The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers. KMT2C (hereafter referred to as MLL3) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in MLL3 within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated MLL3 mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring BAP1 or MLL3 mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in MLL3 and suggests that restoration of a balanced state of Polycomb–COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Morgan, M. A. & Shilatifard, A. Chromatin signatures of cancer. Genes Dev. 29, 238–249 (2015).

  2. 2.

    Plass, C. et al. Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. Nat. Rev. Genet. 14, 765–780 (2013).

  3. 3.

    Hamamoto, R. et al. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat. Cell Biol. 6, 731–740 (2004).

  4. 4.

    Kotake, Y. et al. pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16 INK4α tumor suppressor gene. Genes Dev. 21, 49–54 (2007).

  5. 5.

    Mazur, P. K. et al. SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer. Nature 510, 283–287 (2014).

  6. 6.

    Piunti, A. & Shilatifard, A. Epigenetic balance of gene expression by Polycomb and COMPASS families. Science 352, aad9780 (2016).

  7. 7.

    Ferguson, A. D. et al. Structural basis of substrate methylation and inhibition of SMYD2. Structure 19, 1262–1273 (2011).

  8. 8.

    Herz, H. M. et al. Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling. Science 345, 1065–1070 (2014).

  9. 9.

    Klaus, C. R. et al. DOT1L inhibitor EPZ-5676 displays synergistic anti-proliferative activity in combination with standard-of-care drugs and hypomethylating agents in MLL-rearranged leukemia cells. J. Pharmacol. Exp. Ther. 350, 646–656 (2014).

  10. 10.

    Kubicek, S. et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol. Cell 25, 473–481 (2007).

  11. 11.

    Mohan, M., Herz, H. M. & Shilatifard, A. SnapShot: histone lysine methylase complexes. Cell 149, 498–498.e1 (2012).

  12. 12.

    Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).

  13. 13.

    Shilatifard, A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis. Annu. Rev. Biochem. 81, 65–95 (2012).

  14. 14.

    Miller, T. et al. COMPASS: a complex of proteins associated with a trithorax-related SET domain protein. Proc. Natl Acad. Sci. USA 98, 12902–12907 (2001).

  15. 15.

    Morgan, M. A. & Shilatifard, A. Drosophila SETs its sights on cancer: Trr/MLL3/MLL4 COMPASS-like complexes in development and disease. Mol. Cell. Biol. 33, 1698–1701 (2013).

  16. 16.

    Hu, D. & Shilatifard, A. Epigenetics of hematopoiesis and hematological malignancies. Genes Dev. 30, 2021–2041 (2016).

  17. 17.

    Yu, B. D., Hanson, R. D., Hess, J. L., Horning, S. E. & Korsmeyer, S. J. MLL, a mammalian trithorax-group gene, functions as a transcriptional maintenance factor in morphogenesis. Proc. Natl Acad. Sci. USA 95, 10632–10636 (1998).

  18. 18.

    Ortega-Molina, A. et al. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development. Nat. Med. 21, 1199–1208 (2015).

  19. 19.

    Zhang, J. et al. Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis. Nat. Med. 21, 1190–1198 (2015).

  20. 20.

    Herz, H. M. et al. Enhancer-associated H3K4 monomethylation by trithorax-related, the Drosophila homolog of mammalian MLL3 and MLL4. Genes Dev. 26, 2604–2620 (2012).

  21. 21.

    Hu, D. et al. The MLL3 and MLL4 branches of the COMPASS family function as major histone H3K4 monomethylases at enhancers. Mol. Cell. Biol. 33, 4745–4754 (2013).

  22. 22.

    Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013).

  23. 23.

    Fujimoto, A. et al. Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators. Nat. Genet. 44, 760–764 (2012).

  24. 24.

    Ellis, M. J. et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature 486, 353–360 (2012).

  25. 25.

    Li, W. D. et al. Exome sequencing identifies an MLL3 gene germ line mutation in a pedigree of colorectal cancer and acute myeloid leukemia. Blood 121, 1478–1479 (2013).

  26. 26.

    Gui, Y. et al. Frequent mutations of chromatin-remodeling genes in transitional cell carcinoma of the bladder. Nat. Genet. 43, 875–878 (2011).

  27. 27.

    Chen, C. et al. MLL3 is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia. Cancer Cell 25, 652–665 (2014).

  28. 28.

    Chaudhuri, A. R. et al. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387 (2016).

  29. 29.

    Bailey, P. et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52 (2016).

  30. 30.

    Paoletti, A. C. et al. Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc. Natl Acad. Sci. USA 103, 18928–18933 (2006).

  31. 31.

    Ramón-Maiques, S. et al. The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine 4 and arginine 2. Proc. Natl Acad. Sci. USA 104, 18993–18998 (2007).

  32. 32.

    Carbone, M. et al. BAP1 and cancer. Nat. Rev. Cancer 13, 153–159 (2013).

  33. 33.

    Dey, A. et al. Loss of the tumor suppressor BAP1 causes myeloid transformation. Science 337, 1541–1546 (2012).

  34. 34.

    Tripathi, S. et al. Meta- and orthogonal integration of influenza “OMICs” data defines a role for UBR4 in virus budding. Cell Host Microbe 18, 723–735 (2015).

  35. 35.

    Cieply, B., Farris, J., Denvir, J., Ford, H. L. & Frisch, S. M. Epithelial–mesenchymal transition and tumor suppression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2. Cancer Res. 73, 6299–6309 (2013).

  36. 36.

    Li, Y. et al. Downregulation of RBMS3 is associated with poor prognosis in esophageal squamous cell carcinoma. Cancer Res. 71, 6106–6115 (2011).

  37. 37.

    Nguyen, T. M. H. et al. Loss of ITM2A, a novel tumor suppressor of ovarian cancer through G2/M cell cycle arrest, is a poor prognostic factor of epithelial ovarian cancer. Gynecol. Oncol. 140, 545–553 (2016).

  38. 38.

    Cha, Y., Kim, D. K., Hyun, J., Kim, S. J. & Park, K. S. TCEA3 binds to TGF-β receptor 1 and induces SMAD-independent, JNK-dependent apoptosis in ovarian cancer cells. Cell. Signal. 25, 1245–1251 (2013).

  39. 39.

    Cui, T. et al. The p53 target gene desmocollin 3 acts as a novel tumor suppressor through inhibiting EGFR–ERK pathway in human lung cancer. Carcinogenesis 33, 2326–2333 (2012).

  40. 40.

    Qin, S., Zhang, Z., Li, J. & Zang, L. FRZB knockdown upregulates β-catenin activity and enhances cell aggressiveness in gastric cancer. Oncol. Rep. 31, 2351–2357 (2014).

  41. 41.

    Wang, S. et al. DACT2 is a functional tumor suppressor through inhibiting Wnt–β-catenin pathway and associated with poor survival in colon cancer. Oncogene 34, 2575–2585 (2015).

  42. 42.

    LaFave, L. M. et al. Reply to “Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status”. Nat. Med. 22, 578–579 (2016).

  43. 43.

    LaFave, L. M. et al. Loss of BAP1 function leads to EZH2-dependent transformation. Nat. Med. 21, 1344–1349 (2015).

  44. 44.

    Schoumacher, M. et al. Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status. Nat. Med. 22, 577–578 (2016).

  45. 45.

    Takahashi, Y. H. et al. Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human. Proc. Natl Acad. Sci. USA 108, 20526–20531 (2011).

  46. 46.

    McDonald, E. R. et al. Project DRIVE: a compendium of cancer dependencies and synthetic lethal relationships uncovered by large-scale, deep RNAi screening. Cell 170, 577–592 (2017).

  47. 47.

    Toska, E. et al. PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D. Science 355, 1324–1330 (2017).

  48. 48.

    Lu, Z. H. et al. Mammalian target of rapamycin activator RHEB is frequently overexpressed in human carcinomas and is critical and sufficient for skin epithelial carcinogenesis. Cancer Res. 70, 3287–3298 (2010).

  49. 49.

    Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).

  50. 50.

    Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013).

  51. 51.

    Wang, L. et al. A cytoplasmic COMPASS is necessary for cell survival and triple-negative breast cancer pathogenesis by regulating metabolism. Genes Dev. 31, 2056–2066 (2017).

  52. 52.

    Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

  53. 53.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  54. 54.

    Saldanha, A. J. Java Treeview—extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

  55. 55.

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

  56. 56.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

  57. 57.

    Shen, L., Shao, N., Liu, X. & Nestler, E. ngs.plot: quick mining and visualization of next-generation sequencing data by integrating genomic databases. BMC Genomics 15, 284 (2014).

  58. 58.

    McLean, C. Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010).

  59. 59.

    Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR–Cas9 knockout screens. Genome Biol. 15, 554 (2014).

Download references


We would like to thank F. Zhang (MIT) for the kind gifts of the Px330 and lentiCRISPR v2 vectors. L.W. is supported by the Training Program in Signal Transduction and Cancer (T32 CA070085). Z.Z. is supported by the Robert H. Lurie Comprehensive Cancer Center—Translational Bridge Program Fellowship in Lymphoma Research. E.R.S. is supported by NCI grant R50CA211428. Studies in J.N.S.'s laboratory are supported by NIDCD grant DC013805, and studies related to COMPASS in A.S.'s laboratory are supported by NCI's Outstanding Investigator Award R35CA197569.

Author information


  1. Simpson Querrey Center for Epigenetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    • Lu Wang
    • , Zibo Zhao
    • , Patrick A. Ozark
    • , Damiano Fantini
    • , Stacy A. Marshall
    • , Emily J. Rendleman
    • , Marc A. Morgan
    • , Yoh-hei Takahashi
    • , Edwin R. Smith
    • , Panagiotis Ntziachristos
    • , Lihua Zou
    • , Joshua J. Meeks
    •  & Ali Shilatifard
  2. Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    • Lu Wang
    • , Zibo Zhao
    • , Patrick A. Ozark
    • , Stacy A. Marshall
    • , Emily J. Rendleman
    • , Kira A. Cozzolino
    • , Marc A. Morgan
    • , Yoh-hei Takahashi
    • , Clayton K. Collings
    • , Edwin R. Smith
    • , Panagiotis Ntziachristos
    • , Lihua Zou
    • , Rintaro Hashizume
    • , Joshua J. Meeks
    •  & Ali Shilatifard
  3. Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    • Nundia Louis
    • , Xingyao He
    •  & Rintaro Hashizume
  4. Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    • Jeffrey N. Savas


  1. Search for Lu Wang in:

  2. Search for Zibo Zhao in:

  3. Search for Patrick A. Ozark in:

  4. Search for Damiano Fantini in:

  5. Search for Stacy A. Marshall in:

  6. Search for Emily J. Rendleman in:

  7. Search for Kira A. Cozzolino in:

  8. Search for Nundia Louis in:

  9. Search for Xingyao He in:

  10. Search for Marc A. Morgan in:

  11. Search for Yoh-hei Takahashi in:

  12. Search for Clayton K. Collings in:

  13. Search for Edwin R. Smith in:

  14. Search for Panagiotis Ntziachristos in:

  15. Search for Jeffrey N. Savas in:

  16. Search for Lihua Zou in:

  17. Search for Rintaro Hashizume in:

  18. Search for Joshua J. Meeks in:

  19. Search for Ali Shilatifard in:


L.W. and A.S. designed the study; L.W. and Z.Z. performed the majority of the experiments and part of the analyses, and wrote the first draft of the manuscript; R.H., N.L., X.H., L.W. and A.S. designed the in vivo studies; R.H., N.L. and X.H. performed and analyzed the in vivo experiments; S.A.M. and E.J.R. generated and sequenced the NGS libraries; K.A.C. and J.N.S. performed the mass spectrometry experiments and analyzed the results; C.K.C. performed the initial bioinformatics analyses on the studies related to the role of BAP1 and MLL3 at enhancers; P.A.O. performed all of the other bioinformatics analyses; J.J.M. provided clinical supervision in the interpretation of data; L.Z. and D.F. performed clinical data analysis; P.N. helped with the UTX ChIP-seq; Y.T. performed size-exclusion chromatography; L.W., Z.Z., M.A.M., E.R.S. and A.S. revised the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Ali Shilatifard.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–20

  2. Reporting Summary

  3. Supplementary Table 1

    Raw data for mass spectrometry of MLL3-NTD purification

  4. Supplementary Table 2

    Raw data for MLL3 mutations in human cancers

  5. Supplementary Table 3

    Genes that are significantly regulated by MLL3, BAP1 and UTX

  6. Supplementary Table 4

    Genes that are upregulated by MLL3 for more than twofold

  7. Supplementary Table 5

    Genes that are involved in epithelial cell differentiation pathway

  8. Supplementary Table 6

    GSEA pathway analysis for genes upregulated by MLL3

  9. Supplementary Table 7

    Genes that are upregulated by MLL3 and BAP1 and GSEA pathway analysis

  10. Supplementary Table 8

    Genes that are up-regulated by MLL3, BAP1 and UTX and GSEA pathway analysis

About this article

Publication history






Further reading