LETTERdoi:10.1038/nature13045

ZMYND11 links histone H3.3K36me3 to transcriptionelongation and tumour suppressionHong Wen1,2*, Yuanyuan Li3,4*, Yuanxin Xi5*, Shiming Jiang1, Sabrina Stratton1, Danni Peng1, Kaori Tanaka1, Yongfeng Ren3,4,Zheng Xia5, Jun Wu6, Bing Li6, Michelle C. Barton1,2,7, Wei Li5, Haitao Li3,4 & Xiaobing Shi1,2,7

Recognition of modified histones by ‘reader’ proteins plays a crit-ical role in the regulation of chromatin1. H3K36 trimethylation(H3K36me3) is deposited onto the nucleosomes in the transcribedregions after RNA polymerase II elongation. In yeast, this mark inturn recruits epigenetic regulators to reset the chromatin to a relativelyrepressive state, thus suppressing cryptic transcription2. However,much less is known about the role of H3K36me3 in transcription regu-lation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies3.Here we show that the candidate tumour suppressor ZMYND11specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and reg-ulates RNA polymerase II elongation. Structural studies show thatin addition to the trimethyl-lysine binding by an aromatic cage withinthe PWWP domain, the H3.3-dependent recognition is mediated bythe encapsulation of the H3.3-specific ‘Ser 31’ residue in a compositepocket formed by the tandem bromo–PWWP domains of ZMYND11.Chromatin immunoprecipitation followed by sequencing shows agenome-wide co-localization of ZMYND11 with H3K36me3 andH3.3 in gene bodies, and its occupancy requires the pre-depositionof H3.3K36me3. Although ZMYND11 is associated with highlyexpressed genes, it functions as an unconventional transcription co-repressor by modulating RNA polymerase II at the elongation stage.ZMYND11 is critical for the repression of a transcriptional pro-gram that is essential for tumour cell growth; low expression levelsof ZMYND11 in breast cancer patients correlate with worse prognosis.Consistently, overexpression of ZMYND11 suppresses cancer cellgrowth in vitro and tumour formation in mice. Together, this studyidentifies ZMYND11 as an H3.3-specific reader of H3K36me3 thatlinks the histone-variant-mediated transcription elongation controlto tumour suppression.

ZMYND11(also known as BS69) is an adenovirus early region 1A (E1A)-interacting protein that functions as a co-repressor of E1A and cellulartranscription factors including the c-Myb and ETS-2 oncoproteins4–6.It contains several histone ‘reader’ modules, including a plant homeo-domain (PHD), a bromodomain and a PWWP domain (Fig. 1a), indi-cating a role in chromatin regulation. To determine whether these domainscan recognize histone modification(s), we used a histone peptide array(Supplementary Table 1) and found that the recombinant PHD–bromo–PWWP (PBP) domains specifically bound H3K36me3 peptides (Fig. 1b),which was also confirmed by peptide pull-down assays (Fig. 1c). Inaddition, ZMYND11 PBP also bound H3K36me3 on bulk histones andthe reconstituted H3K36-specific methyl-lysine analogue-containingnucleosomes (Extended Data Fig. 1a, b). Several PWWP domains canrecognize methylation on histones H3K36 or H3K79 (refs 7–9); thereforewe asked whether ZMYND11 PWWP alone was able to bind H3K36me3.The results showed that neither the isolated PWWP nor the PHD andbromodomain bound H3K36me3, whereas the tandem bromo–PWWP

retained a weak interaction (Fig. 1d and Extended Data Fig. 1c). Iso-thermal titration calorimetry (ITC) showed a dissociation constant (Kd)of 56mM for PBP and 71mM for bromo–PWWP to the H3K36me3peptide, whereas weak or no binding to the di-, mono- and unmethylatedH3K36 peptides (Fig. 1e and Extended Data Fig. 1d). Furthermore,deletion of the PWWP, bromo or PHD domains greatly diminishedfull-length ZMYND11 binding to H3K36me3 peptide and chromatin,whereas deletion of the MYND domain had no effect (Extended DataFig. 1e, f).

To gain molecular insights into the reader function of ZMYND11,we solved the crystal structure of the tandem bromo–PWWP domain inits free state and in complex with H3K36me3 peptide at 1.95 A and 2.0 A,respectively (Extended Data Table 1 and Supplementary Discussion).Successful crystallization of the bromo–PWWP–H3K36me3 complexwas facilitated by alanine mutation of D234 and E236 within the bro-modomain to disrupt unfavourable packing contacts without affect-ing protein folding and histone binding (Extended Data Fig. 2a–e). Thetandem bromo–PWWP domain adopts a V-shaped structure impartedby a ‘kink’ around a previously uncharacterized zinc finger (ZnF) motif(Fig. 1f, g). Several structural features imply that ZMYND11 bromo-domain is unlikely to be a histone acetyl-lysine-binding module (ExtendedData Fig. 2f–l). The PWWP domain adopts a five-bladed b-barrel foldwith an extended carboxy (C)-terminal a-helix (aP), with a conservedH3K36me3-binding aromatic cage7–9 formed by F291 and W294 oftheb1–b2 loop and F310 of theb3–b4 loop (Fig. 1h and Extended DataFig. 3a–c). Alanine mutations abrogated ZMYND11 binding to theH3K36me3 peptide and chromatin without affecting the overall proteinfolding (Figs 1i, 2i and Extended Data Fig. 3d–f). Despite extensiveinteractions observed in crystals for the histone peptide, the relativelyweak binding affinity suggests that an alternative binding mechanismis plausible. Indeed, the PWWP domain was previously identified as aDNA-binding motif10. Consistent with a recent report11, we found thatZMYND11 bromo–PWWP bound to DNA at low micromolar level(Extended Data Fig. 3g). The higher binding affinity for DNA than thatfor histone peptides is in agreement with observations for some otherPWWP domains12,13. Although not detected in our pull-downs usingpeptides and oligonucleotides, all these features suggest that a synergybetween histone and DNA may contribute to ZMYND11 binding atthe nucleosomal level (Supplementary Discussion).

The H3K36me3 peptides used in the biochemical and structuralstudies were histone H3.3-based peptides (Fig. 2a). The histone variantH3.3 possesses a signature motif ‘S31 … A87AIG90’ that is distinct fromthe ‘A31 … S87AVM90’ sequence signature of the canonical histoneH3.1/2 (ref. 3). Our structure showed that the H3.3-specific ‘S31’ res-idue and its neighbouring T32 were encapsulated at the bottom of the‘bromo–ZnF–PWWP’ valley (Fig. 2b), contributing to the ZMYND11–H3 interactions in addition to the binding of K36me3 by the PWWP

*These authors contributed equally to this work.

1Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 2Center for Cancer Epigenetics, Center for Genetics and Genomics,and Center for Stem Cell and Developmental Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. 3MOE Key Laboratory of Protein Sciences, Center for StructuralBiology, School of Life Sciences, Tsinghua University, Beijing 100084, China. 4Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China. 5Dan L. DuncanCancer Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA. 6Department of Molecular Biology, The University of Texas Southwestern MedicalCenter, Dallas, Texas 75390, USA. 7Genes and Development Graduate Program, The University of Texas Graduate School of Biomedical Sciences, Houston, Teaxs 77030, USA.

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aromatic cage. Indeed, the binding of ZMYND11 to H3.3K36me3 wasmuch stronger than its binding to the H3.1 counterpart, with a seven-to eightfold difference in binding affinity (Fig. 2c, d and Extended DataFig. 4a, b). Furthermore, Flag-tagged histone H3.3, but not H3.1, stronglyco-immunoprecipitated ZMYND11 in cells (Extended Data Fig. 4c).Together, these results indicate that the H3.3-specific S31 residue is indis-pensable for ZMYND11–H3K36me3 interaction.

We alsosolved the structure of ZMYND11bromo–PWWP–H3.1K36me3complex at 2.3 A for comparison (Extended Data Table 1 and ExtendedData Fig. 4d). Several hydrogen-bonding interactions around the ‘A29–G33’ segment in the bromo–PWWP–H3.3 complex were lost in thebromo–PWWP–H3.1 complex after the ‘Ser to Ala’ conversion at position

31 (Fig. 2e, f and Extended data Fig. 4e, f). Superimposition of the complexstructures showed a conformational adjustment of the H3.1 ‘A29–G33’segment (Fig. 2e), indicating a non-ideal encapsulation. As highlightedin the LIGPLOT diagram14, eight direct hydrogen bonds and two setsof water-mediated hydrogen bonding were detected upon H3.3 variantbinding (Fig. 2g), whereas only three direct hydrogen bonds and twosets of water-mediated hydrogen bonding were observed for H3.1 recog-nition (Fig. 2h). Notably, residues from all three modules, bromo, ZnFand PWWP, contributed to the A29–G33 segment recognition throughthe formation of a composite pocket, calling attention to the theme of‘module integration’ for extended reader function15. Unique hydrogen-bonding interactions for the H3.3-specific readout were particularly

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Figure 1 | Molecular basis for H3.3K36me3 recognition by ZMYND11bromo–PWWP domains. a, Schematic representation of ZMYND11 proteinstructure. b, Histone peptide microarray (details in Supplementary Table 1)probed with glutathione S-transferase (GST)–ZMYND11 PBP domains.c, d, Western blot analysis of histone peptide pull-downs with GST–ZMYND11PBP (c) or individual domains (d) and the indicated biotinylated peptides; aa,amino acids. e, ITC curves of histone H3.3K36me peptides titrated intoZMYND11 PBP domains; ND, not detectable. f, Cartoon view of ZMYND11bromo–PWWP domains in the free state. g, Structure of ZMYND11 bromo–PWWP in complex with H3.3K36me3 peptide. Bromo–PWWP is shown ascharge distribution surface. Blue, positive charges; red, negative charges. The

2Fo 2 Fc omit maps around H3 peptide, polyethylene glycol (PEG) andphosphate (PO4) are shown as green mesh contoured at the 1s level. h, Detailsof the K36me3 recognition by the PWWP domain. Close-up views: left, thehydrogen-bonding network around G34–H39; right, conformationaladjustment of the PWWP aromatic cage upon H3K36me3 peptide binding.ZMYND11 bromo–PWWP in the free state is shown in light grey and bromo–PWWP in the complex state in dark blue; histone peptide is shown in yellow;small red balls indicate water. i, Western blot analysis of peptide pull-downswith GST–PBP or mutants and the indicated peptides. All the H3K36 peptidesused in these experiments were H3.3-based.

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supplied by ‘R168–A29’, ‘E251–S31’, ‘E254–T32’ and ‘N266–S31’ ofthe ZMYND11–H3.3 pair along with an adaptive flipping of the R168side chain (Fig. 2e, g), and mutations diminished the bindings (Fig. 2iand Extended Data Fig. 4g). Together, the results strongly support a roleof ZMYND11 as an H3.3-specific reader of H3K36me3.

We next sought to determine whether ZMYND11 co-localizes withH3K36me3 and the H3.3 variant in the genome by chromatin immuno-precipitation coupled with high-throughput sequencing (ChIP-seq)analysis. We identified 9,759 ZMYND11 peaks distributed in 3,353genes in human osteosarcoma U2OS cells (Supplementary Table 2). Thepeaks were highly enriched in introns and exons (herein defined as thegene body) but not promoters (Fig. 3a). Strikingly, the average distri-bution of all ZMYND11-bound peaks recapitulated the genomic dis-tribution of H3K36me3 (ref. 16) (Fig. 3b and Supplementary Table 3):absent from the transcription start site (TSS), but gradually increasingin the gene body towards the 39 end and peaking at the transcriptiontermination site (TTS).

We also determined the genome-wide distribution of histone variantH3.3 by ChIP-seq analysis of Flag–H3.3 stably expressed in U2OS cells

(Extended Data Fig. 5a and Supplementary Table 4). In accordance withprevious observations17–19, H3.3 was distributed in both the promoterand the body of active genes (Extended Data Fig. 5b). The gene-body-associated H3.3 showed a strong co-occupancy with H3K36me3(Fig. 3c). Two-thirds of all the ZMYND11-bound genes overlappedwith H3K36me3 and H3.3-co-occupied genes (P , 1 3 102465) (Fig. 3c),as also represented by a genome-browser view of the ChIP-seq signals(Fig. 3d). High ZMYND11 occupancies were observed in genes enrichedwith both H3K36me3 and H3.3, whereas ZMYND11 occupancies weremuch lower in genes only decorated with H3K36me3 or H3.3 (Fig. 3e–g).Depletion of ZMYND11 by short hairpin RNAs (shRNAs) drasticallydiminished the ChIP-seq signals (Extended Data Fig. 5c–g), suggestingthat the ChIP data obtained using our ZMYND11 antibody reflect theendogenous ZMYND11 chromatin-binding pattern.

ZMYND11 densities in gene bodies were directly correlated withH3K36me3 levels (Extended Data Fig. 6a). We then asked whetherZMYND11 occupancy was H3K36me3 dependent. We found thatZMYND11 binding on target genes was reduced along with diminishedH3K36me3 levels in the SETD2 (ref. 20) knockdown cells (Fig. 3h and

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Figure 2 | H3.3 variant-specific K36me3 recognition by ZMYND11.a, Sequence alignment of H3.3 and H3.1 spanning amino acids 19–42.The yellow line denotes the visible segment in the complex structures.b, Encapsulation of the ‘S31–T32’ segment and ‘K36me3’ by the paired bromo(pale green), ZnF (salmon) and PWWP (blue) modules of ZMYND11 renderedas solvent-accessible surface. The ‘S31–T32’ segment, K36me3 and two water(Wat) molecules are highlighted in spacing-filling mode. PO4, phosphate.c, Western blot analysis of peptide pull-downs with ZMYND11 PBP and theH3.3 or H3.1 variant peptides bearing K36me. d, ITC curves of H3.3K36me3and H3.1K36me3 peptides titrated into ZMYND11 PBP or bromo–PWWP(BP) domains. e, f, Recognition of the ‘A29–G33’ segment of H3.3 (e) or H3.1(f) by ZMYND11 at the junction of bromo (pale green), ZnF (salmon) and

PWWP (blue). Panel e is prepared in stereo view with H3.1 complex (grey)superimposed for comparison. Histone peptides are highlighted as yellowsticks. Hydrogen-bonding interactions are shown as dashes with cyan for thosebetween H3 peptide and bromo–PWWP, and magenta for those within H3peptide or bromo–PWWP. Small red balls, waters. g, h, LIGPLOT diagramsshowing critical contacts between ZMYND11 bromo–PWWP and the‘A29-G33’ segment of H3.3 (g) and H3.1 (h). H3 segment and key hydrogen-bonding residues from ZMYND11 bromo–PWWP are depicted in ball-and-stick mode. Cyan circle, water; blue dashed line, hydrogen bonds; curved brush,hydrophobic contact. i, ITC curves of ZMYND11 bromo–PWWP domains andthe indicated point mutants with the H3.3K36me3 peptide.

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Extended Data Fig. 6b–d), but not in the NSD2 (an H3K36me2 methyl-transferase)21 knockdown cells where the H3K36me3 levels remainedlargely unchanged (Extended Data Fig. 6e, f). Furthermore, the H3.3K36me3recognition-deficient mutants of ZMYND11 were severely impaired inbinding on target genes (Fig. 3i and Extended Data Fig. 6g, h). Takentogether, these results strongly suggest that ZMYND11 co-localizeswith H3K36me3 and H3.3 in the gene body, and its occupancy requiresthe pre-deposition of trimethylation on H3.3K36.

To identify ZMYND11-regulated genes genome-wide, we performedRNA-seq analysis in U2OS cells. We found that 268 genes were upre-gulated and 370 genes downregulated upon ZMYND11 depletion (Fig. 4a,Extended Data Fig. 7a–c and Supplementary Table 5). Gene ontologyanalysis showed that the upregulated genes in ZMYND11 knockdowncells were specifically enriched in small cell lung cancer and focal adhe-sion pathways; in contrast, the downregulated genes were not enrichedin any pathways (Fig. 4b and Supplementary Table 5). Ingenuity path-way analysis (IPA) of disease association of the upregulated genes alsoshowed a high enrichment in cancer (Fig. 4b). These data suggest thatZMYND11 has a role in both transcriptional repression and activation,but it specifically represses oncogene expression.

Because ZMYND11 is localized in the gene body, we postulated thatZMYND11 regulates gene expression at the transcription elongationrather than the initiation step. The gene body RNA polymerase II (Pol II)density correlates well with transcription elongation rate22,23; we there-fore determined the changes of Pol II density upon ZMYND11 deple-tion. ZMYND11 occupancies were enriched in genes with high Pol IIdensities (P , 1 3 102401) (Extended Data Fig. 8a, b and Supplemen-tary Table 6). Upon ZMYND11 depletion, we observed increased Pol IIoccupancy specifically on ZMYND11-repressed genes (Fig. 4c, d and

Extended Data Fig. 8c). Notably, only the amount of the intragenicPol II, especially Pol II near the 39 end of the gene body, was markedlyincreased, whereas the promoter-associated Pol II remained largelyunchanged (Fig. 4c, d). Similar increases were also observed for theelongation-specific Pol II that is phosphorylated at serine 2 (S2P)24 andthe intragenic H3K36me3 levels (Fig. 4c and Extended Data Fig. 8d, e).The phenomenon of specific increase of Pol II in the gene body wasfurther supported by the quantified Pol II travelling ratio22 (ExtendedData Fig. 8f). Together, these data suggest that ZMYND11 occupancycorrelates with Pol II density in the gene body, and ZMYND11 repressesgene expression by preventing the transition of paused Pol II to elongation.

ZMYND11 is downregulated in several human cancers includingbreast cancer (Extended Data Fig. 9a), indicating that ZMYND11 is a can-didate tumour suppressor25. Overexpression of the wild-type ZMYND11inhibited tumour cell growth, whereas the H3.3K36me3 binding-deficientmutants showed severe defects in suppressing cell proliferation andsurvival in both U2OS and the invasive MDA-MB 231 breast cancercells (Fig. 4e and Extended Data Fig. 9b–e). Furthermore, in a mousexenograft study using MDA-MB 231 cells, we found that comparedwith mice injected with control cells, tumour formation was stronglysuppressed in mice injected with cells expressing wild-type ZMYND11,whereas the H3.3K36me3-binding deficient mutant W294A was severelyimpaired in suppressing tumour growth in vivo (Fig. 4f). Interestingly,D307N, a missense mutation identified in patients with colon and rec-tum adenocarcinoma (Extended Data Fig. 9f), diminished ZMYND11binding to H3.3K36me3 (Fig. 2i and Extended Data Fig. 9g–i) and itstumour suppressor function in mice (Fig. 4f). Taken together, these resultsindicate that ZMYND11 suppresses phenotypes of cancer cells in amanner that depends on its H3.3K36me3-binding activity. Consistent

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Figure 3 | ZMYND11 co-localizes with H3K36me3 and H3.3 in the genebody. a, Genomic distribution of ZMYND11 ChIP-seq peaks in U2OS cells.The peaks are enriched in introns and exons. P , 1 3 1021208 (binomial test).b, Average genome-wide occupancies of ZMYND11 and H3K36me3 along thetranscription unit. The gene body length is aligned by percentage from the TSSto TTS. Five kilobases upstream of TSS and 5 kb downstream of TTS are alsoincluded. c, Venn diagram showing the overlap of ZMYND11-, H3K36me3-and Flag–H3.3-occupied genes. P , 1 3 102465 (three-way Fisher’s exact test).d, Genome-browser view of ChIP-seq peaks in chromosome 16p13.3 regions.TSSs are indicated by arrows. e, Heatmaps of normalized density of ChIP-seqtags in a 20 kb window centred on TTS. f, Average genome-wide ZMYND11

occupancy on genes enriched with both H3K36me3 and H3.3 (red), genes withH3K36me3 (blue) or H3.3 (green) only, or genes with no H3K36me3 and H3.3enrichment (black). Genes were aligned as described in b, and were groupedaccording to the H3K36me3 and Flag–H3.3 ChIP-seq normalized tag numbers.10 (high) or ,2 (none). g, Quantitative PCR (qPCR) analysis of ZMYND11,H3K36me3 and Flag–H3.3 ChIP. h, qPCR analysis of ZMYND11 andH3K36me3 ChIP in MYC gene body in SETD2 knockdown cells. IgG ChIP isshown as a negative control. i, qPCR analysis of Flag ChIP in the cells stablyexpressing Flag-tagged wild type (WT) or mutant ZMYND11. In g–i, all errorbars represent s.e.m. of three experiments. *P , 0.05.

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with these experimental results, low ZMYND11 expression levels inbreast cancer patients correlate with worse disease-free survivals (Fig. 4gand Extended Data 9j, k).

In summary, we have identified ZMYND11 as an H3.3-specificreader of H3K36me3. To our knowledge, it represents the first variant-specific reader of methylated histones. The combination of H3K36me3and H3.3 establishes a unique epigenetic state that defines the genomicdistribution of ZMYND11, offering a spatiotemporal control of geneexpression for both normal and neoplastic growth. Because both H3K36me3and H3.3 are deposited into the chromatin along with elongating Pol II,ZMYND11 is likely to be recruited to gene bodies only after one orseveral rounds of transcription when sufficient H3.3K36me3 is accu-mulated. We propose that, rather than as an on/off switch, ZMYND11mainly functions in ‘fine-tuning’ gene expression by modulating Pol IIelongation.

Our findings also emphasize the importance of the histone variantH3.3 in tumour suppression. Frequent mutations of the H3.3-encodingH3F3A gene have recently been identified in paediatric glioblastoma26,27.Unlike the K27M mutation in a single copy of the H3.3-encoding genethat affects the global H3K27 methylation amounts, the G34V/R muta-tions only alter the H3K36me3 levels at certain genes28,29. Interestingly,the G34V/R mutations impaired ZMYND11 binding to the H3.3K36me3peptide (Extended data Fig. 9l). It remains an open question whether dis-ruption of the binding of ZMYND11, or other yet unknown H3.3K36me3readers, has a physiological impact on the pathogenesis of paediatricglioblastoma.

METHODS SUMMARYPeptide microarray and binding assays were performed in buffer containing 50 mMTris-HCl 7.5, 300 or 200 mM NaCl, 0.1% NP-40 overnight followed by threewashes. Crystallizations of bromo–PWWP and bromo–PWWP–peptide com-plexes were performed by the hanging-drop vapour diffusion method at 18 uC.

Diffraction data were collected at synchrotron beamlines under cryoconditions.The initial phase was solved by zinc single-wavelength anomalous dispersion.

Tumour xenograft was performed by subcutaneously injecting 5 3 106 MDA-MB231 cells into nude mice. Survival analyses were performed as described previously30.Details are described in Methods.

Online Content Any additional Methods, Extended Data display items and SourceData are available in the online version of the paper; references unique to thesesections appear only in the online paper.

Received 27 January 2013; accepted 20 January 2014.

Published online 2 March 2014.

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Figure 4 | ZMYND11 modulates Pol II elongation and represses oncogeneexpression and tumour growth. a, Heatmap representation of differentiallyexpressed genes in control and ZMYND11 knockdown cells. Blue indicatesrelative low expression; yellow indicates high expression (details inSupplementary Table 5). b, KEGG and IPA pathway analyses of upregulatedgenes in ZMYND11 knockdown cells. Genes are shown as the percentage ofgenes (KEGG) or number of genes (IPA) within a functional group. Falsediscovery rate (FDR) and P values are shown at the right. c, The genomebrowser view of Pol II and Pol II-S2P ChIP-seq signals in MYC gene in controland ZMYND11 knockdown cells. TSS is indicated with arrow. d, Average Pol IIoccupancy on ZMYND11-repressed direct target genes in control (blue) andZMYND11 knockdown (red) cells. The left panel shows the entire gene

averages; the right panel shows the close-up view of the elongating Pol II nearthe TTS. P , 2.47 3 1025 (two-way unpaired Student’s t-test). e, Cellproliferation assay in MDA-MB 231 cells overexpressing wild-type ZMYND11or the indicated mutants. Cells (mean 6 s.e.m., n 5 4) were counted for 6 daysafter seeding. f, Volumes of tumours (mean 6 s.e.m., n 5 5) derived fromMDA-MB 231 cells overexpressing wild-type ZMYND11 or the indicatedmutants over 8 weeks after subcutaneous xenograft transplants inimmunodeficient nude mice. In e, f, P , 0.001 (one-way repeated-measuresanalysis of variance). g, Kaplan–Meier curves of overall recurrence-free survivalof 120 patients with triple-negative molecular subtype of breast cancer30.Log-rank P 5 0.0032 (x2 test).

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13. van Nuland, R. et al. Nucleosomal DNA binding drives the recognition of H3K36-methylated nucleosomes by the PSIP1-PWWP domain. Epigenet. Chromatin 6, 12(2013).

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19. Ray-Gallet, D. et al. Dynamics of histone H3 deposition in vivo reveal a nucleosomegap-filling mechanism for H3.3 to maintain chromatin integrity. Mol. Cell 44,928–941 (2011).

20. Edmunds, J. W., Mahadevan, L. C. & Clayton, A. L. Dynamic histone H3 methylationduring gene induction: HYPB/Setd2 mediates all H3K36 trimethylation. EMBO J.27, 406–420 (2008).

21. Kuo, A. J. et al. NSD2 links dimethylation of histone H3 at lysine 36 to oncogenicprogramming. Mol. Cell 44, 609–620 (2011).

22. Rahl, P. B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445(2010).

23. Danko, C. G. et al. Signaling pathways differentially affect RNA polymerase IIinitiation, pausing, and elongation rate in cells. Mol. Cell 50, 212–222 (2013).

24. Sims,R. J., III, Belotserkovskaya,R.&Reinberg,D. ElongationbyRNApolymerase II:the short and long of it. Genes Dev. 18, 2437–2468 (2004).

25. Ansieau, S. & Sergeant, A. BS69 and RACK7, a potential novel class of tumorsuppressor genes. Pathol. Biol. (Paris) 51, 397–399 (2003).

26. Schwartzentruber, J. et al. Driver mutations in histone H3.3 and chromatinremodelling genes in paediatric glioblastoma. Nature 482, 226–231 (2012).

27. Sturm, D. et al. Hotspot mutations in H3F3A and IDH1 define distinct epigeneticand biological subgroups of glioblastoma. Cancer Cell 22, 425–437 (2012).

28. Lewis, P. W. et al. Inhibition of PRC2 activity by a gain-of-function H3 mutationfound in pediatric glioblastoma. Science 340, 857–861 (2013).

29. Bjerke, L. et al. Histone H3.3 mutations drive pediatric glioblastoma throughupregulation of MYCN. Cancer Discov. 3, 512–519 (2013).

30. Gyorffy, B. et al. An online survival analysis tool to rapidly assess the effect of22,277 genes onbreast cancerprognosis using microarraydata of1,809 patients.Breast Cancer Res. Treat. 123, 725–731 (2010).

Supplementary Information is available in the online version of the paper.

Acknowledgements We thank J. Lipsick, Y. Shi, P. Chi, C.D. Allis, D.J. Patel, S.R. Dent,J. Tyler, M. Galko, T. Westbrook, M. Lee, T. Yao and E. Guccione for comments andreagents. We thank the staff at beamlines 1W2B of the Beijing Synchrotron RadiationFacility and BL17U of the Shanghai Synchrotron Radiation Facility for their assistancein data collection. We thank J. Munch for editing the manuscript. This work wassupportedbygrants toX.S. (CPRITRP110471,WelchG1719, AmericanCancerSocietyRSG-13-290-01-TBE, and National institutes of Health (NIH)/MDACC CCSGCA016672), H.L. (The Major State Basic Research Development Program in China,2011CB965300 and Program for New Century Excellent Talents in University),W.L. (CPRIT RP110471, NIH R01HG007538), B.L. (NIH R01GM090077, Welch I1713),Y.L. (China Postdoctoral Science Foundation, 2012M510413) and H.W. (MD AndersonIRG, Center for Cancer Epigenetics pilot grant). W.L. is a recipient of a Duncan ScholarAward and X.S. is a recipient of a Kimmel Scholar Award.

Author Contributions H.W., H.L. and X.S. conceived this study. H.W. performed thebiochemical and cellular studies; Y.L. and H.L. performed structural and calorimetricstudies; Y.X. and Z.X. performed bioinformatic analysis; S.J. and S.S. performed thexenograft study; D.P., K.T. and Y.R. provided technical assistance; J.W. and B.L.performed nucleosome binding experiments; X.S., H.L., H.W., W.L. and M.C.B. analyseddata and wrote the paper.

Author Information Structure data have been deposited in Protein Data Bank underaccession numbers 4N4G (free bromo–PWWP), 4N4H (bromo–PWWP–H3.1K36me3complex) and 4N4I (bromo–PWWP–H3.3K36me3 complex). The ChIP-seq andRNA-seq data been deposited in the Gene Expression Omnibus database underaccession number GSE48423. Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper. Correspondenceand requests for materials should be addressed to X.S. (xbshi@mdanderson.org),H.L. (lht@tsinghua.edu.cn), W.L. (WL1@bcm.edu), or H.W. (hwen@mdanderson.org).

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METHODSMaterials. Human and mouse ZMYND11 complementary DNA (cDNA) werecloned into pENTR3C and subsequently cloned into pBABE-Flag, p3Flag, pCAG-Myc and pGEX destination vectors using Gateway techniques (Invitrogen). ThecDNA encoding the PHD–Bromo–PWWP (PBP, amino acids 74–371) and bromo–PWWP (amino acids 154–371) of mouse ZMYND11 was cloned into the pGEX-6P1and pET-28a vectors (Novagen). Point mutations were generated using a site-directed mutagenesis kit (Stratagene). Histone peptides bearing different modifica-tions were synthesized at the W.M. Keck Facility at Yale University31,32 and SciLightBiotechnology. The anti-ZMYND11 antibody was provided by J. Lipsick5. Anti-histone antibodies including anti-H3 (Ab1791), anti-H3K36me3 (Ab9050), anti-H3K36me2 (Ab9049), anti-H3K4me3 (Ab8580) antibodies and anti-Pol II S2Pwere obtained from Abcam. Anti-Flag (M2) and anti-tubulin antibodies were fromSigma; anti-NSD2 was from Epicypher, anti-GST and anti-total Pol II antibodieswere from Santa Cruz; and anti-MYC antibodies were from Cell Signaling. shRNAconstructs were purchased from Sigma. shRNA sequences were as follows: ZMYND11:59-GAAGGGAAATACCGAAGTTAT and 59-CCTGACAACTGGTTCTGTTAT;SETD2: 59-AGTAGTGCTTCCCGTTATAAA and TGATAGCCATGATAGTATTAAC; NSD2: 59-CGGAAAGCCAAGTTCACCTTT.Peptide microarray and peptide pull-down assay. Peptide microarray and pep-tide pull-down assays were performed as described previously32,33. Briefly, bioti-nylated histone peptides were printed in triplicate onto a streptavidin-coated slide(PolyAn) using a VersArray Compact Microarrayer (Bio-Rad). After a short block-ing with biotin (Sigma), the slides were incubated with the GST-fused ZMYND11PBP or other desired proteins in binding buffer (50 mM Tris-HCl 7.5, 300 mMNaCl, 0.1% NP-40, 1 mM PMSF, 20% fetal bovine serum) overnight at 4 uC withgentle agitation. After being washed with the same buffer, the slides were probedwith an anti-GST primary antibody and then a fluorescein-conjugated secondaryantibody and visualized using a GenePix 4000 scanner (Molecular Devices). Forthe peptide pull-down assays, 1mg of biotinylated histone peptides with differentmodifications were incubated with 1–2mg of GST-fused proteins in bindingbuffer (50 mM Tris-HCl 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF) overnight.Streptavidin beads (Amersham) were added to the mixture, and the mixture wasincubated for 1 h with rotation. The beads were then washed three times andanalysed using SDS–PAGE and western blotting. To preserve the weak bindingbetween ZMYND11 PBP and histone H3.1K36me3, mild buffer (50 mM Tris-HCl7.5, 200 mM NaCl, 0.05% NP-40, 1 mM PMSF) was used for binding and washes.Other steps are essentially the same as described above.Histone binding, nucleosome preparation and electrophoretic mobility shiftassay (EMSA). Full-length histone pull-down assays were performed as describedpreviously34. Briefly, 10mg of full-length histones purified from calf thymus (Worming-ton) were incubated with 25mg of recombinant proteins for 4 h to overnight in abinding solution containing 50 mM Tris pH 7.5, 300 mM NaCl and 0.05% NP-40.Glutathione Sepharose 4B beads (Amersham) were added to the solution for 1 h.The beads were then washed six times and bound histones were detected usingwestern blot analysis.

Recombinant Xenopus histones were individually purified and reconstitutedinto histone octamers using a previously described protocol35. All histone H3K36methylated histones were prepared using the methyl-lysine analogue (MLA)approach36. The chemical reactions result in almost all lysines being convertedto methylation mimics, as was confirmed by mass spectrometry analysis and west-ern blotting.

EMSA for nucleosome binding was as described previously35. EMSA reactionswere performed in a 15-ml system containing 10 mM HEPES pH 7.8, 50 mM KCl,4 mM MgCl2, 5 mM DTT, 0.25 mg ml21 BSA, 5% glycerol and 0.1 mM PMSF.Primer pairs (59-ACAGGATGTATATATCTGACACGTGCCTGG-39) and (59-TGACCAAGGAAAGCATGATTCTTCACAC-39) were designed to generate the216 base-pair mono-nucleosome probe by using plasmid pBL386 as templatefollowed by P32 exchange reaction for labelling. MLA-nucleosomes and indicatedproteins were pre-mixed in EMSA buffer and incubated at 30 uC for 1 h. Then, thetotal reactions were directly loaded onto a 4% native polyacrylamide gel (37.5:1) in0.3 3 TBE. Electrophoresis was performed at 4 uC for 3.5 h.Crystallization, data collection and structural determination. ZMYND11 bromo–PWWP domains were cloned into pET28 vector and expressed with amino (N)-terminal His-tag in Escherichia coli strain BL21 (DE3) (Novagen) in the presenceof 0.5 mM IPTG and 50mM ZnCl2. Overnight-induced cells were collected bycentrifugation and re-suspended in lysis buffer: 300 mM NaCl, 50 mM Na2HPO4,5 mM imidazole, pH 7.0 and 1 mM PMSF. Then cells were lysed with an EmulsiflexC3 (Avestin) high-pressure homogenizer. After centrifugation at 16,770g, the super-natant was applied to a HisTrap column (GE Healthcare), and the protein waseluted with a linear imidazole gradient from 50 to 500 mM. The resultant proteinwas further purified by cation-exchange chromatography using a HiTrap SP col-umn (GE Healthcare). The peaks eluted under the buffer condition 600 mM NaCl,

50 mM Na2HPO4, pH 7.0 were pooled and concentrated to approximately20 mg ml–1. D234A/E236A double mutant bromo–PWWP (BPDM) was generatedby a QuickChange site-direct mutagenesis kit (Stratagene), expressed and purifiedusing essentially the same procedure as for the wild-type protein.

Crystallization was performed by the hanging-drop vapour diffusion methodunder 18 uC by mixing equal volumes (1–2ml) of protein and reservoir solution.Free bromo–PWWP (8–10 mg ml–1) crystals were grown in the solution contain-ing 8% (w/v) polyethylene glycol 3000, and 0.1 M imidazole, pH 7.8. For complexcrystallization, BPDM (8–10 mg ml–1) was first incubated with histone peptides(SciLight Biotechnology) at molar ratio of 1:10 for about 2 h. Crystals of bothH3.319–42K36me3 and H3.121–42K36me3 peptide-bound BPDM could be obtainedunder 25% (w/v) polyethylene glycol 4000, 0.1 M Tris-HCl, pH 8.3, and 0.2 MLi2SO4.

Crystals were briefly soaked in a cryo-protectant composed of reservoir solutionsupplemented with 20% glycerol, and were flash frozen in liquid nitrogen for datacollection at 100 K. The wild-type free bromo–PWWP data set was collected atbeamline 1W2B at the Beijing Synchrotron Radiation Facility at 1.2825 A, the zincabsorption edge. Complex data sets were collected at beamline BL17U at theShanghai Synchrotron Radiation Facility at 0.9793 A for the H3.3K36me3 complexand at 1.0076 A for the H3.1K36me3 complex. All data were indexed, integratedand merged using the HKL2000 software package37. Data collection statistics areshown in Extended Data Table 1.

The phase of free bromo–PWWP was solved by the zinc single-wavelengthanomalous dispersion method using PHENIX software38. The BPDM–H3 peptidecomplex structures were solved by molecular replacement using MOLREP39 withthe free bromo–PWWP structure as search model. All structures were refinedusing PHENIX23, with iterative manual model building using COOT40. Modelgeometry was analysed with PROCHECK41. Ramachandran plot analysis showed92.6%, 95.0% and 93.7% residues in most favoured regions, and 7.4%, 5.0% and6.3% residues in allowed regions, for free bromo–PWWP, BPDM–H3.3 and BPDM–H3.1 complexes, respectively. Detailed structural refinement statistics are shown inExtended Data Table 1. All structural figures were created using PYMOL (http://www.pymol.org/).Circular dichroism spectroscopy. Purified proteins were prepared in buffer 25 mMNa2HPO4 pH 7.0, 500 mM (NH4)2SO4 by overnight dialysis. Circular dichroismspectra were recorded at a protein concentration of 0.1 mg ml–1 under 15 uC usingan Applied Photo-physics Pistar p-180 spectropolarimeter with a 1 mm path-lengthcell and a bandwidth of 3.0 nm. Spectra were scanned from 190 to 260 nm at aninterval of 1 nm and were repeated three times. Each reported circular dichroismcurve was the average of three scans after the subtraction of buffer control.ITC and biolayer interferometry assays. For ITC measurement, synthetic his-tone H3.319–42K36me3 and H3.119–42K36me3 peptides (SciLight Biotechnology)and the wild-type and mutant bromo–PWWP or PBP proteins were extensivelydialysed against ITC buffer: 0.5 M (NH4)2SO4 and 25 mM Na2HPO4, pH 7.0.Protein and peptide concentrations were measured based on their respectiveultraviolet absorption at 280 nm. The titration was performed using a MicroCaliTC200 system (GE Healthcare) at 15 uC. The use of high salt (0.5 M (NH4)2SO4)and low temperature (15 uC) is critical to prevent protein precipitation during theITC titration. Each ITC titration consisted of 17 successive injections with 0.4mlfor the first and 2.42ml for the rest. Usually, H3 peptides at 1.5 mM were titratedinto bromo–PWWP or PBP at 0.07–0.1 mM. Corresponding ‘peptide to buffer’ or‘buffer to protein’ controls were performed for background correction. The res-ultant ITC curves were processed using Origin 7.0 software (OriginLab) accordingto the ‘One Set of Sites’ fitting model. Owing to the low Wiseman c value of mosttitrations, the stoichiometry (n value) was fixed at 1 for valid curve fitting42.

GST and GST-tagged ZMYND11 bromo–PWWP proteins were immobilizedonto anti-GST antibody-coated biosensors (Fortebio, Pall Life Sciences). Afterbeing washed with binding buffer (25 mM NaH2PO4 pH7.0, 150 mM KCl and2 mM MgCl2), the sensors were dipped into binding buffers containing methyla-ted or unmethylated double-strand DNA at various concentrations for 5 min forcomplete association followed by 5 min disassociation in binding buffer withoutDNA. Kinetics were recorded using Octet RED96 (Fortebio, Pall Life Sciences) at1,000 r.p.m. vibration at 25 uC. Steady-state analysis used the following equationwith Octet data analysis 7.0: response 5 Rmax 3 concentration/(Kd 1 concentration),where Rmax is the maximum binding signal and Kd is the dissociation constant.DNA sequences (derived from the Widom 601 sequence43) used in these assayswere as follows: unmethylated dsDNA, 59-CAGCTGAACATCGCTTTTGATG-39;methylated dsDNA, 59-CAGCTGAACAT[5medC]GCTTTTGATG-39.Cell culture, viral transduction and RNA interference. Human U2OS, HEK293T and MDA-MB-231 cells (ATCC) were maintained in DMEM (Cellgro)supplemented with 10% fetal bovine serum (Sigma). Retroviral transductionwas performed as described previously32. Briefly, 293T cells were co-transfectedwith pVPack-VSV-G, pVPack-GP (Stratagene) and pBABE-Flag-ZMYND11,

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pQCXIP-ZMYND11 or control vectors, and viral supernatants were collectedafter 48 h. For lentiviral shRNA packaging, 293T cells were co-transfected withpMD2.G, pPAX2 (Addgene) and pLKO-shRNA constructs. For infections, thecells were incubated with viral supernatants in the presence of 8mg ml21 poly-brene; after 48 h, infected cells were selected with puromycin (2mg ml21). Cellproliferation and colony formation assays were performed as previously described31.Affinity purification, immunoprecipitation and co-immunoprecipitation. Nuclearextracts were prepared as described previously31,44. The nuclear extracts werediluted with low salt buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.2% TritonX-100, 10% glycerol, 1 mM DTT) to a final NaCl concentration of 120 mM. Twohundred microlitres of 50% M2 Flag-agarose beads were added to the nuclearextracts and incubated at 4 uC overnight. The beads were then washed with washbuffer (20 mM Tris-HCl pH8.0, 250 or 500 mM KCl, 1.5 mM MgCl2, 0.2 mMEDTA, 10% glycerol, 0.2 mM PMSF, 1 mM DTT and protease inhibitors) threeto six times, and eluted with 300ml of 0.3 mg ml21 Flag peptide in dialysis buffer(20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT) at 4 uC for 4 h three times.The bound proteins were separated in SDS–PAGE and sent for mass spectrometricanalysis. Liquid chromatography/tandem mass spectrometry was done at theHarvard Taplin MS core facility and at the University of Texas Medical BranchMS core facility.

For immunoprecipitation or co-immunoprecipitation experiments, cells werelysed in cell lysis buffer containing 50 mM Tris-HCl pH 7.4, 250 mM NaCl, 0.5%Triton X-100, 10% glycerol, 1 mM DTT and a complete protease inhibitor tablet(Roche). Antibodies conjugated with protein A/G beads (Millipore) or anti-FlagM2-conjugated agarose beads (Sigma) were incubated with lysates overnight at4 uC. The beads were then washed three to six times with cell lysis buffer, and thebound proteins were eluted in SDS buffer and analysed using western blotting.ChIP and ChIP-seq analysis. ChIP analysis was essentially as previously described32.Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temper-ature, and the reaction was stopped with 125 mM glycine. Nuclei were isolated byre-suspending the cells in swelling buffer containing 5 mM PIPES pH 8.0, 85 mMKCl, 1% NP-40 and a complete protease inhibitor for 20 min at 4 uC. The isolatednuclei were re-suspended in nuclei lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA,1% SDS) and sonicated using a Bioruptor Sonicator (Diagenode). Samples wereimmunoprecipitated with the appropriate antibodies overnight at 4 uC. After incu-bation with Staph A cells for 15 min, immunoprecipitates were washed twice withdialysis buffer (50 mM Tris pH 8.0, 2 mM EDTA, 0.2% Sarkosyl) and four timeswith immunoprecipitation wash buffer (100 mM Tris pH 8.0, 500 mM LiCl, 1%NP-40 and 1% deoxycholic acid sodium salt). After reverse crosslinking was per-formed, DNA was eluted, purified using a PCR purification kit (Qiagene) andsubjected to qPCR on the ABI 7500-FAST System using the Power SYBR GreenPCR Master Mix (Applied Biosystems).

ChIP-seq was done essentially as described above, except using proteinA/Gbeads instead of Staph A cells for immunoprecipitation. Samples were sequencedusing the Illumina Solexa Hiseq 2000. The raw reads were mapped to humanreference genome NCBI 36 (hg18) by Solexa data processing pipeline, allowingup to two mismatches. The genome ChIP-seq profiles were generated using MACS1.3.6 (ref. 45) with only unique mapped reads. Clonal reads were automaticallyremoved by MACS. The ChIP-seq profiles were normalized to 10,000,000 total tagnumbers, and peaks were called at P # 1 3 1028. For ZMYND11 and H3K36me3ChIP samples, peaks were annotated by REFSEQ genes that had overlaps withinTSS to TTS. For Pol II samples, peaks were annotated by REFSEQ genes that hadoverlaps within TSS-3K to TTS. The distribution statistics of the ChIP-seq peakswere performed using the Cis-regulatory element annotation system46. All ChIP-seq data are summarized in Supplementary Table 7.Reverse transcription PCR, northern blotting, qPCR and RNA-seq analysis.Reverse transcription PCR and real-time PCR were performed as previously described32.Total RNA was prepared using the RNeasy Plus kit (Qiagen) and reverse-tran-scribed using the First Strand Synthesis kit (Invitrogen). Northern blotting andhybridization used a Northern Max-Gly kit (Invitrogen) following the manufac-turer’s instructions. Twenty micrograms of total RNA were used for each lane.qPCR was performed on the ABI 7500-FAST Sequence Detection System using thePower SYBR Green PCR Master Mix (Applied Biosystems). Gene expressions werecalculated following normalization to GAPDH amounts using the comparative Ct(cycle threshold) method. Primer sequences are available upon request.

RNA-seq samples were sequenced using the Illumina Hiseq 2000, and raw readswere mapped to human reference genome (hg18) and transcriptome using the

RNA-Seq unified mapper47. The expression levels of ZMYND11 KD and controlexpressed as reads per kilobase per million were compared using EdgeR. Geneswith fewer than five reads per kilobase per million in both the ZMYND11 KD andcontrol samples were removed. Genes with a fold change $ 1.5 were selected asdifferential genes. Gene ontology analysis used the DAVID Bioinformatics Resource6.7 (ref. 48).Expression signatures and survival analysis. Survival analyses were performedas described previously30. The database consisting of 13 breast cancer expressionprofiling data sets from Gene Expression Omnibus was used in the ‘consolidated’survival analysis through the Kaplan–Meier plotter central server. Data wereloaded into the R statistical environment, where calculations were performed.The package ‘survival’ was used to calculate and plot Kaplan–Meier survival curves,and the number-at-risk was indicated below the main plot. P values were calculatedusing the log-rank test. For data validation, independent analysis of Gene Expres-sion Omnibus data sets GSE6532 and GSE7390 were performed as previouslydescribed49.Tumour xenograft. All animal studies were in compliance with ethical regula-tions at the University of Texas MD Anderson Cancer Center. Female athymicnude mice (age 6–8 weeks) were obtained from the National Cancer Institute atFrederick and animals were housed under pathogen-free conditions. Five millionMDA-MB 231 cells stably expressing control pQCXIP vector, wild-type ZMYND11or mutants were suspended in 100ml of serum-free DMEM and injected subcuta-neously into the mice. The growth of tumours was monitored by weekly examinationuntil the largest tumour reached tumour burden. Tumour sizes were measured usinga calliper, and tumour volume was calculated according to the following equation:tumour volume (mm3) 5 (length (mm) 3 width2 (mm2)) 3 0.5. Representative datawere obtained from five mice per experimental group. Statistical analyses wereperformed with one-way repeated-measures analysis of variance.

31. Shi, X. et al. ING2 PHD domain links histone H3 lysine 4 methylation to active generepression. Nature 442, 96–99 (2006).

32. Wen, H. et al. Recognition of histone H3K4 trimethylation by the planthomeodomain of PHF2 modulates histone demethylation. J. Biol. Chem. 285,9322–9326 (2010).

33. Fish, P. et al. Identification of a chemical probe for BET bromodomain inhibitionthrough optimization of a fragment-derived hit. J. Med. Chem. 55, 9831–9837(2012).

34. Mendez, J. & Stillman, B. Chromatin association of human origin recognitioncomplex, cdc6, and minichromosome maintenance proteins during the cell cycle:assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20,8602–8612 (2000).

35. Li, B. et al. Combined action of PHD and chromo domains directs the Rpd3SHDACto transcribed chromatin. Science 316, 1050–1054 (2007).

36. Simon, M. D. et al. The site-specific installation of methyl-lysine analogs intorecombinant histones. Cell 128, 1003–1012 (2007).

37. Zeitlinger, J. et al. RNA polymerase stalling at developmental control genes in theDrosophila melanogaster embryo. Nature Genet. 39, 1512–1516 (2007).

38. Boettiger, A. N. & Levine, M. Synchronous and stochastic patterns of geneactivation in the Drosophila embryo. Science 325, 471–473 (2009).

39. Nechaev, S. & Adelman, K. Promoter-proximal Pol II: when stalling speeds thingsup. Cell Cycle 7, 1539–1544 (2008).

40. Naar, A. M., Lemon, B. D. & Tjian, R. Transcriptional coactivator complexes. Annu.Rev. Biochem. 70, 475–501 (2001).

41. Luo, Z., Lin, C. & Shilatifard, A. The super elongation complex (SEC) family intranscriptional control. Nature Rev. Mol. Cell Biol. 13, 543–547 (2012).

42. Turnbull, W. B. & Daranas, A. H. On the value of c: can low affinity systems bestudied by isothermal titration calorimetry? J. Am. Chem. Soc. 125, 14859–14866(2003).

43. Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding tohistone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276,19–42 (1998).

44. Abmayr, S. M., Yao, T., Parmely, T. & Workman, J. L. Preparation of nuclear andcytoplasmic extracts from mammalian cells. Curr. Protoc. Mol. Biol. 12.11, (2006).

45. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137(2008).

46. Eswaran, J. et al. Structure and functional characterization of the atypical humankinase haspin. Proc. Natl Acad. Sci. USA 106, 20198–20203 (2009).

47. Akue-Gedu, R. et al. Synthesis, kinase inhibitory potencies, and in vitroantiproliferative evaluation of new Pim kinase inhibitors. J. Med. Chem. 52,6369–6381 (2009).

48. Barr, A. J. et al. Large-scale structural analysis of the classical human proteintyrosine phosphatome. Cell 136, 352–363 (2009).

49. Kessler, J. D. et al. A SUMOylation-dependent transcriptional subprogram isrequired for Myc-driven tumorigenesis. Science 335, 348–353 (2012).

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Extended Data Figure 1 | The PBP domains are required for ZMYND11binding to H3.3K36me3. a, Western blot analysis of H3K36me3 levels incalf thymus histones from pull-downs with GST or GST–ZMYND11 PBP.Bottom: GelCode Blue staining of input proteins. b, EMSA of nucleosomesreconstituted from recombinant H3K36-methyl-lysine analogue (MLA)histones incubated with Flag-tagged full-length ZMYND11 proteins. FreeDNA, mono nucleosome (Nu) and ZMYND11-bound nucleosome areindicated. c, Western blot analysis of histone peptide pull-downs with

GST–ZMYND11 PHD finger. d, ITC curves of the H3.3K36me peptidestitrated into ZMYND11 bromo–PWWP (BP) domains. e, Western blotanalysis of histone peptide pull-downs with Flag-tagged full-length ZMYND11or the indicated domain-deletion mutants and biotinylated peptides.Schematics of the deletion mutants are shown in the left panel. f, Western blotanalysis of protein-ChIP assays using the anti-Flag antibody in HEK 293 cellstransfected with Flag-tagged full-length ZMYND11 and the indicated deletionmutants.

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Extended Data Figure 2 | Crystal structures of ZMYND11 bromo–PWWPdomains. a, Sequence and structure-based secondary structural assignment ofthe ZMYND11 tandem bromo–PWWP (BP) domains. Dashed box (magenta)and dashed line (black): unmodelled sequence in the free and complexstructures, respectively; cyan shading: basic residues within the ZA loop (LZA);magenta shading: residues corresponding to canonical acetyl-lysinerecognition motif; black box: residues mutated to facilitate co-crystalformation. Black dot: aromatic caging residues; aster: H3 hydrogen-bondingresidues with magenta unique to H3.3 variant. b, Burial of the K36me3-bindingaromatic cage by an adjacent bromodomain during crystal packing. Two acidicresidues, D234 and E236, contribute to such packing contacts throughelectrostatic interaction with the positive surface patch (blue) of PWWP. c, Anoverall view of superimposed free and H3K36me3 peptide-bound ZMYND11bromo–PWWP double mutant. d, Western blot analysis of histone peptidepull-downs with wild-type ZMYND11 PBP and the PBP-D234A/E236Amutant. e, ITC curves of the histone H3.3K36me3 peptide titrated intoZMYND11 bromo–PWWP-D234A/E236A mutant. f, Solvent-accessiblesurface representations of ZYMND11 bromo–ZnF–PWWP in its free state.Note the tight integration of the paired modules. g, Ribbon view of

bromo–ZnF–PWWP with basic lysine/arginine clusters highlighted as yellowsticks. h, Superimposition of ZMYND11 bromodomain with H4K16ac-boundBPTF bromodomain (Protein Data Bank (PDB) accession number 3QZT).i, Surface representation of BPTF bound to H4K16ac and its comparison withZMYND11 bromodomain. The Kac pocket is missing in ZMYND11 bromoowing to the occurrence of Y231. Note the positive residue clusters around thepeptide-binding surface of ZMYND11 bromo (right). j, Zinc coordinationsphere of the newly identified ZnF motif of ZMYND11. Note the burial of non-zinc-coordinating C274 at the hydrophobic interface between ZnF (salmon)and PWWP (blue) of ZMYND11. k, Encapsulation of ZnF (salmon) by bromo(green) and PWWP (blue) in stereo view. Dashed line denotes hydrogenbonding or zinc coordinating interactions. Note the burial of hydrophobicresidues including F262, L264, F273, C274 and Y275 from ZnF at the bromo–ZnF and ZnF–PWWP interfaces. l, Structural alignment of ZMYND11 ZnF–PWWP (salmon and blue) with Pdp1 PWWP (PDB accession number 2L89)(magenta) and BRPF1 PWWP (PDB accession number 2X4W) (cyan) showingthe structural overlaps of ZMYND11 ZnF with Pdp1 a3 and BRPF1 b2–b3insertion.

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Extended Data Figure 3 | Comparison of ZMYND11 PWWP with otherKme3-binding PWWP domains. a, b, Stereo view of ZMYND11 PWWPdomain in superimposition with H3K36me3-bound BRPF1 PWWP (PDBaccession number 2X4W) (a) and H3K79me3-bound HDGF PWWP (PDBaccession number 3QJ6) (b). c, Sequence alignment of Kme3-binding PWWPdomains. Conserved residues are in the blue box; identical residues are shadedin red. Underscored dots: residues forming the aromatic cage. Sequencealignment was produced using the ESPript. d, Western analysis of histonepeptide pull-downs with indicated point mutants in the context of Flag-taggedfull-length ZMYND11 and biotinylated peptides. e, Western blot analysis of the

protein-ChIP assays in cells expressing Flag-tagged full-length ZMYND11 orthe indicated mutants. f, Point mutations in ZMYND11 bromo–ZnF–PWWPdomains do not affect protein folding. Circular dichroism spectroscopy analysisof the wild-type ZMYND11 bromo–PWWP domains and indicated mutantsused in this study. g, Steady-state analysis of the biolayer interferometrysensorgrams of ZMYND11 bromo–PWWP binding to unmethylated (blacksquare) and fully methylated (blue triangle) 22-base oligonucleotide duplexDNA derived from the Widom 601 sequence. Unmethylated duplex DNA:59-CAGCTGAACATCGCTTTTGATG-39; fully methylated duplex DNA:59-CAGCTGAACAT[5medC]GCTTTTGATG-39.

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Extended Data Figure 4 | Crystal structure of ZMYND11 bromo–PWWP incomplex with H3.1K36me3 and its comparison with the H3.3K36me3-bound complex. a, ITC curves of H3.3K36me3 or H3.1K36me3 peptidestitrated into ZMYND11 bromo–PWWP and PBP domains. Titration c valueswere 1.41 for bromo–PWWP–H3.3, 0.15 for bromo–PWWP–H3.1, 1.26 forPBP–H3.3 and 0.16 for PBP–H3.1, respectively. The ‘n’ value was fixed at 1 forcurve fitting. b, Western blot analysis of peptide pull-downs under stringentbinding conditions. c, Western blot analysis of Flag immunoprecipitation incells co-expressing Flag–H3.3 or H3.1 and Myc-ZMYND11. d, Structure ofZMYND11 bromo–PWWP in complex with H3.1K36me3 peptide. Bromo–PWWP is in surface representation with bromo, ZnF and PWWP, colouredgreen, salmon and blue, respectively. The 2Fo 2 Fc omit maps around H3peptide, polyethylene glycol (PEG) and phosphate (PO4) are shown as cyanmesh contoured at the 1s level. e, f, Simulated annealing Fo 2 Fc omit mapcountered at 2.5s level around the histone segments containing H3.1Ala31

(e) or H3.3Ser31 (f) in complex with ZMYND11 bromo–PWWP. ResiduesR168, H250, E251, E254, N266, R268, R309, R317 of bromo–PWWP, abridging water and segment ‘Ala29–Val35’ of histone H3.1 or H3.3 peptideswere omitted for simulated annealing (starting temperature 2,500 K and 500cooling steps) map calculation by the Phenix program. Magenta dashes:hydrogen bonds. Note that the Ne atom of R268 side chain (e) and side chainsof R168 (e, f) showed poor densities, suggesting their conformational flexibility.g, Western analysis of histone peptide pull-downs with indicated point mutantsand biotinylated peptides. h, Structural alignment of ZMYND11 bromo–PWWP–H3.3K36me3 (blue), ZMYND11 bromo–PWWP–H3.1K36me3(salmon) and BRPF1 PWWP–H3.1K36me3 (PDB accession number 2X4W)(yellow). i, Structural alignment of H3K36me3-bound ZMYND11 PWWP(blue), PHF19 Tudor (PDB accession number 4BD3) (red) and PHF1 Tudor(PDB accession number 4HCZ) (cyan). Both bromo–PWWP and H3 peptidesare presented as backbone coils, with key residues depicted as sticks.

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Extended Data Figure 5 | Analysis of Flag–H3.3 ChIP-seq and ZMYND11ChIP-seq data. a, Western blot analysis of U2OS cells stably expressing Flag–H3.3 with the indicated antibodies. The arrow indicates the ectopic Flag–H3.3protein. b, Average occupancy of Flag–H3.3 along the transcription unit ongenes with high, intermediate and low expression levels. The gene expressionlevels were grouped according to the RNA-seq reads per kilobase per millionvalue as low (,1), medium (1–20) or high (.20). The gene body length isaligned by percentage from the TSS to TTS as in Fig. 3b. c, Western analysis ofZMYND11 protein and H3K36me3 levels in control and ZMYND11

knockdown U2OS cells. The asterisk indicates a non-specific band. d, Genomebrowser view of ZMYND11 occupancy in the chromosome 16p13.3 regions (asshown in Fig. 3d) in control and ZMYND11 knockdown cells. e, The averagegenome-wide occupancy of ZMYND11 along the transcription unit in cells asin Fig. 3b. f, qPCR analysis of ZMYND11 ChIP in the gene bodies of theindicated genes in cells as in c. Error bars, s.e.m. of three experiments. P , 0.01(Student’s t-test). g, qPCR analysis of Flag ChIP in cells stably expressing Flag–ZMYND11 and control U2OS cells. Error bars, s.e.m. of three independentexperiments.

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Extended Data Figure 6 | ZMYND11 occupancy in gene body dependson SETD2-mediated H3K36me3. a, Average genome-wide ZMYND11occupancy on genes with high, intermediate or low amounts of H3K36me3.Genes were aligned as described in Fig. 3b, and were grouped according to theH3K36me3 ChIP-seq normalized tag numbers as low (,2), intermediate(2–10) or high (.10). ZMYND11 ChIP-seq occupancies were normalized to10 million total tag numbers. b, qPCR analysis of the expression of ZMYND11target genes and SETD2 in control and SETD2 knockdown cells. c, Western blotanalysis of H3K36me3 and ZMYND11 amounts in SETD2 knockdown cells.Asterisks indicate non-specific bands. Bottom panel: western blot analysis ofFlag–SETD2-expressing cells co-transfected with SETD2-targeting shRNAs

using the M2 anti-Flag antibody, indicating efficient knockdowns. d, qPCRanalysis of the ZMYND11 and H3K36me3 ChIP in the intragenic regions of theNFKB2 gene in control and SETD2 knockdown cells. e, Western blot analysis ofNSD2 and H3K36me2 in NSD2 knockdown cells. f, qPCR analysis ofH3K36me2, H3K36me3 and ZMYND11 ChIP in MYC and NFKB2 genes inNSD2 knockdown cells. g, Western blot analysis of ZMYND11 expressionlevels in U2OS cells stably expressing the wild-type ZMYND11 and theindicated H3.3K36me3 binding-deficient mutants. h, qPCR analysis of FlagChIP in the NFKB2 gene in cells as in g. In b, d, f, h, error bars indicate the s.e.m.of three independent experiments. *All P values , 0.05 (Student’s t-test).

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Extended Data Figure 7 | ZMYND11 has a role in both transcriptionalactivation and repression. a, Western blot analysis of ZMYND11 and MYCprotein levels in control and ZMYND11 knockdown cells. The asteriskindicates a non-specific band. b, qPCR analysis of the expression of ZMYND11and ZMYND11 target genes in control and ZMYND11 knockdown U2OS cells.Signals were normalized to GAPDH expression. Error bars represent the s.e.m.

of three experiments. *Two-tailed unpaired Student’s t-tests, P , 0.01. c, Nocryptic transcripts observed in ZMYND11 knockdown cells. Northern blotanalysis of indicated ZMYND11 direct target genes on the total RNA extractedfrom control and ZMYND11 knockdown U2OS cells. GAPDH was used as aloading control.

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Extended Data Figure 8 | ZMYND11 knockdown increases the occupanciesof total Pol II and Pol II S2P in gene bodies. a, Venn diagram showing theoverlap of ZMYND11- and Pol II-occupied genes. P , 1 3 102322 (Fisher’sexact test). A comprehensive list of Pol II ChIP-seq peaks is given inSupplementary Table 6. b, Average genome-wide ZMYND11 occupancy ongenes with high, intermediate and low levels of Pol II occupancy. Genes werealigned as described in Fig. 3b, and were grouped according to the Pol II ChIP-seq normalized tag numbers as low (,2), intermediate (2–10) or high (.10).ZMYND11 ChIP-seq occupancies were normalized to 10 million total tagnumbers. c, Average genome-wide occupancies of Pol II along the transcriptionunit of all ZMYND11-activated direct target genes (left) and all

non-ZMYND11 target genes (right) in control and ZMYND11 knockdowncells as in Fig. 4d. d, Average genome-wide occupancies of Pol II S2P near theTTS of all ZMYND11-repressed direct target genes in control and ZMYND11knockdown cells. e, qPCR analysis of the ZMYND11, H3K36me3 and Pol IIS2P ChIP in the intragenic regions of MYC and NFKB2 genes in control andZMYND11 knockdown cells. f, Pol II travelling ratio (TR) on ZMYND11-repressed direct target genes in control and ZMYND11 knockdown cells.Lower travelling ratio values indicate a higher degree of elongating Pol II. Theleft panel shows the schematic representation of the calculation of Pol IItravelling ratio21. The right panel shows the whisker plot of Pol II travellingratio. P 5 2.5 3 1025 (Student’s t-test).

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Extended Data Figure 9 | ZMYND11 suppresses tumour cell growth andis downregulated and mutated in human cancers. a, ZMYND11 isdownregulated in cancers. ZMYND11 gene expression in approximately40,000 tumour or normal tissue samples from three data sets (Gene ExpressionOmnibus, ArrayExpress and Expression Project for Oncology) were analysedusing GENT. N, normal; C, cancer. All P , 0.0001. b, Cell proliferation assay ofU2OS cells (mean 6 s.e.m., n 5 3) with knockdown (KD) or overexpression(OE) of ZMYND11. c, Cell proliferation assay of U2OS cells stably expressingthe wild-type or mutant ZMYND11 proteins (mean 6 s.e.m., n 5 5). Cells werecounted 6 days after seeding. d, Colony formation assay of cells as in c. Cellcolonies (mean 6 s.e.m., n 5 3) were counted 2 weeks after seeding. e, Westernblot analysis of ZMYND11 expression levels in stable MDA-MB 231 cells usedin Fig. 4e. f, Schematic representation of ZMYND11 missense somatic

mutations in colon and rectum adenocarcinoma identified in the TCGAdatabase. g, Peptide pull-downs of wild-type ZMYND11 PBP and the D307Nmutant with H3.3K36me peptides. h, Western blot analysis of Flag-taggedwild-type ZMYND11 and the D307N mutant stably expressed in MDA-MB231 cells. i, qPCR analysis of Flag ChIP in MYC and NFKB2 genes in the stablecells as in h. Error bars, s.e.m. of three independent experiments *P , 0.01(Student’s t-test). j, k, Low ZMYND11 expression levels in patients with breastcancer correlate with worse disease-free survivals. Kaplan–Meier survivalcurves of patients with breast cancer from cohort studies National Institutes ofHealth Gene Expression Omnibus GSE6532 (j) and GSE7390 (k). P values werecalculated by x2 test. l, ITC curves of the histone H3.3(G34R)K36me3 andH3.3(G34V)K36me3 peptides titrated into ZMYND11 bromo–PWWPdomains.

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Extended Data Table 1 | Data collection and refinement statistics

One crystal was used for each structure.*Highest resolution shell is shown in parenthesis.{Ligands/ions are zinc and phosphate for free bromo–PWWP, and peptide, zinc, phosphate, PEG for bromo–PWWP complexes.{Data processing and refinement statistics in this column are based on anomalous scaling.1 A high-resolution cutoff at 2.3 A was made owing to ice ring.

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