MOLECULAR BIOLOGY

Histone demethylase KDM6A directlysenses oxygen to control chromatinand cell fateAbhishek A. Chakraborty1, Tuomas Laukka2, Matti Myllykoski2, Alison E. Ringel3,Matthew A. Booker4, Michael Y. Tolstorukov4, Yuzhong Jeff Meng1,5,6,7,8,Samuel R. Meier5, Rebecca B. Jennings9, Amanda L. Creech5,Zachary T. Herbert10, Samuel K. McBrayer1, Benjamin A. Olenchock11,Jacob D. Jaffe5, Marcia C. Haigis3, Rameen Beroukhim1,5,7, Sabina Signoretti9,Peppi Koivunen2*, William G. Kaelin Jr.1,12*

Oxygen sensing is central to metazoan biology and has implications for humandisease. Mammalian cells express multiple oxygen-dependent enzymes called2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in theiroxygen affinities and hence their ability to sense oxygen. The 2-OGDD histonedemethylases control histone methylation. Hypoxia increases histone methylation,but whether this reflects direct effects on histone demethylases or indirect effectscaused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcriptionfactor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, wereport that hypoxia promotes histone methylation in a HIF- and 2-HG–independentmanner. We found that the H3K27 histone demethylase KDM6A/UTX, but notits paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27demethylation and blocked cellular differentiation. Restoring H3K27 methylationhomeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affectschromatin regulators to control cell fate.

Oxygen’s appearance in Earth’s atmospherewas a watershed that created the evolu-tionary selection pressure for a conservedpathway used by metazoans to sense andrespond to changes in ambient oxygen that

converges on the heterodimeric HIF (hypoxia-inducible factor) transcription factor. Undernormoxic conditions, the HIFa subunit is prolylhydroxylated by the EglN (egg-laying defectivenine) isoenzymes of the 2-OGDD dioxygenase

family. Hydroxylated HIFa is marked for de-gradation by the von Hippel-Lindau (VHL) ubi-quitin ligase complex. Hypoxia inactivates theEglNs and thereby stabilizes HIFa, which thenassociates with HIF1b [also called ARNT (arylhydrocarbon nuclear translocator)] and transcrip-tionally activates genes that promote adaptationto inadequate oxygen (1).The 2-OG-dependent dioxygenase family also

includes the collagen prolyl hydroxylases, theJmjC (Jumonji C) domain histone lysine de-methylases (KDMs), the ten-eleven transloca-tion (TET) DNA hydroxylases, and ~50 otherenzymes that are relatively understudied (2). Incontrast to the high-oxygen-affinity (low KM)collagen prolyl hydroxylases, the EglNs exhibitlow oxygen affinities (high KM) (1), which en-ables them to sense physiological changes inoxygen.Several previous studies have suggested that

oxygen regulates histone methylation. CertainKDMs display low oxygen affinities in vitro (3).Moreover, many KDMs are transcriptionallyactivated by hypoxia and HIF (4), perhaps tocompensate for a decrease in their enzymaticspecific activity. Finally, hypoxia can induce his-tone hypermethylation (5). However, in theseprevious studies, it was unclear whether histonehypermethylation reflected a direct effect of hy-poxia on KDMs or was confounded by indirectconsequences of hypoxia (andHIF). For example,in some cells hypoxia increases the L-enantiomerof 2-hydroxyglutarate (L-2HG), which is an endo-genous inhibitor of 2-OG–dependent dioxyge-

nases (6–8). Moreover, HIF can potentially affectchromatin inmanyways, such as by alteringKDMprotein levels (see above), by inducing chromatin-modifying enzymes other thanKDMs [e.g., TETs(9) and DNA methyltransferases (10)], or by up-regulating transcription factors that enforce anepithelial-mesenchymal transition (EMT) and ac-companying epigenetic reprogramming (11).To rigorously address whether hypoxia has a

direct or indirect effect on histone methylation,we lentivirally transduced anArnt-defective (HIF-inactive) mouse hepatoma cell line (mHepa-1 c4)to express either green fluorescent protein (GFP),wild-type (WT) ARNT, or a functionally inactiveARNT mutant (D414) that is missing 414 basepairs from its N terminus, thereby eliminating itsbasic helix-loop-helix domain and ability to hete-rodimerize (Fig. 1A and fig. S1, A to C). mHepa-1c4 did not tolerate prolonged growth in 1%oxygen, which is the oxygen concentration typi-cally used tomodel hypoxia ex vivo.We thereforeused more modest levels of hypoxia (2 to 5%) tostudy these cells. As expected, canonical HIF-target genes (e.g., Egln3 and Ndrg1) were trans-criptionally induced by 5% oxygen in the cellsexpressing WT ARNT but not in the cells ex-pressing ARNT (D414) or GFP (fig. S1D).We next used a multiplexed mass spectro-

metric assay (12) to quantify changes in histonemethylation in response to hypoxia in the iso-genic [GFP, ARNT(WT), ARNT(D414)] mHepa-1c4 cells. Unsupervised clustering of histonemod-ification patterns revealed that the isogenic celllines clustered primarily based on oxygen avail-ability during growth and not HIF status (Fig.1B). Consistent with prior reports (5), hypoxiapromoted the dimethylation and trimethylation(me3) of H3K4 (histone 3, lysine 4), H3K9, andH3K27 (Fig. 1B). Hypermethylation of H3K9 andH3K27 was confirmed by immunoblot analysis(Fig. 1C). We also observed a concomitant de-crease in hypomethylated H3K27 (me0 andme1 states) and acetylated H3K27 in responseto hypoxia (Fig. 1B), which is consistent withthe knowledge that histone methylation andacetylation are reciprocally regulated. The H3K27hypermethylation was not explained by increasedexpression of EZH2 (enhancer of zeste homo-log 2), which controls bulk H3K27 methylation,or decreased expression of the primary H3K27histone demethylases KDM6A [also calledubiquitously transcribed TPR protein on theX chromosome (UTX)] and KDM6B (fig. S2).Hypoxia also promoted histone hypermethyl-

ation in VHL−/− RCC4 human renal carcinomacells (fig. S3). Thus, hypoxia promotes histonehypermethylation both in cells that cannotmount a HIF response (mHepa-1 c4 cells)and in cells that constitutively overproduce HIF(RCC4 cells), arguing that hypoxia’s effects onhistone methylation are not caused by changesin HIF activity.We next explored whether hypoxia’s effects

on histone methylation in mHepa-1 c4 cellswere caused by metabolic changes that caninhibit KDMactivity, such as increased L-2HG ordecreased 2-OG (6–8, 13). In the isogenic mHepa-1

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1Department of Medical Oncology, Dana-Farber CancerInstitute and Brigham and Women’s Hospital, HarvardMedical School, Boston, MA 02215, USA. 2BiocenterOulu, Faculty of Biochemistry and Molecular Medicine,Oulu Center for Cell-Matrix Research, University ofOulu, FIN-90014 Oulu, Finland. 3Department ofCell Biology, Harvard Medical School, Boston, MA02115, USA. 4Department of Informatics, Dana-FarberCancer Institute, Harvard Medical School, Boston, MA02215, USA. 5Broad Institute of Harvard and MIT,Cambridge, MA 02142, USA. 6Biological and BiomedicalSciences, Harvard Medical School, Boston, MA 02115,USA. 7Department of Cancer Biology, Dana-FarberCancer Institute, Harvard Medical School, Boston, MA02115, USA. 8The Harvard-MIT Program in HealthSciences and Technology, Harvard Medical School,Boston, MA 02115, USA. 9Department of Pathology,Brigham and Women’s Hospital, Harvard MedicalSchool, Boston, MA 02115, USA. 10Molecular BiologyCore Facility, Dana-Farber Cancer Institute and Brighamand Women’s Hospital, Harvard Medical School, Boston,MA 02215, USA. 11Division of Cardiovascular Medicine,Department of Medicine, The Brigham and Women'sHospital, Boston, MA 02115, USA; Harvard MedicalSchool, Boston, MA 02115, USA. 12Howard HughesMedical Institute, Chevy Chase, MD 20815, USA.*Corresponding author. Email: william_kaelin@dfci.harvard.edu(W.G.K.); peppi.karppinen@oulu.fi (P.K.)

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c4 cells, 5% oxygen did not significantly induceeither total 2-HG (fig. S4A) or enantiomer-specific2-HG (fig. S4, B and C) and actually increased2-OG levels (fig. S4D). 2-HG was modestly in-duced in parental mHepa-1 c4 cells by more pro-found levels of hypoxia (0.5 to 2% oxygen) (fig.S4, C and E), albeit as D-2HG rather than L-2HG(fig. S4C). The importance of the latter findingis unclear. Even under these more extreme con-ditions, the 2-HG levels achieved were ~100-foldbelow both the intracellular levels in mutant iso-citrate dehydrogenase 1 cells (fig. S4F), wherein2-HG serves as an oncometabolite (14), and theintracellular levels required to promote histonemethylation inmHepa-1 c4 cells treatedwith cell-permeable versions of D-2HG or L-2HG (fig. S4,G andH). The latter observation is consistent withthe biochemical D-2HG and L-2HGhalf-maximalinhibitory concentration values for the KDMs (15).

Hypoxia can incite reactive oxygen species(ROS), which can inhibit 2-OG-dependent dioxy-genases (16). Treating mHepa-1 c4 cells with theROS-inducer tert-Butyl hydroperoxide showedthat intracellular ROS levels ~10-fold higher thanthose observed after exposure to 2% oxygen wererequired to induce histone methylation (fig. S5).These findings suggested that theHIF-independenteffects of hypoxia on KDM activity were notcaused by increased L-2HG, decreased 2-OG, orincreasedROSbut insteadwere caused by a directeffect of hypoxia on the enzymatic activities ofspecific KDMs.In support of this idea, we found that recom-

binantKDM4B, KDM5A, andKDM6A [also calledubiquitously transcribed TPR protein on the Xchromosome (UTX)] have relatively low oxygenaffinities that are comparable to the EglN family,whereas recombinant KDM4A, KDM5B, KDM5C,

KDM5D, andKDM6Bhave high oxygen affinities(figs. S6 to S8). Finally, like full-length KDM6A,the isolated KDM6A catalytic domain also hadlow oxygen affinity compared to its KDM6Bcounterpart (Fig. 1, D to F, and fig. S8).We focused our attention on theKDM6H3K27

demethylases because hypoxia and histoneH3K27methylation have been independently linked tothe control of cellular differentiation (17–19),because this histone mark can be manipu-lated with drugs, and because KDM6A has thelowest oxygen affinity of the KDMs tested todate. We first confirmed that hypoxia inducedH3K27 methylation in additional human celllines including 293T embryonic kidney cells,MCF7 breast cells, and SK-N-BE(2) neuroblas-toma cells (fig. S9). Moreover, histological analy-sis showed elevated H3K27 methylation inmouse tissues that are known to be hypoxic, such

Chakraborty et al., Science 363, 1217–1222 (2019) 15 March 2019 2 of 6

Fig. 1. Hypoxia causes HIF-independent histone hypermethylation.(A to C) Vector schematic (A), histone modification profiling bymass spectrometry (B), and histone immunoblot analysis (C) ofArnt-deficient mouse hepatoma (mHepa-1 c4) cells that were lentivirallytransduced to produce the indicated V5-tagged proteins and culturedat the indicated oxygen levels for 4 days. In (B), rows represent twobiological replicates of the indicated samples, and the color in each cellrepresents log2 fold change relative to all samples in the column,

normalized for total histone by using an internal control peptide(histone H3: residues 41 to 49). CMV, cytomegalovirus promoter.(D to F) Coomassie blue staining (D) and kinetic analysis of baculovir-ally expressed and purified JmjC catalytic domains of KDM6A(KDM6A*) (E) and KDM6B (KDM6B*) (F). KM values are mean ± SD(N = 3 independent biochemical measurements). (G) Immuno-histochemical (IHC) analysis of the indicated tissues derived fromrepresentative male and female age-matched mice.

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as the kidney (20), splenic germinal centers (21),and thymus (22), but not in well-oxygenated tis-sues such as the heart (Fig. 1G). Similarly, H3K27methylation is increased in hypoxic regions ofmouse tumors (23–25) (fig. S10). Finally, Gene SetEnrichmentAnalysis (26) of ~2000human tumorsthat were previously annotated as normoxic orhypoxic on the basis of their HIF signature (27)(table S1) revealed that hypoxic tumors hadtranscriptional signatures indicative of H3K27hypermethylation (fig. S11 and tables S2 and S3).To examine the effect of hypoxia on cell dif-

ferentiation in vitro, we studied C2C12 murine

myoblasts. C2C12 differentiate intomyosin heavychain (MyHC)–positive multinucleated myotubeswhen shifted from serum-rich growth media(GM) to nutrient-poor differentiationmedia (DM).Hypoxia blocks C2C12 differentiation (fig. S12, Ato D) (28, 29). Hypoxic C2C12 cells grown in DMentered a quiescence-like state but more readilyproliferated when returned to GM under nor-moxic conditions, compared with their normoxiccounterparts (fig. S12, E to G). Similarly, hypoxiablocked the myogenic differentiation of mouseembryo fibroblasts (MEFs) that conditionallyexpress MyoD (fig. S13).

Eliminating ARNT in C2C12 cells by usingCRISPR-Cas9 blocked (rather than accentuated)their ability to differentiate under normoxic con-ditions (fig. S14, A to C) and did not rescue theirability to differentiate under hypoxia (fig. S14D).Moreover, expression of stabilized versions ofHIF1a or HIF2a did not block normoxic C2C12differentiation (fig. S14, E and F). Thus, the dif-ferentiation block exhibited by hypoxia in C2C12cells is not due to HIF activation.Total 2-HG was not induced, and L-2HG was

induced only about twofold (fig. S15, A to C) inC2C12 cells by 2% oxygen. C2C12 cells that were

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Fig. 2. Regulation ofmyogenic differentia-tion by the KDM6AH3K27 demethylase.(A to B) Immuno-fluorescence analysisof C2C12 cells that werelentivirally transducedto express the shRNAstargeting Kdm6A (A)or Kdm6B (B) and thencultured in DM for 4 days.DAPI, 4′,6-diamidino-2-phenylindole. (C toE) Immunoblot analysisof C2C12 cells lentivirallytransduced to expressthe indicated sgRNAs andcultured for 4 days eitherin GM or DM, as indi-cated. (E) Immunoblotanalysis of C2C12 cellsexpressing, where indi-cated, Kdm6a sg2[described in (C) and(D)] that were lentivirallytransduced to produceeither GFP (control)or wild-type humanKDM6A [6A(WT)] andthen cultured in DMat the indicated oxygenconcentrations for4 days. The mouseKdm6A sg2 targetsequence is notconserved in humanKDM6A. Con sg, Non-targeting control sgRNA.(F) Immunoblot analysisof histones fromC2C12 cells expressingthe indicated sgRNA thatwere cultured at theindicated oxygenconcentrations for3 days. (G to J) Heatmaprepresenting mRNAlevels determined byRNA sequencing (RNA-seq) (G) and H3K27me3 levels determined by chromatin immunoprecipitation sequencing (ChIP-seq) analysis at theActc1 (H), Myl1 (I), and the Myog (J) loci from two biological replicates (A and B) of C2C12 cells cultured in the indicated media for 4 days at theindicated oxygen concentration.

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pharmacologically or genetically manipulated tohave intracellular L-2HG levels three- to fivefoldhigher than levels observed under hypoxia stilldifferentiated in DM (fig. S15, D to G). Therefore,hypoxia’s effects on C2C12 differentiation werenot caused by 2-HG.Similar to hypoxia, treatment of C2C12 cells

with the KDM6 family inhibitor GSK-J4 promotedH3K27 hypermethylation and blocked myogenicdifferentiation (fig. S16). Similar results were ob-tained with MEFs expressing MyoD (fig. S13). Incontrast, KDM-C70, a KDM5 family inhibitor, didnot block C2C12 differentiation (fig. S17).The differential oxygen affinities of KDM6A

and KDM6B suggested that the ability of hy-

poxia to promote H3K27 methylation and blockdifferentiation is caused specifically by a loss ofKDM6A activity. Indeed, down-regulating KDM6A,but not KDM6B, with different short hairpinRNAs (shRNAs) phenocopied the effects of hy-poxia on differentiation (Fig. 2, A and B, andfig. S18, A to D) (30). Moreover, eliminatingKDM6A in C2C12 cells with CRISPR-Cas9 blockedtheir ability to differentiate unless they were res-cued with a single guide RNA (sgRNA)–resistantKDM6A cDNA (Fig. 2, C to E). In contrast, elimi-nating KDM6B had no effect (fig. S18, E and F),and eliminating KDM5A promoted differenti-ation (fig. S19). Bulk H3K27me3 was oxygen-insensitive in the Kdm6a-deficient C2C12 cells,

consistent with KDM6A being the primary oxy-gen sensor amongst the KDM6 paralogs (Fig. 2F).Previous work showed that KDM6A is directly

recruited to myogenic targets during differenti-ation (30). We therefore investigated whetherdifferentiation programs driven by KDM6A activ-ity involve transcriptional changes that dependon H3K27me3 elimination. Comparison of tran-scriptional signatures of normoxic C2C12 cellsgrown in GM versus DM revealed profounddifferences in transcriptional output, particu-larly of muscle-specific target genes (Fig. 2G,fig. S20, and table S5). Hypoxia (and the con-sequent differentiation block), however, bluntedthe transcriptional differences between these

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Fig. 3. Restoring the balance of H3K27 methyltransferase activityto H3K27 demethylase activity rescues myogenic differentiationunder hypoxic conditions. (A) Model for control of H3K27methylation by the indicated opposing demethylases (“erasers”)and methyltransferases (“writers”). (B) Immunoblot analysis of C2C12cells lentivirally transduced to express the indicated sgRNAs andcultured under the indicated conditions. Vinc, Vinculin. (C) Structuralmodels of the KDM6A (pink) and KDM6B (cyan) catalytic pockets.The nonconserved M1190 (KDM6A) → T1434 (KDM6B) and theE1335 (KDM6A) → D1579 (KDM6B) are highlighted. Peptidic H3K27me3

substrate (yellow), Fe+2 (orange), 2-OG (green), and Zn+2 (darkpurple) are shown. (D and E) Michaelis-Menten plots (insetLineweaver-Burk plot) and measured oxygen KM and Vmax values(mean ± SD, N = 3) of recombinant KDM6A wild type and theMT/ED mutant. (F and G) Real-time quantitative polymerase chainreaction analysis (mean ± SD, N = 3) of the indicated genes (F) andimmunofluorescence analysis (G) of C2C12 cells transduced toproduce WT human KDM6A or the KDM6A MT/ED variant and thencultured in DM at the indicated oxygen concentrations for 4 days.*P < 0.05 ns, not significant; Rel., relative.

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two conditions (fig. S20A), which was associ-ated with a failure of these cells to inducemuscle-specific markers in DM (Fig. 2G andfig. S20B). H3K27me3 status typically repressestranscription. The inability of C2C12 cells grownin DM to activate late myogenic genes (e.g.,Actc, Myl1, and Myog) under hypoxia corre-lated with a failure to erase H3K27me3 at thoseloci (Fig. 2, H to J, and fig. S21), presumablybecause of inactivation of KDM6A. This wasspecific because H3K4me3 decreased at latemyogenic genes under hypoxia (fig. S21).Loss of cellular differentiation is a hallmark

of cancer, and KDM6A is a human tumor sup-pressor gene that is inactivated in a variety ofcancers, including leukemia, kidney cancer, andbladder cancer (31). Gene Set EnrichmentAnalysis showed that a myogenic differentia-tion gene set, which presumably also containsgenes linked to differentiation in other con-texts, is more highly expressed in KDM6A WTbladder cancers compared with KDM6A mutanttumors (fig. S22 and table S6).These data suggest that KDM6A inactivation

by hypoxia promotes the persistence of H3K27me3and prevents the transcriptional reprogrammingrequired for differentiation. If true, the effects ofhypoxia on differentiation should be redressed byinhibiting H3K27 methyltransferase activity (Fig.3A). Like mHepa-1 c4 cells, hypoxia did not alterthe protein levels of the EZH H3K27 methyl-transferases in C2C12 cells (fig. S23A). Inhibit-ing the H3K27 methyltransferase EZH2 withCRISPR-Cas9 (Fig. 3B and fig. S23B) or withthe drug GSK126 (fig. S23, C and D) reducedH3K27me3 levels and partially rescued the abil-ity of C2C12 cells to differentiate under hypoxicconditions. By contrast, rescue with the G9a andGLP methyltransferase inhibitor UNC638 wasineffective (fig. S23, C and D). Finally, GSK126rescued the hypoxia-induced differentiationblock in human primary myoblasts and inMEFs conditionally expressing MyoD (fig. S23,E and F).Hypoxia can also, in a HIF-independent man-

ner, alter the differentiation of human mammaryepithelial cells (25), causing an EMT (fig. S24,A and B) and up-regulation of the cancer stem-like marker CD44 (fig. S24C). Similar to our find-ings with C2C12 cells, these hypoxia-associatedchanges were phenocopied by pharmacologic(GSK-J4) (fig. S24, A to C) or genetic (CRISPR-Cas9) disruption of KDM6A (fig. S24, D to F)and rescued by the EZH inhibitor GSK126 (fig.S24, A to C).Finally, we tried to directly increase KDM6A’s

oxygen affinity. We reasoned that certain non-conserved residues in the catalytic domains ofKDM6A and KDM6B caused their vastly differ-ent oxygen affinities. We overlaid published mod-els of the catalytic JmjC domains of KDM6Awith KDM6B (32, 33) and noted two noncon-served residues, M1190 (KDM6A)→ T1434 (KDM6B)and E1335 (KDM6A) → D1579 (KDM6B), liningthe 2-OG– and Fe+2-binding pocket (Fig. 3C). Aspredicted, a KDM6A variant that harbored thesetwo KDM6B-like changes (MT/ED: M1190 → T

and E1335→ D) displayed a twofold increasedaffinity for oxygen in vitro, albeit at the cost ofa decreased Vmax (Fig. 3, D and E). WT KDM6Aand the MT/ED variant were comparably in-sensitive to L-2HG and to ROS (fig. S25, A toD), which was induced less than twofold by2% oxygen in C2C12 cells (fig. S25E). Both wild-type KDM6A and the MT/ED rescued the abilityof KDM6A-deficient C2C12 cells to differen-tiate under normoxia (fig. S26A). The doublemutant, however, was superior to WT KDM6Aat rescuing differentiation under hypoxic con-ditions in both parental and KDM6A-deficientC2C12 cells, presumably because of its enhancedoxygen affinity (Fig. 3, F and G, and fig. S26, Bto D).Independent lines of research have shown

that oxygen and H3K27 methylation eachregulate embryological development, cellulardifferentiation, stemness, and malignant trans-formation (17–19). We propose that these obser-vations are linked. Specifically, we argue thatoxygen has both direct and indirect effectson chromatin and that the former involvesenzymes such as KDM6A, which couple changesin oxygen availability to changes in H3K27methylation and the transcriptional controlof cell fate.The observed H3K4 hypermethylation and

H3K9hypermethylation in hypoxicArnt-deficientmHepa-1 c4 cells, together with our biochemicalstudies, suggest that at least one H3K4 and oneH3K9 histone demethylase also act as oxygen sen-sors. For example, our biochemical data, togetherwith the data in the accompanying manuscript(34), argue thatKDM5Aplays such a role. Profoundhypoxia can also inhibit other 2-OG–dependentenzymes, including TETs, leading to DNA hyper-methylation (27).Mammalian embryological development oc-

curs in a hypoxic environment and mamma-lian stem cells are maintained in hypoxic niches.It is well established that hypoxia can affectstemness and cellular differentiation by acti-vating HIF and HIF-target genes such as Oct4(19). Such effects are not mutually exclusivewith direct effects of oxygen on histone methyl-ation and might serve to reinforce one another.Hypoxia promotes stemness in both metazoansand plants, but the HIF pathway is only presentin the former (35). It is possible that oxygensensing by histone demethylases evolutionar-ily preceded the emergence of oxygen-sensitivetranscription factors.

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ACKNOWLEDGMENTS

We thank H. Zhao, D. Lambrechts, and P. Carmeliet forsharing their TCGA tumor annotations. We thank R. Looperfor synthesizing and sharing the esterified 2-HG, M. K. Koski andR. Wierenga for help with structural modeling, and T. Aatsinkiand E. Lehtimaki for technical assistance. We thank theBroad GDAC group for help with the GDAC-GSEA source code.We thank M. Oser for sharing mouse KDM5A sgRNAs andV. Koduri for assistance in acquiring microscopic images.Funding: W.G.K. was supported by grants from theNIH (R01CA068490, P50CA101942, and R35CA210068).A.A.C. was supported by grants from the Friends of Dana-Farberand the NIH (Cancer Biology Training grant: T32CA009361and the DF/HCC Kidney SPORE CEP and DRP award:P50CA101942). P.K. was supported by Academy of Finlandgrants (266719 and 308009), the S. Juselius Foundation, theJane and Aatos Erkko Foundation, and the Finnish CancerOrganizations. T.L. was supported by the Finnish MedicalFoundation and, with P.K., the Emil Aaltonen Foundation. S.K.M.is supported by an American Cancer Society postdoctoralfellowship (PF-14-144-01-TBE) and by a Career EnhancementProject award from the Dana-Farber/Harvard Cancer CenterBrain SPORE. W.G.K. is an HHMI Investigator. Authorcontributions: A.A.C., P.K., and W.G.K. conceived experimentsand wrote the manuscript; A.A.C., T.L., M.M., A.E.R., A.L.C.,and R.B.J. performed experiments. A.A.C., T.L., M.M, A.E.R.,M.A.B., M.Y.T., Y.J.M., S.R.M., S.K.M., B.A.O., Z.T.H., J.D.J., S.S.,M.C.H., R.B., P.K., and W.G.K. analyzed data. Competinginterests: W.G.K. has financial interests related to LillyPharmaceuticals (Board of Directors), Agios Pharmaceuticals[scientific advisory board (SAB)], Cedilla Therapeutics(founder), Fibrogen (SAB), Nextech Invest (SAB), PelotonTherapeutics (SAB), Tango Therapeutics (founder), and TraconPharmaceuticals (SAB). W.G.K. is a coinventor on a patent(US6855510B2) covering pharmaceuticals and methods fortreating hypoxia, which has been licensed to Fibrogen. S.S. has

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a consulting or advisory role in AstraZeneca/MedImmuneand Merck; patents, royalties, and other intellectual propertyfrom Biogenex Laboratories; and research funding fromAstraZeneca, Exelixis, and Bristol-Myers Squibb. R.B. receivesresearch funding from Novartis. Data and materialsavailability: All data are available in the manuscript or the

supplementary material. RNA-seq and ChIP-seq data have beenuploaded to the GEO (GSE114086).

SUPPLEMENTARY MATERIALSwww.sciencemag.org/content/363/6432/1217/suppl/DC1Materials and Methods

Figs. S1 to S26Tables S1 to S6References (36–55)

5 May 2018; accepted 15 January 201910.1126/science.aaw1026

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Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate

G. Kaelin Jr.Benjamin A. Olenchock, Jacob D. Jaffe, Marcia C. Haigis, Rameen Beroukhim, Sabina Signoretti, Peppi Koivunen and WilliamYuzhong Jeff Meng, Samuel R. Meier, Rebecca B. Jennings, Amanda L. Creech, Zachary T. Herbert, Samuel K. McBrayer, Abhishek A. Chakraborty, Tuomas Laukka, Matti Myllykoski, Alison E. Ringel, Matthew A. Booker, Michael Y. Tolstorukov,

DOI: 10.1126/science.aaw1026 (6432), 1217-1222.363Science 

, this issue p. 1217, p. 1222; see also p. 1148Scienceof genes that govern cell fate.oxygen. In cell-culture models, hypoxia diminished the activity of these enzymes and caused changes in the expressionchromatin regulators. Certain histone demethylases, such as KDM6A and KDM5A, were found to be direct sensors of

now show that hypoxia can also affect gene expression through direct effects onet al. and Batie et al.Chakraborty regulate the activity of a transcription factor called hypoxia-inducible factor (see the Perspective by Gallipoli and Huntly).studies examining the way in which hypoxia alters gene expression have focused on oxygen-sensing enzymes that

The cellular response to hypoxia (oxygen deficiency) is a contributing factor in many human diseases. PreviousOxygen sensing revisited

ARTICLE TOOLS http://science.sciencemag.org/content/363/6432/1217

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2019/03/13/363.6432.1217.DC1

CONTENTRELATED

http://science.sciencemag.org/content/sci/363/6432/1222.fullhttp://science.sciencemag.org/content/sci/363/6432/1148.full

REFERENCES

http://science.sciencemag.org/content/363/6432/1217#BIBLThis article cites 55 articles, 14 of which you can access for free

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Science. No claim to original U.S. Government WorksCopyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of

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