ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression

Abstract

Recognition of modified histones by ‘reader’ proteins plays a critical role in the regulation of chromatin1. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions after RNA polymerase II elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state, thus suppressing cryptic transcription2. However, much less is known about the role of H3K36me3 in transcription regulation 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 ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates RNA polymerase II elongation. Structural studies show that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific ‘Ser 31’ residue in a composite pocket formed by the tandem bromo–PWWP domains of ZMYND11. Chromatin immunoprecipitation followed by sequencing shows a genome-wide co-localization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed 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 program that is essential for tumour cell growth; low expression levels of ZMYND11 in breast cancer patients correlate with worse prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumour formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone-variant-mediated transcription elongation control to tumour suppression.

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Figure 1: Molecular basis for H3.3K36me3 recognition by ZMYND11 bromo–PWWP domains.
Figure 2: H3.3 variant-specific K36me3 recognition by ZMYND11.
Figure 3: ZMYND11 co-localizes with H3K36me3 and H3.3 in the gene body.
Figure 4: ZMYND11 modulates Pol II elongation and represses oncogene expression and tumour growth.

Accession codes

Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

Structure data have been deposited in Protein Data Bank under accession numbers 4N4G (free bromo–PWWP), 4N4H (bromo–PWWP–H3.1K36me3 complex) and 4N4I (bromo–PWWP–H3.3K36me3 complex). The ChIP-seq and RNA-seq data been deposited in the Gene Expression Omnibus database under accession number GSE48423.

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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 and reagents. We thank the staff at beamlines 1W2B of the Beijing Synchrotron Radiation Facility and BL17U of the Shanghai Synchrotron Radiation Facility for their assistance in data collection. We thank J. Munch for editing the manuscript. This work was supported by grants to X.S. (CPRIT RP110471, Welch G1719, American Cancer Society RSG-13-290-01-TBE, and National institutes of Health (NIH)/MDACC CCSG CA016672), 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 Anderson IRG, Center for Cancer Epigenetics pilot grant). W.L. is a recipient of a Duncan Scholar Award and X.S. is a recipient of a Kimmel Scholar Award.

Author information

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Authors

Contributions

H.W., H.L. and X.S. conceived this study. H.W. performed the biochemical and cellular studies; Y.L. and H.L. performed structural and calorimetric studies; Y.X. and Z.X. performed bioinformatic analysis; S.J. and S.S. performed the xenograft 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. analysed data and wrote the paper.

Corresponding authors

Correspondence to Hong Wen or Wei Li or Haitao Li or Xiaobing Shi.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The PBP domains are required for ZMYND11 binding to H3.3K36me3.

a, Western blot analysis of H3K36me3 levels in calf thymus histones from pull-downs with GST or GST–ZMYND11 PBP. Bottom: GelCode Blue staining of input proteins. b, EMSA of nucleosomes reconstituted from recombinant H3K36-methyl-lysine analogue (MLA) histones incubated with Flag-tagged full-length ZMYND11 proteins. Free DNA, mono nucleosome (Nu) and ZMYND11-bound nucleosome are indicated. c, Western blot analysis of histone peptide pull-downs with GST–ZMYND11 PHD finger. d, ITC curves of the H3.3K36me peptides titrated into ZMYND11 bromo–PWWP (BP) domains. e, Western blot analysis of histone peptide pull-downs with Flag-tagged full-length ZMYND11 or the indicated domain-deletion mutants and biotinylated peptides. Schematics of the deletion mutants are shown in the left panel. f, Western blot analysis of protein-ChIP assays using the anti-Flag antibody in HEK 293 cells transfected with Flag-tagged full-length ZMYND11 and the indicated deletion mutants.

Extended Data Figure 2 Crystal structures of ZMYND11 bromo–PWWP domains.

a, Sequence and structure-based secondary structural assignment of the ZMYND11 tandem bromo–PWWP (BP) domains. Dashed box (magenta) and dashed line (black): unmodelled sequence in the free and complex structures, respectively; cyan shading: basic residues within the ZA loop (LZA); magenta shading: residues corresponding to canonical acetyl-lysine recognition motif; black box: residues mutated to facilitate co-crystal formation. Black dot: aromatic caging residues; aster: H3 hydrogen-bonding residues with magenta unique to H3.3 variant. b, Burial of the K36me3-binding aromatic cage by an adjacent bromodomain during crystal packing. Two acidic residues, D234 and E236, contribute to such packing contacts through electrostatic interaction with the positive surface patch (blue) of PWWP. c, An overall view of superimposed free and H3K36me3 peptide-bound ZMYND11 bromo–PWWP double mutant. d, Western blot analysis of histone peptide pull-downs with wild-type ZMYND11 PBP and the PBP-D234A/E236A mutant. e, ITC curves of the histone H3.3K36me3 peptide titrated into ZMYND11 bromo–PWWP-D234A/E236A mutant. f, Solvent-accessible surface 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 yellow sticks. h, Superimposition of ZMYND11 bromodomain with H4K16ac-bound BPTF bromodomain (Protein Data Bank (PDB) accession number 3QZT). i, Surface representation of BPTF bound to H4K16ac and its comparison with ZMYND11 bromodomain. The Kac pocket is missing in ZMYND11 bromo owing to the occurrence of Y231. Note the positive residue clusters around the peptide-binding surface of ZMYND11 bromo (right). j, Zinc coordination sphere 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 hydrogen bonding or zinc coordinating interactions. Note the burial of hydrophobic residues 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) showing the structural overlaps of ZMYND11 ZnF with Pdp1 α3 and BRPF1 β2–β3 insertion.

Extended Data Figure 3 Comparison of ZMYND11 PWWP with other Kme3-binding PWWP domains.

a, b, Stereo view of ZMYND11 PWWP domain in superimposition with H3K36me3-bound BRPF1 PWWP (PDB accession number 2X4W) (a) and H3K79me3-bound HDGF PWWP (PDB accession number 3QJ6) (b). c, Sequence alignment of Kme3-binding PWWP domains. Conserved residues are in the blue box; identical residues are shaded in red. Underscored dots: residues forming the aromatic cage. Sequence alignment was produced using the ESPript. d, Western analysis of histone peptide pull-downs with indicated point mutants in the context of Flag-tagged full-length ZMYND11 and biotinylated peptides. e, Western blot analysis of the protein-ChIP assays in cells expressing Flag-tagged full-length ZMYND11 or the indicated mutants. f, Point mutations in ZMYND11 bromo–ZnF–PWWP domains do not affect protein folding. Circular dichroism spectroscopy analysis of the wild-type ZMYND11 bromo–PWWP domains and indicated mutants used in this study. g, Steady-state analysis of the biolayer interferometry sensorgrams of ZMYND11 bromo–PWWP binding to unmethylated (black square) and fully methylated (blue triangle) 22-base oligonucleotide duplex DNA derived from the Widom 601 sequence. Unmethylated duplex DNA: 5′-CAGCTGAACATCGCTTTTGATG-3′; fully methylated duplex DNA: 5′-CAGCTGAACAT[5medC]GCTTTTGATG-3′.

Extended Data Figure 4 Crystal structure of ZMYND11 bromo–PWWP in complex with H3.1K36me3 and its comparison with the H3.3K36me3-bound complex.

a, ITC curves of H3.3K36me3 or H3.1K36me3 peptides titrated into ZMYND11 bromo–PWWP and PBP domains. Titration c values were 1.41 for bromo–PWWP–H3.3, 0.15 for bromo–PWWP–H3.1, 1.26 for PBP–H3.3 and 0.16 for PBP–H3.1, respectively. The ‘n’ value was fixed at 1 for curve fitting. b, Western blot analysis of peptide pull-downs under stringent binding conditions. c, Western blot analysis of Flag immunoprecipitation in cells co-expressing Flag–H3.3 or H3.1 and Myc-ZMYND11. d, Structure of ZMYND11 bromo–PWWP in complex with H3.1K36me3 peptide. Bromo–PWWP is in surface representation with bromo, ZnF and PWWP, coloured green, salmon and blue, respectively. The 2Fo − Fc omit maps around H3 peptide, polyethylene glycol (PEG) and phosphate (PO4) are shown as cyan mesh contoured at the 1σ level. e, f, Simulated annealing Fo − Fc omit map countered at 2.5σ level around the histone segments containing H3.1Ala31 (e) or H3.3Ser31 (f) in complex with ZMYND11 bromo–PWWP. Residues R168, H250, E251, E254, N266, R268, R309, R317 of bromo–PWWP, a bridging water and segment ‘Ala29–Val35’ of histone H3.1 or H3.3 peptides were omitted for simulated annealing (starting temperature 2,500 K and 500 cooling steps) map calculation by the Phenix program. Magenta dashes: hydrogen bonds. Note that the Nε atom of R268 side chain (e) and side chains of R168 (e, f) showed poor densities, suggesting their conformational flexibility. g, Western analysis of histone peptide pull-downs with indicated point mutants and 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 peptides are presented as backbone coils, with key residues depicted as sticks.

Extended Data Figure 5 Analysis of Flag–H3.3 ChIP-seq and ZMYND11 ChIP-seq data.

a, Western blot analysis of U2OS cells stably expressing Flag–H3.3 with the indicated antibodies. The arrow indicates the ectopic Flag–H3.3 protein. b, Average occupancy of Flag–H3.3 along the transcription unit on genes with high, intermediate and low expression levels. The gene expression levels were grouped according to the RNA-seq reads per kilobase per million value as low (<1), medium (1–20) or high (>20). The gene body length is aligned by percentage from the TSS to TTS as in Fig. 3b. c, Western analysis of ZMYND11 protein and H3K36me3 levels in control and ZMYND11 knockdown U2OS cells. The asterisk indicates a non-specific band. d, Genome browser view of ZMYND11 occupancy in the chromosome 16p13.3 regions (as shown in Fig. 3d) in control and ZMYND11 knockdown cells. e, The average genome-wide occupancy of ZMYND11 along the transcription unit in cells as in Fig. 3b. f, qPCR analysis of ZMYND11 ChIP in the gene bodies of the indicated 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 independent experiments.

Extended Data Figure 6 ZMYND11 occupancy in gene body depends on SETD2-mediated H3K36me3.

a, Average genome-wide ZMYND11 occupancy on genes with high, intermediate or low amounts of H3K36me3. Genes were aligned as described in Fig. 3b, and were grouped according to the H3K36me3 ChIP-seq normalized tag numbers as low (<2), intermediate (2–10) or high (>10). ZMYND11 ChIP-seq occupancies were normalized to 10 million total tag numbers. b, qPCR analysis of the expression of ZMYND11 target genes and SETD2 in control and SETD2 knockdown cells. c, Western blot analysis of H3K36me3 and ZMYND11 amounts in SETD2 knockdown cells. Asterisks indicate non-specific bands. Bottom panel: western blot analysis of Flag–SETD2-expressing cells co-transfected with SETD2-targeting shRNAs using the M2 anti-Flag antibody, indicating efficient knockdowns. d, qPCR analysis of the ZMYND11 and H3K36me3 ChIP in the intragenic regions of the NFKB2 gene in control and SETD2 knockdown cells. e, Western blot analysis of NSD2 and H3K36me2 in NSD2 knockdown cells. f, qPCR analysis of H3K36me2, H3K36me3 and ZMYND11 ChIP in MYC and NFKB2 genes in NSD2 knockdown cells. g, Western blot analysis of ZMYND11 expression levels in U2OS cells stably expressing the wild-type ZMYND11 and the indicated H3.3K36me3 binding-deficient mutants. h, qPCR analysis of Flag ChIP 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).

Extended Data Figure 7 ZMYND11 has a role in both transcriptional activation and repression.

a, Western blot analysis of ZMYND11 and MYC protein levels in control and ZMYND11 knockdown cells. The asterisk indicates a non-specific band. b, qPCR analysis of the expression of ZMYND11 and 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, No cryptic transcripts observed in ZMYND11 knockdown cells. Northern blot analysis of indicated ZMYND11 direct target genes on the total RNA extracted from control and ZMYND11 knockdown U2OS cells. GAPDH was used as a loading control.

Extended Data Figure 8 ZMYND11 knockdown increases the occupancies of total Pol II and Pol II S2P in gene bodies.

a, Venn diagram showing the overlap of ZMYND11- and Pol II-occupied genes. P < 1 × 10−322 (Fisher’s exact test). A comprehensive list of Pol II ChIP-seq peaks is given in Supplementary Table 6. b, Average genome-wide ZMYND11 occupancy on genes with high, intermediate and low levels of Pol II occupancy. Genes were aligned 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 tag numbers. c, Average genome-wide occupancies of Pol II along the transcription unit of all ZMYND11-activated direct target genes (left) and all non-ZMYND11 target genes (right) in control and ZMYND11 knockdown cells as in Fig. 4d. d, Average genome-wide occupancies of Pol II S2P near the TTS of all ZMYND11-repressed direct target genes in control and ZMYND11 knockdown cells. e, qPCR analysis of the ZMYND11, H3K36me3 and Pol II S2P ChIP in the intragenic regions of MYC and NFKB2 genes in control and ZMYND11 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. The left panel shows the schematic representation of the calculation of Pol II travelling ratio21. The right panel shows the whisker plot of Pol II travelling ratio. P = 2.5 × 10−5 (Student’s t-test).

Extended Data Figure 9 ZMYND11 suppresses tumour cell growth and is downregulated and mutated in human cancers.

a, ZMYND11 is downregulated in cancers. ZMYND11 gene expression in approximately 40,000 tumour or normal tissue samples from three data sets (Gene Expression Omnibus, ArrayExpress and Expression Project for Oncology) were analysed using GENT. N, normal; C, cancer. All P < 0.0001. b, Cell proliferation assay of U2OS cells (mean ± s.e.m., n = 3) with knockdown (KD) or overexpression (OE) of ZMYND11. c, Cell proliferation assay of U2OS cells stably expressing the wild-type or mutant ZMYND11 proteins (mean ± s.e.m., n = 5). Cells were counted 6 days after seeding. d, Colony formation assay of cells as in c. Cell colonies (mean ± s.e.m., n = 3) were counted 2 weeks after seeding. e, Western blot analysis of ZMYND11 expression levels in stable MDA-MB 231 cells used in Fig. 4e. f, Schematic representation of ZMYND11 missense somatic mutations in colon and rectum adenocarcinoma identified in the TCGA database. g, Peptide pull-downs of wild-type ZMYND11 PBP and the D307N mutant with H3.3K36me peptides. h, Western blot analysis of Flag-tagged wild-type ZMYND11 and the D307N mutant stably expressed in MDA-MB 231 cells. i, qPCR analysis of Flag ChIP in MYC and NFKB2 genes in the stable cells 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 breast cancer correlate with worse disease-free survivals. Kaplan–Meier survival curves of patients with breast cancer from cohort studies National Institutes of Health Gene Expression Omnibus GSE6532 (j) and GSE7390 (k). P values were calculated by χ2 test. l, ITC curves of the histone H3.3(G34R)K36me3 and H3.3(G34V)K36me3 peptides titrated into ZMYND11 bromo–PWWP domains.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion that describes in detail the crystal structures of ZMYND11 BP domains in complex with H3.3K36me3, and in free state. It also contains Supplementary Tables 1 and 7. (PDF 204 kb)

Supplementary Data

This file contains Supplementary Tables 2-6. (XLSX 13741 kb)

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Wen, H., Li, Y., Xi, Y. et al. ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression. Nature 508, 263–268 (2014). https://doi.org/10.1038/nature13045

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