spinal curve progression in some IS patients,even after the onset and clinical diagnosis ofscoliosis.

REFERENCES AND NOTES

1. J. C. Cheng et al., Nat. Rev. Dis. Prim. 1, 15030 (2015).2. M. M. Janssen, R. F. de Wilde, J. W. Kouwenhoven,

R. M. Castelein, Spine J. 11, 347–358 (2011).3. K. F. Gorman, F. Breden, Med. Hypotheses 72, 348–352

(2009).4. M. Hayes et al., Nat. Commun. 5, 4777 (2014).5. J. G. Buchan et al., Dev. Dyn. 243, 1646–1657 (2014).6. S. A. Patten et al., J. Clin. Invest. 125, 1124–1128 (2015).7. S. Sharma et al., Nat. Commun. 6, 6452 (2015).8. M. Hayes, M. Naito, A. Daulat, S. Angers, B. Ciruna,

Development 140, 1807–1818 (2013).9. T. J. Park, B. J. Mitchell, P. B. Abitua, C. Kintner,

J. B. Wallingford, Nat. Genet. 40, 871–879 (2008).10. A. Borovina, S. Superina, D. Voskas, B. Ciruna, Nat. Cell Biol.

12, 407–412 (2010).11. A. Caron, X. Xu, X. Lin, Development 139, 514–524

(2012).12. V. Singla, J. F. Reiter, Science 313, 629–633 (2006).13. S. C. Goetz, K. V. Anderson, Nat. Rev. Genet. 11, 331–344

(2010).14. J. J. Essner et al., Nature 418, 37–38 (2002).15. M. J. Simon, J. J. Iliff, Biochim. Biophys. Acta 1862, 442–451

(2016).16. L. Lee, J. Neurosci. Res. 91, 1117–1132 (2013).17. W. J. Wang et al., J. Pediatr. Orthop. 31, S14–S27

(2011).18. V. G. Engesaeth, J. O. Warner, A. Bush, Pediatr. Pulmonol. 16,

9–12 (1993).19. S. P. Choksi, G. Lauter, P. Swoboda, S. Roy, Development 141,

1427–1441 (2014).20. A. Becker-Heck et al., Nat. Genet. 43, 79–84 (2011).21. R. Hjeij et al., Am. J. Hum. Genet. 95, 257–274 (2014).22. A. Tarkar et al., Nat. Genet. 45, 995–1003 (2013).23. C. Austin-Tse et al., Am. J. Hum. Genet. 93, 672–686

(2013).24. K. M. Jaffe et al., Cell Rep. 14, 1841–1849 (2016).25. A. Chuma et al., Spine 22, 589–594, discussion 595

(1997).26. M. Turgut, E. Cullu, A. Uysal, M. E. Yurtseven, B. Alparslan,

Neurosurg. Rev. 28, 289–297 (2005).27. T. H. Milhorat et al., Neurosurgery 44, 1005–1017

(1999).28. R. A. Ozerdemoglu, F. Denis, E. E. Transfeldt, Spine 28,

1410–1417 (2003).29. M. Verhoef et al., Dev. Med. Child Neurol. 46, 420–427

(2004).30. C. M. Karner, F. Long, L. Solnica-Krezel, K. R. Monk, R. S. Gray,

Hum. Mol. Genet. 24, 4365–4373 (2015).

ACKNOWLEDGMENTS

We gratefully acknowledge E. Lee, D. Holmyard, P. Paroutis,and L. Yu for technical assistance; A. Morley, A. Ng, C. Hasty,D. Bosco, and P. Johnson for zebrafish care; andJ. Schottenfeld-Roames and T. Ku for initial observations ofspine curvatures in c21orf59TS mutant fish. This work wassupported in part by funding from the Canada Research Chairsprogram to R.M.H. and B.C.; National Institute of Child Healthand Development grant 2R01HD048584 to R.D.B.; and CanadianInstitutes of Health Research (MOP-111075) and March of DimesFoundation (#1-FY13-398) grants to B.C. D.T.G., C.W.B., andN.F.C.M. performed all experiments characterizing scoliosisphenotypes. C.W.B. cloned and characterized foxj1a transgeniclines. N.F.C.M. generated the dyx1c1pr13 allele. C.W.B. and R.M.H.performed mCT. B.C. and C.W.B. performed SEM and CSF flowanalyses. D.T.G., C.W.B., N.F.C.M., R.D.B., and B.C. analyzed thedata. D.T.G., C.W.B. and B.C. wrote the manuscript, and all authorsedited the manuscript.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6291/1341/suppl/DC1Materials and MethodsFigs. S1 to S5Movies S1 to S6References (31–33)

8 March 2016; accepted 16 May 201610.1126/science.aaf6419

CANCER

The histone H3.3K36M mutationreprograms the epigenomeof chondroblastomasDong Fang,1* Haiyun Gan,1* Jeong-Heon Lee,1,2* Jing Han,1* Zhiquan Wang,1*Scott M. Riester,3 Long Jin,3 Jianji Chen,4 Hui Zhou,1 Jinglong Wang,5,6

Honglian Zhang,1 Na Yang,5 Elizabeth W. Bradley,3 Thai H. Ho,7 Brian P. Rubin,8

Julia A. Bridge,9 Stephen N. Thibodeau,10 Tamas Ordog,2,11,12 Yue Chen,4

Andre J. van Wijnen,1,3 Andre M. Oliveira,3,10 Rui-Ming Xu,5,6

Jennifer J. Westendorf,1,3 Zhiguo Zhang1,2†

More than 90% of chondroblastomas contain a heterozygous mutation replacing lysine-36with methionine-36 (K36M) in the histone H3 variant H3.3. Here we show that H3K36methylation is reduced globally in human chondroblastomas and in chondrocytes harboringthe same geneticmutation, due to inhibition of at least twoH3K36methyltransferases,MMSETand SETD2, by the H3.3K36Mmutant proteins. Genes with altered expression as well asH3K36 di- and trimethylation in H3.3K36M cells are enriched in cancer pathways. In addition,H3.3K36M chondrocytes exhibit several hallmarks of cancer cells, including increased abilityto form colonies, resistance to apoptosis, and defects in differentiation.Thus, H3.3K36Mproteins reprogram the H3K36 methylation landscape and contribute to tumorigenesis,in part through altering the expression of cancer-associated genes.

Chondroblastomas are locally recurrent pri-mary bone tumors (1). Recently, it has beenreported that one allele of theH3F3B gene,one of two genes encoding histone H3 var-iant H3.3 (2, 3), is frequently mutated in

chondroblastoma (4). In addition, global reduc-tions of di- and trimethylation of histone H3 atlysine-36 (H3K36me2 and H3K36me3) of endog-enoushistoneH3 inmammalian cells exogenouslyexpressing the H3.3 Lys36→Met36 (H3.3K36M)mutant protein have been observed (5, 6). How-ever, the mechanism by which the mutant pro-teins exert their effects on H3K36 methylation

of endogenous histones and how the H3.3K36Mmutation promotes tumorigenesis of this poorlystudied tumor are largely unknown.We used H3K36me2- and H3K36me3-specific

antibodies (fig. S1) to analyze the levels ofH3K36me2and H3K36me3 in three primary human chon-droblastomas harboring the H3.3K36Mmutationand in three giant cell tumorswith theH3.3G34W(Gly34→Trp34) mutation (table S1). H3K36me2and H3K36me3 were globally reduced in eachchondroblastoma specimen, but not in giant celltumors or normal bone tissues (Fig. 1A).We used the CRISPR/Cas9 system (7) to intro-

duce the H3.3K36M mutation into one H3F3Ballele (fig. S2) of T/C28a2 cells, which are im-mortalized human chondrocytes (8). The levelsof H3K36me1, H3K36me2, and H3K36me3 werereduced in two independent mutant cell lines, ascompared with methylation levels in parental T/C28a2 cells (Fig. 1B and fig. S3). As determinedby mass spectrometric analysis, H3K36me2 wasreducedmore substantially thanH3K36me3 (fig.S3, A and B). No apparent changes were observedinH3K4me3,H3K9me3,H3K27me3, orH4K20me3(Fig. 1B). These results demonstrate that the glo-bal reduction of H3K36 methylation in tumortissues results from the expression of H3.3K36Mmutant proteins.To understand how H3.3K36M mutant pro-

teins globally reduce H3K36 methylation, we firsttested the ability of aH3.3K36Mpeptide to inhibitthe enzymatic activities of four human H3K36methyltransferases—SETD2, ASH1L, MMSET/WHSC1, and NSD1—which catalyze H3K36me1,H3K36me2, andH3K36me3 (9–11). The purifiedcatalytic domains of each enzyme exhibitedmethyl-transferaseactivitiesagainstH3.3-containingmono-nucleosomes. TheH3.3K36Mpeptide inhibited the

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1Department of Biochemistry and Molecular Biology, MayoClinic College of Medicine, 200 First Street SW, Rochester,MN 55905, USA. 2Epigenomics Program, Center ofIndividualized Medicine, Mayo Clinic College of Medicine, 200First Street SW, Rochester, MN 55905, USA. 3Department ofOrthopedic Surgery, Mayo Clinic College of Medicine, 200First Street SW, Rochester, MN 55905, USA. 4Department ofBiochemistry, Molecular Biology, and Biophysics, Universityof Minnesota at Twin Cities, Minneapolis, MN 55455, USA.5National Laboratory of Biomacromolecules, Institute ofBiophysics, Chinese Academy of Sciences, 5 Datun Road,Beijing 100101, China. 6University of Chinese Academy ofSciences, 19A Yuquan Road, Beijing 100049, China. 7Divisionof Hematology/Oncology, Mayo Clinic Arizona, 13400 EastShea B., Scottsdale, AZ 85259, USA. 8Robert J. TomsichPathology and Laboratory Medicine Institute and Departmentof Cancer Biology, Cleveland Clinic and Lerner ResearchInstitute, L2 9500 Euclid Avenue, Cleveland, OH 44195, USA.9Departments of Pathology and Microbiology, Pediatrics, andOrthopaedic Surgery and Rehabilitation. 10Department ofLaboratory Medicine and Pathology, Mayo Clinic College ofMedicine, 200 First Street SW, Rochester, MN 55905, USA.11Department of Physiology and Biomedical Engineering,Division of Gastroenterology and Hepatology, Mayo ClinicCollege of Medicine, 200 First Street SW, Rochester, MN55905, USA. 12Interdisciplinary Health Science Initiative, 1110Micro and Nanotechnology Laboratory, M/C 249, Universityof Illinois Urbana-Champaign, Urbana, IL 61801, USA.*These authors contributed equally to this work. †Correspondingauthor. Email: zhang.zhiguo@mayo.edu

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activities of MMSET [median inhibitory con-centration (IC50)=67mM]andSETD2(IC50=39mM)in a dose-dependent manner, as compared withthe wild-type (WT) H3 peptide (Fig. 1C and fig.S4A). Moreover, H3.3K36M-containing mono-nucleosomes inhibited the enzymatic activities ofMMSET and SETD2 (Fig. 1D). In contrast, neitherthe H3.3K36M peptide nor the H3.3K36Mmono-nucleosomes exhibited an inhibitory effect onthe activities of ASH1L and NSD1 in vitro (fig.S4). We also observed that MMSET—but notASH1L, NSD1, or two subunits of the H3K27methyltransferase complex PRC2 (Ezh2 andSuz12)—was enriched in H3.3K36M-containingmononucleosomes compared with WT H3.3-containing mononucleosomes (Fig. 1E). Finally,in a peptide pull-down assay,MMSETandSETD2bound to the H3.3K36M peptide more efficientlyunder higher-salt conditions than to the corre-sponding normal H3 peptide (fig. S5A). Theseresults indicate that the H3.3K36M mutant pro-tein inhibits at least two mammalian H3K36methyltransferases, MMSET and SETD2.

We used chromatin immunoprecipitationcoupled with next-generation sequencing (ChIP-seq) (12) to identify 29,250 and 13,694 H3K36me2peaks in T/C28a2 cells and H3.3K36M lines, re-spectively (Fig. 2, A and B). H3K36me2 peakspresent in only the H3.3K36M cells were signi-ficantly enriched at promoters, gene bodies, andtranscription end sites (TESs) ± 2 kilo–base pairs(kbp) (P < 0.01) but showed significant depletionat intergenic regions (P < 0.01), as compared withH3K36me2 peaks in T/C28a2 cells (Fig. 2, A to C).On average, levels of H3K36me2 in intergenicregions (Fig. 2D) and gene bodies (Fig. 2E) werereduced in each of the H3.3K36M mutant linescompared with the WT T/C28a2 cells. The reduc-tion of H3K36me2 in four selected genes andtwo intergenic regions was confirmed by ChIP–polymerase chain reaction (PCR) (Fig. 2F).Depletion of SETD2 had no apparent effect on

H3K36me2. In contrast, depletion of MMSETalone or in combination with SETD2 resulted inamarked reduction of H3K36me2, but to a lesserextent than in cells expressing H3.3K36M, as de-

termined from Western blot analysis (fig. S5, Band C). Depletion of SETD2 or MMSET did notaffect the expression of the three other H3K36methyltransferases that we tested (fig. S5D). Fi-nally, H3K36me2 ChIP-PCR andChIP-seq resultsshow that depletion of MMSET alone or in com-bination with SETD2, but not SETD2 alone, ledto reduction of H3K36me2 in gene bodies andintergenic regions but to a lesser extent than inH3.3K36M mutant cells (Fig. 2G and fig. S5, Eand F). Together, these results support the ideathat the reduction of H3K36me2 in H3.3K36Mcells is mediated, at least in part, through theinhibition ofMMSET by theH3.3K36Mproteins.Similar numbers of H3K36me3 ChIP-seq peaks

were detected in gene bodies from T/C28a2 cellsand H3.3K36Mmutant cells (fig. S6A). However,the amount of H3K36me3 throughout the genebodies was reduced in each of the two H3.3K36Mmutant lines compared with the T/C28a2 cells(Fig. 2H). Additionally, more than 60% of geneswith reduced levels of H3K36me3 also exhibitedreduced H3K36me2 (fig. S6B). The reduction

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Fig. 1. H3K36 methylation is reduced in tumors and cells containing theH3.3K36M mutation. (A and B) H3K36me2 and H3K36me3 levels are re-duced in chondroblastoma tumor samples (A) and in chondrocyte cell linescontaining the H3.3K36M mutation (B). (C) Enzymatic activity of the H3K36methyltransferases MMSETand SETD2 was measured using H3.3-containingrecombinant mononucleosomes in the presence of increasing amounts ofH3.3K36M peptide or its corresponding H3.3 peptide. Data are mean ± SD (N =3 independent replicates). c.p.m., counts per minute. (D) H3.3K36M mono-

nucleosomes inhibit the enzymatic activities of MMSET (top) and SETD2(bottom) in vitro. (E) MMSET is enriched in H3.3K36M-containing mono-nucleosomes compared with H3.3 WTnucleosomes. Mononucleosomes werepurified from human embryonic kidney 293 T (HEK293T) cells (negativecontrol), HEK293Tcells expressing FLAG-tagged WT H3.3, or the H3.3K36Mmutant. Proteins from input and immunoprecipitated (IP) samples wereanalyzed by Western blotting using the indicated antibodies. The intensity ofeach blot was quantified, with blots of both input and IP in WTcells set as 1.0.

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of H3K36me3 is correlated with the reduction ofH3K36me2 (fig. S6C), which suggests that bothH3K36me2 and H3K36me3 are altered to a sim-ilar degree within gene bodies of a large fractionof genes. ChIP-PCR experiments confirmed thereduction of H3K36me3 in gene bodies of fourselected genes in two H3.3K36M mutant lines(fig. S6D). Finally, depletion of SETD2andMMSETreduced levels of H3K36me3 in gene bodies, withMMSET depletion having a lesser effect thanSETD2 depletion (figs. S5, B and C, and S6, E andF). These results indicate that reduced H3K36me3levels in gene bodies of H3.3K36Mmutant chon-drocytes are mainly due to SETD2 inhibition, al-though MMSET inhibition may also contribute.We also used ChIP-seq to analyze H3K36me2

and H3K36me3 levels in primary chondroblas-tomas. The H3K36me2 and H3K36me3 chroma-tin occupancy in gene bodies was reduced in thetwo chondroblastoma samples that we analyzed(fig. S7, A and B). Gene set enrichment analysis(GSEA) (13) indicated that genes with reducedlevels ofH3K36me2 andH3K36me3 in gene bodiesin chondrocyte cell lines were also enriched inthe corresponding gene sets from tumor samples(fig. S7, C and D). These results indicate thatH3.3K36M mutant proteins have similar effectson the reduction of H3K36me2 and H3K36me3in gene bodies in both primary chondroblastomasamples and chondrocyte cell lines containingthe same H3.3K36M mutation.

We used H3.3K36M-specific antibodies (fig.S8A) and three different assays [Western blot,immunofluorescence (IF), and ChIP-seq] to deter-mine howH3.3K36Mmutant proteins associatewith chromatin. Themajority of the H3.3K36Mmutant proteins were detected on chromatin,similarly to WT H3, as evidenced by chromatinfractionation and IF assays (fig. S8, B and C).H3.3K36M ChIP-seq identified 6162 overlappingpeaks between twoH3.3K36Mmutant lines (Fig.3A). H3.3K36M peaks were significantly enrichedat promoters, gene bodies, and TESs ± 2 kbp (P <0.01) but exhibited significant depletion (P < 0.01)at intergenic regions, as compared with randomlyshuffled peaks of the same length (Fig. 3B). Twoof these peaks were confirmed using ChIP-PCR(Fig. 3C). The levels of H3.3K36M mutant pro-teins correlated with gene expression levels (Fig.3D). These results indicate that chromatin local-ization of H3.3K36M proteins is similar to thatof WT H3.3 observed in other cell lines (14, 15).Furthermore, the levels of H3K36M mutant pro-teinswerehigher in geneswith reducedH3K36me2orH3K36me3 levels than in thosewithout changesin H3K36me2 and H3K36me3 (Fig. 3E and fig.S9A). Conversely, the levels of H3K36me2 andH3K36me3 were lower within H3.3K36M peakregions than in the surrounding regions (Fig. 3Fand fig. S9, B and C). The inverse relationshipbetween the levels of H3.3K36Mmutant proteinsand levels of H3K36me2 andH3K36me3 on chro-

matin lends support to the idea that the reductionof H3K36me2 and H3K36me3 is at least partlyattributable to the inhibitionofMMSETandSETD2byH3.3K36Mmutant proteins incorporated intothe chromatin.Gene expression analysis using RNA-seq in-

dicated that the expression of 567 and 799 geneswas elevated and reduced, respectively, in twoH3.3K36M mutant cell lines compared with pa-rental T/C28a2 cells (fig. S10A). In addition, theexpression of intergenic regions with reduced lev-els of H3K36me2 was lower in H3.3K36M cellsthan in WT cells (fig. S10B). GSEA analysis in-dicated that genes with reduced expression inH3.3K36Mmutant chondrocyte lines were signif-icantly enriched among genes with reduced ex-pression in chondroblastoma samples, whereasgenes with increased expressionwere not enriched(fig. S10, C and D). A lack of correlation of geneswith increased expression between H3.3K36Mmutant lines and chondroblastoma samples wasprobably due to heterogeneity of chondroblas-toma samples (table S1). The correlation of geneswith reduced expression between the H3.3K36Mmutant chondrocyte lines and chondroblastomadata sets suggests commonmechanisms linked toH3.3K36M expression and the loss ofH3K36me2and H3K36me3. On the basis of our analysis ofa limited number of gene loci, incorporation ofH3.3K36M mutant proteins into nucleosomesoccurs immediately prior to or simultaneouslywith

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Fig. 2. H3.3K36M re-programs chromatin-bound H3K36me2and H3K36me3.(A) IntegrativeGenomics Viewertracks showingH3K36me2 andH3K36me3 distributionon chromosome 5(chr5) in WT (blue) andtwo H3.3K36M (K36M)cell lines (red). (B) Venndiagram illustration ofH3K36me2 peaks inWTand two H3.3K36Mcell lines and theiroverlap. (C) Genomicdistribution ofH3K36me2 peaksunique to WTcells(23214), peaks sharedby WTand H3.3K36Mcells (6036), and peaksunique to H3.3K36Mcells (7658). (D and E)Normalized tagdistribution profiles ofH3K36me2 for inter-genic regions [(D), *P <0.01] and gene bodies (E) in WTand K36M cell lines. RRPM, reference-adjusted reads per million; TSS, transcription start site. (F) Analysis of H3K36me2 at fourgene loci and two intergenic regions using ChIP-PCR in two H3.3K36M mutant lines. Error bars indicate SD from three independent replicates. (G) Depletion ofMMSET, but not SETD2, reduced H3K36me2 at four gene loci and two intergenic regions. Data are mean ± SD (N = 3 independent replicates, *P < 0.05, **P <0.01). NT, nontarget control. (H) H3K36me3 levels in gene bodies are reduced in two H3.3K36M chondrocyte lines.

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Fig. 3. H3K36M levels in chromatin are inversely correlated with levelsof H3K36me2 and H3K36me3. (A) Venn diagram showing H3K36M ChIP-seqpeaks identified in WTand two H3K36M cell lines. (B) Most H3.3K36M ChIP-seqpeaks are in gene bodies and intergenic regions. (C) Validation ofH3.3K36Mpeaksby ChIP-PCR in H3.3K36M mutant lines. Error bars indicate SD from three inde-pendent replicates. (D)Normalized readdensityofH3K36MChIP-seq signals from

10 kb upstream of the TSS to 10 kb downstream of the TES.Genes were split intofive groups,with 0 to 20% representing geneswith the highest expression. RPKM,readsper kilobasepermillionmapped reads. (E)H3K36MChIP-seq readdensitiesin H3.3K36M cell lines for genes with or without significantly reducedH3K36me2.(F) Genome browser track examples of the occupancy profiles of H3K36me2 andH3.3K36M in chromosome 12 (chr12).The green bar indicates H3.3K36M peaks.

Fig. 4. H3.3K36M alters theH3K36 methylation landscape andexpression of genes linked tocarcinogenesis. (A) Log2 foldoccupancy changes in H3K36me3(left) and H3K36me2 (right) werecalculated for H3.3K36M cells versusWT cells and plotted relative tocorresponding fold changes in mRNAdetected by RNA-seq. Genes withsignificantly changed H3K36me3 orH3K36me2 in gene bodies (P < 10−5)in both cell lines were chosen foranalysis. Each dot represents asingle gene. R, correlation coefficient.(B) H3.3K36M increases colonyformation of T/C 28a2 cells.Representative images are shown atthe top. (C) Annexin V–positive cellswere analyzed and quantified byfluorescence-activated cell sorting.DMSO, dimethyl sulfoxide. (D)Expression of BMP2 was analyzed byreal-time PCR during differentiationconditions and normalized againstglyceraldehyde-3-phosphatedehydrogenase. (E and F) H3K36me2and H3K36me3 ChIP assays in cellsdifferentiated for 0, 3, or 7 days. ChIPDNA was analyzed using primersamplifying the BMP2 gene body. Foldenrichment relative to T/C28a2 cells atthe start of differentiation was calculated. Results in (B) to (F) represent the mean ± SD (N = 3 independent replicates, **P < 0.01, ***P < 0.001).

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the reduction of H3K36me2 and H3K36me3 andalters gene expression (fig. S11, A and B). Finally,we observed that genes with reduced levels ofH3K36me2 or H3K36me3 within gene bodies ex-hibited a significant correlation with changes ingene expression in both cell lines (Fig. 4A) and inchondroblastoma samples (fig. S11C). These re-sults suggest that H3K36me2 and H3K36me3levels are associated with changes in gene expres-sion in both cell lines and tumors. It is known thatH3K36me2andH3K36me3antagonizeH3K27me3(12, 16), raising the possibility that changes in theexpressionof somegenes inH3.3K36Mmutant cellsmay also be linked to deregulation of H3K27me3-repressed genes.We performed Ingenuity Pathway Analysis (IPA)

using three gene sets with altered occupancy ofH3K36me2 (1143 genes) and H3K36me3 (1359genes) and altered gene expression (598 genes)in both chondrocytes and chondroblastoma tumorsamples. Genes associated with the “MolecularMechanisms of Cancer” IPA canonical pathwaywere highly enriched in all three data sets (fig.S12A). Several genes assigned to the “MolecularMechanisms of Cancer” pathway are known tobe involved in DNA repair, differentiation, andapoptosis (fig. S12 and table S2). We investigatedwhether H3.3K36Mmutant chondrocytes displaycancer-associated cellular phenotypes, includingDNA repair, inwhichH3K36me3has a role (17–19).As determinedbyMTTassay, theH3.3K36Mmuta-tions did not affect proliferation of chondrocytecell lines (fig. S13A) but increased the ability ofthese cells to form colonies (Fig. 4B). Moreover,H3.3K36M mutant chondrocyte cells were lesssensitive to staurosporine-induced apoptosis (Fig.4C) and formed densermicromasses when placedinto chondrocyte differentiationmedium (fig. S13B).The effect ofH3.3K36Mmutation on staurosporine-induced apoptosis could be detected at the sametime when H3.3K36M mutant proteins were in-corporated into chromatin (fig. S13C). Finally, the

H3.3K36M mutant cells were also defective inhomologous recombination (fig. S13D) but hadno apparent defects in nonhomologous end join-ing or mismatch repair (fig. S13, E and F). Thus,H3.3K36M mutant cells exhibit several cancer-associated cellular phenotypes.Consistent with differentiation defects, expres-

sion of BMP2 and other genes that regulate chon-drocyte differentiation (20)was reduced inH3.3K36Mcell lines, as determined by analysis of RNA-seqresults (fig. S14A). In the micromass assays, BMP(bonemorphogenetic protein) signaling is requiredfor hypertrophic chondrocyte differentiation(21). The mRNA levels of BMP2 and SOX9 werereduced inmicromass cultures of T/C28a2 cellsexpressing the H3.3K36M mutant protein, ascompared with the parental cell line (Figs. 4Dand fig. S14B). Consistent with the reduction ingene expression, ChIP-PCR results showed thatH3K36me2 and H3K36me3 occupancy of BMP2was reduced during differentiation (Fig. 4, E andF). In addition to genes involved in differentiation(BMP2 and RUNX2), the expression of two genesinvolved in homologous recombination (BRCA1and ATR) was reduced inH3.3K36Mmutant lines,as well as in MMSET- and SETD2-depleted cells(fig. S14, C and D). Thus, the cancer-associatedcellular phenotypes observed inH3.3K36Mmutantcells are caused, at least in part, by alterations inH3K36methylation and expression of key genesthat regulate oncogenesis and chondrogenesis.Our results show that H3.3K36M mutant pro-

teins are incorporated into chromatin in amannersimilar to incorporation ofWTH3.3 and that thesemutant proteins inhibit, at minimum, MMSETand SETD2 to reduce H3K36methylation.More-over, H3.3K36Mmutant proteins affect variousH3K36 methyltransferases differently and pro-mote chondroblastoma tumorigenesis, probablythrough alterations of several cancer-related proc-esses including colony formation, apoptosis, andchondrocytic differentiation.

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ACKNOWLEDGMENTS

We thank M. Goldring for T/C28a2 cells and Z. Lou for plasmids,B. Eckloff and T. Wood for sequencing, and B. Madden for massspectrometry analysis. These studies were supported by the NIH[grants GM81838 and CA157489 (Z.Z.), AR68103 (J.J.W.),AR65397 (E.W.B.), AR049069 (A.J.v.W.), and DK58185 (T.O.)],the Natural Science Foundation of China [grant 31210103914(R.-M.X.)], and the Epigenomics Program (Center for IndividualizedMedicine) of the Mayo Clinic. ChIP-seq and RNA-seq data setshave been deposited into the Gene Expression Omnibus underaccession number GSE7523.

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6 December 2015; accepted 16 May 2016Published online 26 May 201610.1126/science.aae0065

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The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas

ZhangThibodeau, Tamas Ordog, Yue Chen, Andre J. van Wijnen, Andre M. Oliveira, Rui-Ming Xu, Jennifer J. Westendorf and ZhiguoJinglong Wang, Honglian Zhang, Na Yang, Elizabeth W. Bradley, Thai H. Ho, Brian P. Rubin, Julia A. Bridge, Stephen N. Dong Fang, Haiyun Gan, Jeong-Heon Lee, Jing Han, Zhiquan Wang, Scott M. Riester, Long Jin, Jianji Chen, Hui Zhou,

originally published online May 26, 2016DOI: 10.1126/science.aae0065 (6291), 1344-1348.352Science 

, this issue p. 1344Scienceknown cancer-related genes and impart cancer-related characteristics to the chondrocyte cells.with the enzymes that normally methylate this residue. The altered chromatin methylation patterns alter the expression ofmutant histones inhibit the normal methylation of this same residue in wild-type H3 histones. They do so by interfering

show that the K36Met al.methionine (K36M) ''oncohistone'' mutation is seen in almost all chondroblastomas. Fang −to−Mutations in the chromatin protein histone H3 are found in a number of pediatric cancers. The lysine-36

A cancer-promoting histone protein

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REFERENCES

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