GSK-J5-treated ESCs (Fig. 4A, left top panel). GSK-J4 treatment uniformly

increased H3K27me3 signals in ESCs and embryoid bodies,resulting in an overlay of γH2AX and H3K27me3 signals, whichare unlikely to represent true colocalisation. In agreement with this,the quantification of signal overlap in ESCs and embryoid bodies byusing Manders’ colocalisation coefficient (Manders et al., 1993)showed only a moderate overlap of the γH2AX and H3K27me3signals independent of treatment conditions, whereas Pearson’sanalyses for linear dependence of the two signals (Manders et al.,1993) indicated no correlation (Fig. 4C,D). In ESCs and embryoid

Fig. 2. Inhibition of KDM6 induces DNA damage in differentiating ESCs. (A) Heat map of genes differentially expressed in embryoid bodies that had beencultured for 0.75 days in the presence of DMSO, GSK-J5 or GSK-J4. The hierarchical clustering dendrogram (top) indicates the relatedness in total geneexpression. (B) Differentially expressed genes from global gene expression analyses were associated to GO terms using GO-slim software. (C) Western blotanalyses of ESCs and embryoid bodies cultured in the presence of DMSO, GSK-J5 (J5) or GSK-J4 (J4) and probed with antibodies specific for phosphorylated(p-)p53 (ser15), p53, γH2AX and GAPDH (loading control). Molecular masses (kDa) are indicated on the left. (D) Quantification of Comet analyses. 1-day-oldembryoid bodies that had been treated with DMSO, GSK-J5 or GSK-J4 were subjected to Comet assays. As control, embryoid bodies were exposed to 2 Gy or6 Gy ionising radiation. Representativemicrographs are shown on the right. Scale bar: 250 µm. (E). GSK-J4 treatment does not impair homologous recombinationin differentiating ESCs. The frequency of DSB repair was determined using DR-GFP reporter transgenic ESCs. A schematic representation of the reporter systemis indicated. Shown are the frequencies of GFP-positive cells following GSK-J5, GSK-J4 or Mirin (selective MRE11 inhibitor) treatment. (D,E) Data representmeans±s.e.m.; *P≤0.05, **P≤0.01 (two-way Student’s t-test), n=3 independent experiments.

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Fig. 3. Knockdown of KDM6B in KDM6A-KO ESCs results in reduced proliferation and increased DDR signalling upon differentiation. (A) KDM6A-KOESCs were transduced with lentiviral shRNAs coding for a scrambled (scr.) or two different KDM6B sequences (shRNA#1, shRNA#2). After 4 days of antibioticselection, qRT-PCR analyses were performed to determine the expression of KDM6A and KDM6B in ESCs (left). ESCs were transferred to differentiationculture, and KDM6A and KDM6B levels were determined from 3-day-old embryoid bodies (right graph). Error bars represent s.d. Also shown are cumulativepopulation doublings (CPD) of ESCs and population doublings (PD) of embryoid bodies (2×106 ESCs seeded at day 0). Significance is indicated between knockoutshRNA scr. cells, KO shRNA#1 and KO shRNA#2 cells. *P≤0.05, **P≤0.01 (two-way Student’s t-test). Data represent means±s.e.m., n=3 independentexperiments. (B) KDM6A, actin, phosphorylated (p)-p53 (ser15), p53, γH2AXandH2AX-specific western blots. Numbers below blots indicate relative densitometryintensities (phosphorylated to unphosphorylated form of p53 or H2AX respectively). Shown is one representative example, n=2. Asterisks highlight the KDM6Abands. Molecular mass (kDa) is indicated on the left. (C) Quantification of Comet assays. 3-day-old embryoid bodies were subjected to Comet assays. As control,embryoid bodies were exposed to 6 Gy ionising radiation. Representative micrographs are shown on the top. Scale bar: 250 µm. n=2. (D) ChIP qPCR analysis ofwild-type (WT), KO, and KO and KD ESCs at day 3 of differentiation. ChIP was performed with H3K27me3 (me3)-, H3K27me2 (me2)- and H3K27me1 (me1)-specific antibodies and control IgG. DNAwas analysed for enrichment using primers specific for the Sox2, HoxB1 or Pou2f3 promoter regions. (E) Fraction of early(annexinV positive) and late (annexinVpropidium iodide double positive) apoptotic cells inWTshRNAscr., KDM6A-KOshRNAscr., KDM6A-KOKDM6B-shRNA#1and KDM6A-KOKDM6B-shRNA#2 embryoid bodies 24 h post exposure to 2.5 Gy. Error bars in panels D,E indicate means±s.e.m. n=2 independent experiments.

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bodies, KDM6B localised to the nucleus and cytoplasm,independently of treatment (Fig. 4B). Visual inspection orcoefficient analyses (Fig. 4C,D) did not reveal colocalisation ofγH2AX-positive nuclear foci with KDM6B.

Colocalisation of H3K27me3 but not KDM6B or KDM6Awithlaser microirradiation tracks induced γH2AX-positiveregionsAs GSK-J4 could, as part of its DNA damaging action, impinge onthe correct localisation of KDM6A or KDM6B, we assessedwhether H3K27me3 or KDM6A and KDM6B colocalised withγH2AX-positive sites of laser-microirradiation-induced DNAdamage. We exposed 0.74-day embryoid body cells tomicroirradiation. As microirradiation severely damaged GSK-J4-treated embryoid bodies, only GSK-J5-treated cells could be

analysed. Fig. 5A shows that, following microirradiation,H3K27me3 and γH2AX signals colocalised in differentiatingESCs. We next asked whether H3K27me3 accumulated atmicroirradiation-induced DNA damage sites in undifferentiatedESCs. Fig. 5B shows that after microirradiation of ESCs, we did notobserve colocalisation of H3K27me3 with γH2AX-positive areas.Time course analysis of the subnuclear distribution of KDM6A orKDM6B did not reveal co-localisation at γH2AX-positive sites(Fig. 6). In conclusion, and in agreement with previous reports onUV-induced damage (Mosammaparast et al., 2013; Šustáckováet al., 2012), we did not observe colocalisation between KDM6B orKDM6A and γH2AX at sites of laser-microirradiation-inducedDNA damage. These data are consistent with a differentialrequirement for H3K27me3 at sites of DNA damage inundifferentiated and differentiating ESCs.

Fig. 4. Subcellular distribution of H3K27me3, γH2AX foci and localisation of KDM6B following GSK-J4 treatment. (A) Immunofluorescence stainingof ESCs that had been cultured for 2 days or of (B) embryoid bodies that had been cultured for 0.75 days in the presence of GSK-J5 (J5) or GSK-J4 (J4). ESCsand sectioned embryoid bodies (EB) were probed with antibodies specific for H3K27me3 (green) and γH2AX (red) or KDM6B (green) and γH2AX (red). Nucleiwere counter-stained with DAPI (blue). ESCs are shown in left, embryoid bodies in right panels. Overlay images represent segment magnifications of areasmarked by white squares. Scale bars: 10 µm (overlay images); 5 µm ‘overlay/DAPI’ images. n≥2 independent experiments. (C,D) Quantification of signal overlapbetween H3K27me3 and KDM6Bwith γH2AX in ESCs (C) and in embryoid bodies (D). Manders’ coefficient in the range of 0.35 to 0.5 indicates amoderate signaloverlap. Pearson’s coefficient in the range of −0.1 to 0.1 indicates very weak correlation of signal intensities (Manders et al., 1993). Data represent means±s.d.(n=2 independent experiments). White dashed lines mark nuclear perimeters.

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Lack of H3K27me3 attenuates GSK-J4-induced DDR indifferentiating ESCsAs we observed the colocalisation of H3K27me3 and γH2AX atlaser-induced sites of DNA damage, we assessed whether acomplete lack of the H3K27me3 modification influences theDDR in GSK-J4-treated embryoid bodies. To address thisquestion, we employed Eed-KO ESCs which, as previouslyreported (Schoeftner et al., 2006) and shown in Fig. S4A, aredevoid of H3K27me3 owing to the lack of functional PRC2.Eed-KO ESCs can form embryoid bodies and express KDM6Aand KDM6B (Fig. S4B). Similar to WT ESCs, treatment of Eed-KO ESCs with DMSO or GSK-J5 had no effect on ESCdifferentiation. In contrast to WT ESCs, however, Eed-KO ESCdifferentiation in the presence of GSK-J4 was only moderatelyaffected (Fig. 7A). Consistent with this observation, we noticedonly a mild decline of population doubling values of GSK-J4-treated Eed-KO embryoid bodies, whereas population doublingvalues of GSK-J4-treated WT embryoid bodies were stronglyreduced (Fig. 7B). We conclude that the absence of Eedattenuates the GSK-J4 effect on cell numbers. We nextdetermined the frequency of γH2AX-positive foci in WT andEed-KO embryoid bodies following GSK-J5 or GSK-J4treatment as a read out of DSB formation. Consistent withprevious reports showing decreased DSB repair in cells lackingEZH2 (Campbell et al., 2013), an integral component of PRC2,we observed that numbers of γH2AX-positive foci weremarkedly lower in GSK-J4-treated Eed-KO embryoid bodies ascompared to those in treated WT embryoid bodies (Fig. 7C). Insummary, the effects of treatment with GSK-J4 are attenuated indifferentiating Eed-KO ESCs, suggesting that H3K27me3 isrequired to elicit DDR in the presence of GSK-J4.

DISCUSSIONIn this study, by using a KDM6A- and KDM6B-specific enzymaticinhibitor (GSK-J4), and by employing KDM6A-KO KDM6B-KDESCs, we analysed the role of the KDM6 demethylase activityin undifferentiated ESCs and during ESC differentiation.Unexpectedly, we observed that inhibition of KDM6 led to celldeath in early differentiating, but not in undifferentiated, ESCs.Global gene expression analyses in differentiating ESCs revealedthat only a limited set of genes responded to the GSK-J4 treatment,with more genes up- than downregulated. Several of the upregulatedgenes encode factors that are associated with DDR. Consistently,GSK-J4 incubation caused severe DNA damage, which precededG1 arrest and cell death. H3K27me3 but not KDM6A or KDM6Bcolocalised with γH2AX-positive areas that marked DSBs in laser-microirradiated nuclei of differentiating ESCs. In contrast, acolocalisation of H3K27me3 signals with laser-irradiation-induced γH2AX-positive areas was not observed. Finally, weshowed that the GSK-J4-induced DDR was attenuated indifferentiating Eed-KO ESCs.

The H3K27me3 mark is generally associated with gene silencing(Zhou et al., 2011). In agreement, lack of H3K27me3 inundifferentiated ESCs – i.e. due to knockout of Eed results in de-repression of PRC2 target genes (Boyer et al., 2006; Elliott et al.,1998). Although we observed higher global H3K27me3 levels inresponse to GSK-J4 treatment in differentiating ESCs, geneexpression analyses showed that only a small set of genes wasdifferentially expressed. Published whole genome ChIP-seqanalyses show that H3K27me3 forms broad local enrichments(BLOCS) in gene-rich regions (Pauler et al., 2009). BLOCSalternate with H3K27me3-deficient gene-poor regions. It iscurrently not known whether low H3K27me3 levels in gene-poor

Fig. 5. Subcellular distribution ofH3K27me3 and γH2AX in ESCs andembryoid bodies, and localisation ofKDM6B in embryoid bodies followinglaser microirradiation. (A) ESCs werecultured in suspension in embryoid body (EB)medium for 24 h. Embryoid bodies wereattached to microscopy chambers. GSK-J5(J5) was added for 0.75 days. Thereafter,cells were fixed and stained byimmunofluorescence analysis (antibodiesagainst H3K27me3 and γH2AX). Inlaysrepresent segment magnifications of markedareas. Scale bar: 50 µm; 2.5 µm in inset.(B) ESCs were cultured in the presence ofGSK-J5 or GSK-J4 (J4) for 0.75 days.Cells were irradiated and fixed forimmunofluorescence analysis withH3K27me3- and γH2AX-specific antibodies.The exposure time for picture acquisition ofGSK-J4-treated and H3K27me3-stainedcells was reduced for optimal signal-to-noiseratio. Scale bars: 5 µm. (A,B) White dashedlines mark nuclear perimeters. n=2independent experiments.

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regions are actively maintained by KDM-family proteins. If this isthe case, elevated H3K27me3 levels specifically in gene-poorregions in response to GSK-J4 treatment could explain the smallnumber of differentially regulated genes. In support of this notion,we did not find increased H3K27 methylation in the promoterregion of putative KDM6 target genes in KDM6A-KO KDM6-KDcells, despite a global increase in H3K27me3 levels. These results

further support previous findings indicating a demethylase-independent function of KDM6 for the regulation ofdevelopmental gene expression (Kang et al., 2015; Lee et al.,2012). In contrast to what might be expected upon globalupregulation of a repressive chromatin mark, the majority ofdifferentially expressed genes were expressed at higher levelsfollowing GSK-J4 treatment. Mis-expression of cell-differentiation-

Fig. 6. Localisation of KDM6A and KDM6B inembryoid bodies following laser microirradiation.ESCs were cultured in suspension in embryoid bodymedium for 24 h. Embryoid bodies were attached tomicroscopy chambers and cultured for 0.75 days. Cellswere irradiated and fixed directly after ionising radiation(0) or cultured for additional time periods (15 min or35 min). Thereafter, cells were fixed and stained forimmunofluorescence analysis (top: antibodies againstHA, recognising HA–KDM6A; against γH2AX; bottom:antibodies against KDM6B and γH2AX). Inlaysrepresent segment magnifications of marked areas.White dashed lines indicate nuclear perimeters. n=2independent experiments. Scale bar: 10 µm; 2.5 µm ininsets.

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associated genes could lead to failed differentiation and, as a result,to cell death. However, only a minority of the genes that were eitherup- or downregulated were categorised as being associated with celldifferentiation or developmental processes. Incorrect elimination ofsurplus cells during embryonic development could also be aconsequence of ill-executed developmental programs (Coucouvanisand Martin, 1995; Debnath et al., 2002). In analogy to embryonicdevelopment, apoptosis occurs in floating embryoid body culturesduring a process termed cavitation, which forms a proamniotic-likecavity (He et al., 2010; Qi et al., 2012). Bnip3, a BH3-onlyproapoptotic protein, is of key importance for this process (Qi et al.,2015). As Bnip3 was among the downregulated genes in our globalexpression analysis (Table S1), we consider it unlikely that normaldifferentiation was switched to a global proapoptotic program uponinhibition of KDM6 proteins. Our global gene expressionanalyses were performed at a time point when DNA damage wasalready evident in GSK-J4-treated embryoid bodies. It is thereforeconceivable that the underlying cause for the observed geneexpression pattern was cellular stress. In agreement with this,expression of multiple DNA-damage-associated genes wasupregulated following GSK-J4 treatment. We consider it more

likely that DNA damage rather than failed differentiation is theunderlying cause for the observed cell death of differentiating ESCsin response to GSK-J4.

Our experiments using Eed-KO ESCs suggest that theH3K27me3 mark is necessary to elicit a DDR duringdifferentiation under KDM6 inhibition. In the absence of H3K27methylation in Eed-KO ESCs, the frequency of γH2AX-positivefoci under GSK-J4 treatment was comparable to the frequency inuntreated control cells. Nevertheless, the population doublings ofEed-KO embryoid body cultures were not as those in controls. Thisindicates that the GSK-J4 inhibitor has off-target effects. In supportof this interpretation, the effect of combined KDM6A KO andKDM6B KD is less pronounced. In agreement with additionalGSK-J4 targets, it has been reported that GSK-J4 also targetsH3K4-specific KDM5-family members (Heinemann et al., 2014;Kamikawa and Donohoe, 2015). Though the inhibitorconcentration we used did not affect H3K4 methylation,additional, so far unidentified, targets of GSK-J4 are possible.However, part of the GSK-J4 effect has to involve H3K27methylation as we see attenuation of treatment effects indifferentiating Eed-KO ESCs.

Fig. 7. Eed-KO ESC differentiation in the presence of GSK-J4. (A) Representative micrographs of R1 and J1 embryoid bodies and of J1 Eed-KO embryoidbodies differentiated in the presence of DMSO, GSK-J5 (J5) or GSK-J4 (J4) for 3 days. WT, wild type. Scale bar: 500 µm. (B) Analysis of population doublings(PD) of J1 embryoid bodies and J1 Eed-KO embryoid bodies in the presence of GSK-J5 or GSK-J4. (C) γH2AX-positive foci frequencies in nuclei of 0.75-dayembryoid body cells (left). Data show means±s.e.m. *P≤0.05; **P≤0.01; n.s., no significance (two-way Student’s t-test); n=3 independent experiments.Representativemicrographs of γH2AX-specific immunofluorescence staining are shown (right). Examples of nuclei categorised as 1–3, 4–5 or >5 γH2AX-positivefoci per nucleus are shown (inlay images). White dashed lines mark nuclear perimeters. Scale bar: 10 µm; 2.5 µm in insets.

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Recently, Shpargel et al. have reported that KDM6A KDM6Bdouble-KO embryos can develop to term and that repressed geneslose H3K27 methylation and are then expressed (Shpargel et al.,2014). The strategy used in the approach by Shpargel et al.involved the deletion of KDM6B exons 14–21. KDM6B-KO micegenerated using this strategy show perinatal lethality owing todefects in lung development (Satoh et al., 2010). In contrast, thephenotype of mice with a complete loss of KDM6B proteinexpression (due to the deletion of the ATG site and theintroduction of a frame shift mutation) was more severe asembryos died at embryonic day (E)6.5 (Ohtani et al., 2013). Thisindicates that depending on the knockout strategy, the phenotypeof KDM6B KO shows variation. Therefore, it is possible thatKDM6B exerts additional, so far undescribed, functions in earlydevelopment. Our own results show no effect of KDM6Bknockdown on wild-type embryoid body formation. Asdiscussed above, we observed a milder effect of combinedKDM6A KO and KDM6B KD compared to that upon GSK-J4treatment. Therefore, a complete genomic abrogation of KDM6B– e.g. using the strategy described by Ohtani and colleagues –might yield a more severe phenotype.A previous report reveals that members of the PRC2 H3K27

methyltransferase complex and of PRC1 are recruited to sites ofDSBs (Gieni et al., 2011). Data on PRC function during DDRsuggest that PRCs play a role downstream of ATM (Vissers et al.,2012). Consistently, sensitisation to ionising radiation or UV isreported as a consequence of deletions of PRC2 components such asEZH2 (Campbell et al., 2013; Chou et al., 2010). In agreement withan increased sensitivity to DNA damage in the absence ofH3K27me3, we found higher frequencies of γH2AX-positivefoci in control-treated differentiating Eed-KO ESCs than indifferentiating WT ESCs. Although the colocalisation of PRCswith sites of DNA damage is well established, data linkingH3K27me3 directly to DNA damage foci are inconsistent. Tworecent studies show increased H3K27me3 at UV-light-inducedDNA damage sites (Chou et al., 2010; O’Hagan et al., 2008),whereas two other studies have reported no colocalisation inresponse to UV or ionising radiation (Campbell et al., 2013;Šustácková et al., 2012). This discrepancy could derive fromvarying experimental conditions. Our own work showscolocalisation of γH2AX and H3K27me3 marks in differentiatingESCs upon laser microirradiation, indicating that H3K27me3 ispart of the DDR at sites of DNA damage. We did not findcolocalisation of H3K27me3 with laser-microirradiation-inducedγH2AX-positive areas in undifferentiated ESCs, pointing towards adifferential requirement for H3K27me3 at sites of DNA damage inundifferentiated and differentiating ESCs. To what extent thedifferential localisation of H3K27me3 at sites of DNA damage inundifferentiated and differentiating ESCs is linked to the differentialresponse of these cells to KDM6 inhibition remains elusive.In agreement with recent literature (Mosammaparast et al., 2013),

we did not find the colocalisation of KDM6B with γH2AX-positiveDNA damage, and we extend this observation to KDM6A.Although we cannot exclude the possibility that KDM6A andKDM6B are recruited to γH2AX-positive foci at a level below thedetection limit of immunofluorescence microscopy analyses, ourobservations are so far consistent with an indirect role of KDM6activity in DNA repair.A possible cause for the increased DNA damage in

differentiating ESCs upon KDM6 inhibition could be thederegulation of cellular processes such as cell cycle regulation.Our results show that GSK-J4-treated or KDM6A-KO and

KDM6B-KD cells increase G1 cell frequencies. In parallel, cellcycle regulators (p21, RB1) are upregulated. Because RB1 andp21 are downstream effectors of DNA damage (Sperka et al.,2012), we consider it more likely that the elevated frequencies ofG1 cells is a consequence of DNA damage rather than a cause.Another cellular process that might be deregulated upon KDM6inhibition is replication. In this regard, recent data (Piunti et al.,2014) are of interest, which show that the PRC2 core componentEZH2 localises at replication forks and that abrogation of PRC1core component Ring1B expression induces DNA damage. Towhat extent H3K27me2 and H3K27me3 methylation, theproduct of the EZH2 enzymatic activity, are necessary for theprogression of replication forks is an interesting subject of futureanalyses.

Inefficient repair of basal DNA damage could be another causefor the accumulation of DNA lesions. Seminal studies revealed thatundifferentiated ESCs are distinct from differentiated cells withrespect to DDR. In this regard, we showed that the determination ofγH2AX-positive foci frequencies in undifferentiated mouse ESCsdoes not reflect true DNA damage frequencies, because the foci areassociated with global chromatin decondensation rather thanpre-existing DNA damage (Banáth et al., 2009). In our analyses,we confirmed high frequencies of γH2AX-positive foci byimmunofluorescence staining and high levels of γH2AX inwestern blot analyses of undifferentiated ESCs. γH2AX levelsand foci frequencies decline upon differentiation. Another featurethat distinguishes undifferentiated ESCs from differentiating ESCsis the choice of DNA damage repair pathways. ESCs removeionising-radiation-induced DNA damage through homologousrecombination rather than by error-prone non-homologous endjoining (NHEJ) (Fortini et al., 2013). To our knowledge, no detailedexperimental data on the DNA repair mechanism during ESCdifferentiation are available. It is, however, tempting to speculatethat KDM6 plays a role in the transition from the use of homologousrecombination in undifferentiated cells towards the use of NHEJ indifferentiated ESCs. In this context, the co-presence of PTIP,KDM6A and KDM6B in the MLL2 complex might be relevant(Cloos et al., 2008). PTIP is also known for its interaction with53BP1, and it is required for 53BP1-mediated inhibition ofhomologous recombination (Callen et al., 2013). So far however,it is unknown whether KDM6A and/or KDM6B are involved inPTIP-mediated homologous recombination inhibition. Our owndata show that homologous recombination is utilised bydifferentiating ESCs to repair endonuclease-mediated DNAdamage; however, we did not observe reduced homologousrecombination frequencies in the presence of GSK-J4. Furtheranalyses will showwhether NHEJ is inhibited by GSK-J4 treatment.In light of a potential role for KDM6 proteins in switching fromhomologous recombination to NHEJ during differentiation, a reporton the transcriptional regulation of the NHEJ factor Ku80 byDrosophila UTX in response to DNA damage (Zhang et al., 2013)could also be relevant. In addition, under UTX KD and exposure toionising radiation, decreased cell numbers were observed. Our owndata show only moderate effects of ionising radiation on cellsurvival, indicating species- and cell-specific effects of KDM6proteins.

In summary, we observed that inhibition of KDM6 activityresults in increased DNA damage, activation of the DDR andincreased cell death in ESCs that had begun to initiate embryoidbody differentiation. Our findings suggest that KDM6 proteinactivity is necessary to prevent DDR and for survival at the exit fromESC pluripotency.

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MATERIALS AND METHODSESC culture and differentiationESC lines [R1, J1 and J1 Eed KO (Schoeftner et al., 2006)] were cultured onmitomycin C (MitC)-inactivated MEFs in high glucose Dulbecco’smodified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with15% ESC-grade fetal bovine serum (FBS; Biochrom, Berlin, Germany),0.1 mM β-mercaptoethanol (Sigma-Aldrich), 2 mM L-glutamine (GELifesciences), 1 mM sodium pyruvate (GE Lifesciences), 100 U/mlpenicillin (GE Lifesciences) 0.1 mg/ml streptomycin (GE Lifesciences),1% non-essential amino acids (GE Lifesciences) and LIF. For analysis ofcumulative population doublings of ESC cultures, feeder-depleted ESCs(separated by passing over gelatin-coated plates) were seeded onto MitC-inactivated MEFs at a density of 0.5×106 in 6-cm culture dishes. After2 days of culture, cells were trypsinised, and live cell numbers weredetermined by Trypan Blue staining. Subsequently, 0.5×106 ESCs werereseeded and cultured for a total of three passages. The population doublinglevel at each passage was determined according to the following equation:x=log10[log10(N1)−log10(NH)], where N1 is the inoculum number, NH isthe cell harvest number and x is the population doubling value (Cristofaloet al., 1998). New population doubling values were added to previous valuesto calculate cumulative population doubling values. For ESC differentiation,feeder-depleted ESCs were cultured in embryoid body medium (high-glucose DMEM; Sigma-Aldrich), 10% pre-selected FCS (Gibco, LifeTechnologies, Darmstadt, Germany), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 2 mM L-glutamine (GE Lifesciences), 1 mM sodium pyruvate(GE Lifesciences), 100 U/ml penicillin (GE Lifesciences), 0.1 mg/mlstreptomycin (GE Lifesciences), 1% nonessential amino acids (GELifesciences) in non-coated bacteriological Petri dishes. DifferentiatingESCs formed embryoid bodies. For harvesting, embryoid bodies weretransferred to 15-ml Falcon tubes and allowed to sediment. For preparationof single-cell suspensions, medium was replaced by Trypsin-EDTA(Invitrogen, Life Technologies) followed by 3 min of incubation at 37°C.Live cell numbers were determined by Trypan Blue staining. To generateembryoid bodies and determine population doubling levels in embryoidbodies, ESCs were seeded in embryoid body medium at a density of 1×105–2×105 cells/ml. Embryoid bodies were harvested at 1, 2 or 3 days ofdifferentiation, single-cell suspensions were prepared and live cell numberswere determined. Population doubling levels were calculated as describedabove. The GSK-J4 (Tocris Bioscience, Bristol, UK) concentration usedthroughout this study was determined in a dose-finding assaymonitoring R1ESC viability and H3K27me3 levels. Starting from the maximumconcentration (30 µM) for KDM6 protein inhibition (Kruidenier et al.,2012), the concentration was gradually reduced to 0.9 µM (twofold dilutionsteps). A concentration of 1.8 µM yielded a maximum H3K27me3 levelwithout interfering with cell viability. Based on this observation, GSK-J4 orthe control compound GSK-J5 (Cayman Chemical) were both added at aconcentration of 1.8 µM to ESC and embryoid body cultures. Irradiation ofESC and embryoid body cultures was performed in a Faxitron CP-160 X-rayradiation cabinet (160 kV, 6.3 mA, 0.71 Gy/min, filter: 0.5 mm Cu). Unlessotherwise specified, all experiments were performed with R1 ESCs.

Western blot analysisSingle-cell suspensions of ESC or embryoid body cultures were washedtwice with PBS before lysis in 2× Laemmli buffer (0.5 M Tris HCl, pH 6.8,0.4% SDS, 2% glycerol, 0.5% β-mercaptoethanol and Bromophenol Blue).Lysates were adjusted to 106 cells/125 µl and heated (5 min, 95°C). Proteinswere separated by using SDS PAGE and blotted onto nitrocellulosemembranes (Schleicher & Schuell, Dassel, Germany). Membranes wereblocked in PBS, 0.05% Tween, 5% milk powder (30 min, roomtemperature) followed by overnight incubation with primary antibodies.Membranes were washed three times with PBS followed by incubation withsecondary antibodies (1 h, room temperature). After three washes in PBS,membranes were incubated with Amersham ECL Select Western blotDetection Reagent (GE Lifesciences). Chemiluminescence was detectedusing a Chemidoc XRS low light imager (Bio-Rad Laboratories, Munich,Germany). If phospho-specific antibodies were used, PBS was replaced byTBS. The intensity of protein bands was determined using ImageJ software(http://rsb.info.nih.gov/ij/).

Immunofluorescence stainingFor staining of ESCs, cells were grown onMitC-treatedMEFs on cover slipsfor two days. Next, ESCs were fixed with 4% PFA (10 min, roomtemperature) followed by 3 washes with PBS. For preparation of tissuesections, embryoid bodies were transferred to Falcon tubes and allowed tosediment. After 2 washes with PBS embryoid bodies were fixed in 4% PFAfor 3 h and dehydrated overnight in a 20% sucrose solution (4°C). embryoidbodies were embedded in Tissue-Tek (Sakura, Leiden, NL), 5-μm sectionswere cut and mounted onto superfrost microscope slides. Fixed ESC orembryoid body sections were permeabilised (0.1% Triton X-100 in PBS) for30 min at room temperature followed by incubation in blocking solution(20% FCS, 0.1% Triton X-100 in PBS) for 30 min. Subsequently, sampleswere incubated overnight at 4°C with primary antibodies diluted in blockingsolution. Samples were washed three times with PBS before incubation (1 h,room temperature) with secondary antibodies diluted in blocking solution.After incubation, samples were washed three times. DAPI (5 µg/ml) wasadded to the last washing step and was followed by mounting in Mowiol.Microscopy-based inspection and imaging of samples was performed on aninverted confocal laser scanning microscope (LSM780, Zeiss). Fordetermination of γH2AX frequencies, microscopic images of γH2AX-stained embryoid body sections were prepared using an invertedepifluorescence Microscope (Zeiss). Average numbers of γH2AX-positivefoci per nucleus were determined from tissue sections of 15–30 embryoidbodies per condition (total of 100–650 cells).

Flow cytometryAnalyses of cell cycle phase propidium iodide staining of single-cellsuspensions were performed. Briefly, cells were washed twice with coldPBS, permeabilised (70% EtOH, 30 min, −20°C), washed twice with PBSand incubated (30 min, 37°C) with propidium iodide (10 µg/µl) and RNase(10 µg/µl) before fluorescence-activated cell sorting (FACS) analysis.Frequencies of apoptotic cells were determined by annexinV–phycoerythrin Apoptosis Detection Kit I (BD Biosciences). Analyseswere performed on a flow cytometer (FACS Calibur, BD Biosciences). Cellcycle distribution was determined using ModFit software (Verity SoftwareHouse, Topsham, ME).

Alkaline comet assayComet assays were performed with the OxiSelect™ Comet Assay Kit (CellBiolabs, SanDiego, CA). Briefly, after trypsinisation, single-cell suspensionsof embryoid body cultures that were either control or had been treated withGSK-J4were allowed to recover by incubation (15 min, 37°C) in the presenceor absence of inhibitor. For ionising radiation exposure, embryoid bodieswere transferred to embryoid body culture medium without FCS beforeirradiation and immediately kept at 4°C after irradiation to avoid DNA repair.Subsequently, cells werewashed, resuspended in pre-chilled 1× PBS (Sigma-Aldrich) andmixed with liquefied OxiSelect™ comet agarose. 104 cells/wellwere pipetted onto OxiSelect™ Comet well slides and incubated (15 min,4°C, in the dark). Slides were transferred into pre-chilled lysis buffer andincubated (60 min), followed by brief incubation in H2O and finally inalkaline solution (30 min, 4°C, in the dark). Alkaline electrophoresis wasperformed at 4°C with alkaline electrophoresis solution (30 min, 1 V/cm,300 mA). After electrophoresis, slides were washed three times in pre-chilledH2O followed by incubation in ice-cold 70% EtOH (5 min). Slides were air-dried and stained with Vista Green DNA Dye. Microscopy-based inspectionand imaging of samples was performed with an inverted epifluorescencemicroscope (Zeiss). For comet quantification, >50 nuclei were imaged percondition. Comet analyses were performed using CASP software (Koncaet al., 2003) (CASP Lab).

RNA preparation and global gene expression analysesTotal RNA was extracted using RNeasy Mini (Qiagen) or peqGOLD RNAPURE (Peqlab, Erlangen, Germany) kits. RNA quality was assessed with theRNA 6000 nano kit using the Bioanalyzer 2100 instrument (Agilent). Beforemicroarray hybridisation, 100 ng of total RNA was amplified using the WTExpression kit (Ambion, Life Technologies), followed by single stranded (ss)DNA labelling (WT Terminal Labeling Kit; Affymetrix). Samples werehybridised to GeneChip Mouse Gene 2.0 ST arrays (Affymetrix).

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Fluorescence intensities were scanned with a GeneChip Scanner 3000 7G(Affymetrix). Image processing (microarray grid definition, feature intensityreadout) and CEL file generation were performed with GeneChip OperatingSoftware (Affymetrix). Raw microarray data were background corrected,normalised and summarised to probeset expression values using the RobustMicroarray Average (RMA) algorithm (Bolstad et al., 2003; Irizarry et al.,2003).Differentially expressed probe setswere detected byestimating the falsediscovery rates (FDR) with an empirical Bayesian methodology employinglognormal–normal data modelling (Kendziorski et al., 2003). Significantprobe sets (FDR<0.05) were subjected to gene-set enrichment analysis usingWebGestalt (Wang et al., 2013). Analyses were performed in the Renvironment (http://www.r-project.org) using Bioconductor (http://www.bioconductor.org) packages ‘affy’ and ‘embryoid body arrays’. Microarraydata were deposited inMIAME-compliant form at Gene Expression Omnibus(GEO; http://www.ncbi.nlm.nih.gov/geo) under entry GSE49886.

Generation of KDM6A-KO ESCs and lentiviral shRNAtransductionKDM6A-KO ESCs were generated by transfection of R1 KDM6Aconditional mutant ESCs (UTX FD), which contain KDM6A exon 3flanked by loxP sites with the pCAG-CreERT2 vector (Addgene entry14797) followed by 2 days of selectionwith Tamoxifen. Single colonies werepicked and screened by using PCR for successful deletion of exon 3. Forlentiviral small hairpin (sh)RNA transduction, viral particles were producedin 293T cells. Subsequently, ESCs were inoculated two consecutive timeswith lentiviral particles (24 h inoculation time each), followed by puromycin(Invitrogen, Life Technologies, Darmstadt, Germany) (1 µg/ml) selectionfor 4 days. ESCs and embryoid bodies were kept under puromycinselection. Lentiviral pLKO.1-puro vectors (Sigma-Aldrich) were asfollows: SHC002 (Scr-shRNA), NM_001017426.1-5159s1c1 (KDM6B-shRNA1), NM_001017426.1-3013s1c1 (KDM6B-shRNA2).

Generation of DR-GFP reporter ESC and transient SceIexpressionFor generation of the DR-GFP reporter transgenic cells (used to measureDSB homologous repair), R1 ESCs were transfected with the pHPRT-DRGFP construct (Addgene entry 26476) (Pierce et al., 2001), followedby 8 days of selection with puromycin (Invitrogen, Life Technologies)(1 µg/ml). Bulk cultures were used for subsequent experiments. Fortransfection with the pCBASceI construct (Addgene entry 26477)(Richardson et al., 1998), 106 DR-GFP reporter cells were electroporatedwith 2 µg of pCBASceI vector using an Amaxa Nucleofector. After SceItransfection, cells were cultured in ESC medium for 18 h and thentransferred to differentiation medium containing GSK-J4 (1.8 µM), GSK-J5(1.8 µM) or Mirin (100 µM) – a specific MRE11 inhibitor (Dupré et al.,2008). The frequency of GFP-positive cells was assessed with flowcytometry 24 h after the induction of differentiation.

Generation of HA–KDM6A expressing ESCsHA-tagged human KDMA6A (hUTX) was PCR-amplified from thepMSCV-HA-hUTX-puro vector to introduce a 5′NheI and a 3′SacIIrestriction site, respectively. The PCR product was NheI–SacII-digested,followed by ligation into the corresponding restriction sites of the pB-EF1-MCS-IRES-NEO Piggybac vector (System Biosciences, Mountain View,CA) multiple cloning site to generate the pB-EF1-HA-UTX-IRES-NEOvector. The pB-EF1-HA-UTX-IRES-NEO vector was electroporated intoR1 KDM6A-KO ESCs together with the transposase coding pBase vector.Neomycin-selection (Invitrogen, Life Technologies) (800 µg/ml) wasstarted 1 day after transfection. Single colonies were picked after 2 weeksof selection and analysed for HA–UTX expression by western blotting andreverse transcriptase PCR analysis.

Laser microirradiationFor laser microirradiation of adherent embryoid bodies, ESCs were culturedin embryoid body medium for 24 h to allow embryoid body formation. Theembryoid bodies were subsequently transferred to x-well tissue culturechambers (Sarstedt). The culture medium was supplemented with 5 µM

BrdU for 24 h to sensitise the cells to DSB generation by UV-A laser(λ=337 nm) irradiation. Experiments were performed with a Leica ASLMDlaser microdissection microscope (laser power set to 35%). DMSO, GSK-J5or GSK-J4 was added 18 h before irradiation. For laser microirradiation ofESCs, cells were cultured for 24 h in the presence of 5 µMBrdU. GSK-J4 orGSK-J5 was added 18 h prior to irradiation. Cells were seeded into x-welltissue culture chambers 4 h before irradiation. Immediately before laserirradiation, the culture medium was changed to Phenol-Red-free HEPES-buffered embryoid body or ESC medium. Following irradiation of ∼100cells (a procedure taking 10 min), embryoid bodies and ESCs were fixed in3.7% formaldehyde and subjected to immunofluorescence staining. Fortime course analyses of KDM6A or KDM6B localisation after lasermicroirradiation, embryoid bodies were irradiated as described above. Afterirradiation medium had been exchanged for normal embryoid body culturemedium, embryoid bodies were incubated under standard conditions (37°C,5% CO2) for the indicated time periods; 20–30 nuclei were analysed percondition.

qRT-PCR analysisTotal RNAwas extracted using RNeasy Mini (Qiagen) or peqGOLD RNAPURE (Peqlab) kits. 1 µg of total RNAwas used for cDNA synthesis usingthe First Strand cDNA Synthesis Kit (Fermentas, Life Technologies).Quantitative real-time (qRT)-PCR reactions were performed using theABsolute SYBR Green Mix (Thermo Scientific, Life Technologies) in aRoche Light Cycler 480 II (Roche Diagnostics) under the followingconditions: initial denaturation for 15 min at 95°C, followed by repetitivecycles of 10 s at 95°C, 20 s at 60°C and 30 s at 72°C. CT valueswere normalised to two housekeeper genes (GAPDH and RPL4), whichwere pre-selected based on their stable expression using geNormsoftware (Vandesompele et al., 2002). Primer sequences were: KDM6Aforward 5′-GCTGGAACAGCTGGAAAGTC-3′, reverse 5′-GAGTCAA-CTGTTGGCCCATT-3′; KDM6A exon 3 forward 5′-CTGAAGGGAA-AGTGGAGTCTG-3′, reverse 5′-TCGACATAAAGCACCTCCTG-3′;KDM6B forward 5′-GGAAGCCACAGCTACAGGAG-3′, reverse 5′-CC-ACCAGGAACCAGTCAAGT-3′; UTY forward 5′-ATAGTGTCCAGA-CAGCTTCA-3′, reverse 5′-GAGGTAGGAATACGTAAGAA-3′; Oct4forward 5′-AGGCCCGGAAGAGAAAGCGAACTA-3′, reverse 5′-TGG-GGGCAGAGGAAAGGATACAGC-3′; Sox2 forward 5′-GCGGAGTGG-AAACTTTTGTCC-3′, reverse 5′-CGGGAAGCGTGTACTTATCCTT-3′;Nanog forward 5′-TCTTCCTGGTCCCCACAGTTT-3′, reverse 5′-GCA-AGAATAGTTCTCGGGATGAA-3′; GAPDH forward 5′-TGGAGAAA-CCTGCCAAGTATG-3′, reverse 5′-TCATACCAG GAAATGAGCTTGA-3′; RPL4 forward 5′-TTGGGTTGTATTCACTCTGCG-3′, reverse 5′-CA-GACCAGTGCTGAGTCTTGG-3′; Polk forward 5′-AGCTCAAATTAC-CAGCCAGCA-3′, reverse 5′-GGTTGTCCCTCATTTCCACAG-3′; Btg2forward 5′-ATGAGCCACGGGAAGAGAAC-3′, reverse 5′-GCCCTAC-TGAAAACCTTGAGTC-3′; Mdm4 forward 5′-TTCGGAACAAATTA-GTCAGGTGC-3′, reverse 5′-AGTGCATTACCTCTTTCATGGTG-3′;p21CIP forward 5′-CACAGCTCAGTGGACTGGAA-3′, reverse 5′-ACC-CTAGACCCACAATGCAG-3′; FoxA2 forward 5′-TAGCGGAGGCAA-GAAGACC-3′, reverse 5′-CTTAGGCCACCTCGCTTGT-3′; Gata4forward 5′-CCCTACCCAGCCTACATGG-3′, reverse 5′-ACATATCGA-GATTGGGGTGTCT-3′; Sox17 forward 5′-GATGCGGGATACGCCAG-TG-3′, reverse 5′-CCACCACCTCGCCTTTCAC-3′; Nestin forward5′-CAGAGAGGCGCTGGAACAGAGATT-3′, reverse 5′-AGACATAG-GTGGGATGGGAGTGCT-3′; Sox1 forward 5′-ACAGATGCAACCGA-TGCACC-3′, reverse 5′-TGGAGTTGTACTGCAGGGCG-3′; FLk-1forward 5′-CCTGGTCAAACAGCTCATCA-3′, reverse 5′-AAGCGTCT-GCCTCAATCACT-3′, brachyury (T) forward 5′-GCTCTAAGGAACCA-CCGGTCATC-3′, reverse 5′-ATGGGACTGCAGCATGGACAG-3′.

Alkaline phosphatase stainingCells were stained using an Alkaline Phosphatase Detection Kit (Millipore).

Determination of SSEA-1-positive cell frequenciesSingle-cell suspensions of feeder-depleted ESCs were washed with ice-coldFACS buffer (PBS, 0.1% BSA) and stained with an anti-mouse SSEA-1antibody (BioLegend) (45 min, 4°C). Cells were washed with PBS and

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incubated with a secondary PE–Cy7–antibody (BD Biosciences) (45 min).Subsequently cells were washed with PBS and analyses were performed ona flow cytometer (FACSCalibur, BD).

Antibodies for western blot, immunofluorescence and ChIPanalysesFor a list of antibodies used in this study see Table S2.

Chromatin immunoprecipitationChIP experiments were performed as described previously (Dahl andCollas, 2009) with modifications. Briefly, 1.2×106 single-cell suspensionsof embryoid bodies at day 3 were cross-linked with 16% formaldehyde.After nuclei extraction, sonication was performed using a Covaris M220focused-ultrasonicator (20 min; 10% duty factor). 100 µl of chromatinsolution was used as input sample and for immunoprecipitation withantibodies against histone 3 (2.5 μg, Abcam), H3K27me3 (2.5 μg,Diagenode), H3K27me2 (2.5 µg, Abcam), H3K27me1 (2.5 µg,Diagenode) or IgG isotype control (2.5 μg, Abcam). Genomic DNAswere isolated by phenol chloroform extraction and ethanol precipitation.Promoter-specific primers were: Sox2 forward 5′-TTTAGGGTAAGGTA-CTGGGAAGG-3′, reverse 5′-GAGCCCGGGAAATTCTTTTA-3′; Hoxb1forward 5′-CTCTGGTCCCTTCTTTCC-3′, reverse 5′-GGCCAGAGTTT-GGCAGTC-3′; Pou2f3 forward 5′-GGTAAGCGGTTGGAAGGCAAG-3′,reverse 5′-GGCTTCTGCCTAGTTGGGCTC-3′.

Colocalisation analysisColocalisation in image pairs was assessed from scatter plots of midnuclearconfocal images as described previously (von Mikecz et al., 2000) andquantified using the ZEN black software (Zeiss). For each treatmentcondition, at least four different ESC colonies (at least 15 nuclei per colony)or sections of four individual embryoid bodies (at least 20 nuclei per section)were analysed. Background thresholds for each channel were determined asmean intensity value of the image region of interest (ROI) plus two times thestandard deviation. The colocalisation coefficients for two signals weredetermined according to Manders, which provides a value range between 0and 1 (0, no colocalisation; 1, all pixels colocalise) and according toPearson, which provides a value range between −1 and 1 (−1, negativecorrelation of signal intensities; 0, no correlation of signal intensities; 1,positive correlation of signal intensities) (Manders et al., 1993).

AcknowledgementsEed-KO ESCs were kindly provided by Dr A. Wutz (Institute for Molecular HealthSciences, Zurich, Switzerland). Conditional mutant UTX ESCs were kindly providedby Dr Konstantinos Anastassiadis (BIOTEC, Technische Universitat Dresden,Dresden, Germany). The pMSCV-HA-hUTX-puro vector was kindly provided byRobert Slany (Lehrstuhl fur Genetik, Erlangen, Germany).We thankVeronikaHornichUrsula Sauer for excellent technical assistance and Dr Detlef Schindler, Dr ElizabethD. Martinez, Dr Stanislaw Gorski, Dr Almut Horch, Dr Tcholpon Djuzenova, Dr JensVanselow and Dr Peter Hemmerich for helpful discussions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsC.H., J.M.K., H.W. and S.H. collected and assembled data; C.H., A.M.M., M.B. andA.S. conceived and designed experiments; C.H., A.M.M., M.B. and A.S. analysedand interpreted data. A.M.M. and M.B. generated financial support and wrote themanuscript.

FundingThis work was supported by a grant from the German Research Council (DFG)[grant number MU 1228/11 to A.M.M., BE 4563/1 to M.B.].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.175174/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2016) 129, 788-803 doi:10.1242/jcs.175174

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