Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

Abstract

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: UNC1215 is a potent antagonist of L3MBTL3.
Figure 2: X-ray crystal structure of the UNC1215–3MBT complex.
Figure 3: UNC1215 binds a small set of Kme reader proteins with lower affinity than L3MBTL3.
Figure 4: UNC1215 potently antagonizes 3MBT localization in cells.
Figure 5: UNC1215 binds and colocalizes with full-length L3MBTL3.
Figure 6: Identification of BCLAF1 as a new L3MBTL3 protein interactor.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Wang, Z. et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat. Genet. 40, 897–903 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Taverna, S.D., Li, H., Ruthenburg, A.J., Allis, C.D. & Patel, D.J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025–1040 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hughes, R.M., Wiggins, K.R., Khorasanizadeh, S. & Waters, M.L. Recognition of trimethyllysine by a chromodomain is not driven by the hydrophobic effect. Proc. Natl. Acad. Sci. USA 104, 11184–11188 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zacharias, N. & Dougherty, D.A. Cation-π interactions in ligand recognition and catalysis. Trends Pharmacol. Sci. 23, 281–287 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Dawson, M.A. et al. Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 478, 529–533 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Delmore, J.E. et al. BET bromodomain inhibition as a therapeutic strategy to target c-myc. Cell 146, 904–917 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zeng, L. et al. Mechanism and regulation of acetylated histone binding by the tandem PHD finger of DPF3b. Nature 466, 258–262 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Margueron, R. et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Adams-Cioaba, M.A. & Min, J. Structure and function of histone methylation binding proteins. Biochem. Cell Biol. 87, 93–105 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Kuo, A.J. et al. The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome. Nature 484, 115–119 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Li, H. et al. Structural basis for lower lysine methylation state–specific readout by MBT repeats of L3MBTL1 and an engineered PHD finger. Mol. Cell 28, 677–691 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Santiago, C., Nguyen, K. & Schapira, M. Druggability of methyl-lysine binding sites. J. Comput. Aided Mol. Des. 25, 1171–1178 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Herold, J.M. et al. Structure-activity relationships of methyl-lysine reader antagonists. Med. Chem. Commun. 3, 45–51 (2012).

    Article  CAS  Google Scholar 

  15. Herold, J.M. et al. Small-molecule ligands of methyl-lysine binding proteins. J. Med. Chem. 54, 2504–2511 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Frye, S.V. The art of the chemical probe. Nat. Chem. Biol. 6, 159–161 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Bonasio, R., Lecona, E. & Reinberg, D. MBT domain proteins in development and disease. Semin. Cell Dev. Biol. 21, 221–230 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Addou-Klouche, L. et al. Loss, mutation and deregulation of L3MBTL4 in breast cancers. Mol. Cancer 9, 213 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Northcott, P.A. et al. Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma. Nat. Genet. 41, 465–472 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gurvich, N. et al. L3MBTL1 polycomb protein, a candidate tumor suppressor in del(20q12) myeloid disorders, is essential for genome stability. Proc. Natl. Acad. Sci. USA 107, 22552–22557 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Perna, F. et al. Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q- polycythemia vera. Blood 116, 2812–2821 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Honda, H. et al. Hemp, an MBT domain–containing protein, plays essential roles in hematopoietic stem cell function and skeletal formation. Proc. Natl. Acad. Sci. USA 108, 2468–2473 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Arai, S. & Miyazaki, T. Impaired maturation of myeloid progenitors in mice lacking novel Polycomb group protein MBT-1. EMBO J. 24, 1863–1873 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nady, N. et al. Histone recognition by human malignant brain tumor domains. J. Mol. Biol. 423, 702–718 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Bonasio, R., Lecona, E. & Reinberg, D. MBT domain proteins in development and disease. Semin. Cell Dev. Biol. 21, 221–230 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Botuyan, M.V. et al. Structural basis for the methylation state–specific recognition of histone H4–K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wigle, T.J. et al. Screening for inhibitors of low-affinity epigenetic peptide-protein interactions: an AlphaScreenTM-based assay for antagonists of methyl-lysine binding proteins. J. Biomol. Screen. 15, 62–71 (2010).

    Article  CAS  PubMed  Google Scholar 

  28. Gao, C. et al. Biophysical probes reveal a “compromise” nature of the methyl-lysine binding pocket in L3MBTL1. J. Am. Chem. Soc. 133, 5357–5362 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Nady, N. et al. Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein. J. Biol. Chem. 286, 24300–24311 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wang, W.K. et al. Malignant brain tumor repeats: a three-leaved propeller architecture with ligand/peptide binding pockets. Structure 11, 775–789 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kim, J. et al. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7, 397–403 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Takada, Y. et al. Mammalian polycomb Scmh1 mediates exclusion of polycomb complexes from the XY body in the pachytene spermatocytes. Development 134, 579–590 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Trojer, P. et al. L3MBTL2 protein acts in concert with PcG protein–mediated monoubiquitination of H2A to establish a repressive chromatin structure. Mol. Cell 42, 438–450 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Trojer, P. et al. L3MBTL1, a histone-methylation–dependent chromatin lock. Cell 129, 915–928 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Ogawa, H., Ishiguro, K.-I., Gaubatz, S., Livingston, D.M. & Nakatani, Y. A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells. Science 296, 1132–1136 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Nady, N. et al. Histone recognition by human malignant brain tumor domains. J. Mol. Biol. 423, 702–718 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Kim, C.A., Gingery, M., Pilpa, R.M. & Bowie, J.U. The SAM domain of polyhomeotic forms a helical polymer. Nat. Struct. Biol. 9, 453–457 (2002).

    CAS  PubMed  Google Scholar 

  38. Knight, M.J., Leettola, C., Gingery, M., Li, H. & Bowie, J.U. A human sterile α motif domain polymerizome. Protein Sci. 20, 1697–1706 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Haraguchi, T. et al. Emerin binding to Btf, a death-promoting transcriptional repressor, is disrupted by a missense mutation that causes Emery-Dreifuss muscular dystrophy. Eur. J. Biochem. 271, 1035–1045 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Lee, Y.Y., Yu, Y.B., Gunawardena, H.P., Xie, L. & Chen, X. BCLAF1 is a radiation-induced H2AX-interacting partner involved in γH2AX-mediated regulation of apoptosis and DNA repair. Cell Death Dis. 3, e359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hope, H. Cryocrystallography of biological macromolecules: a generally applicable method, Acta Crystallogr. B 44, 22–26 (1988).

    Article  PubMed  Google Scholar 

  42. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Evans, P.R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol. Crystallogr. 67, 282–292 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  PubMed  Google Scholar 

  45. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  46. Berman, H.M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Schüttelkopf, A.W. & van Aalten, D.M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  PubMed  Google Scholar 

  48. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  51. Yguerabide, J., Schmidt, J.A. & Yguerabide, E.E. Lateral mobility in membranes as detected by fluorescence recovery after photobleaching. Biophys. J. 40, 69–75 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Vedadi, G. Wasney and F. Li for support with the protein lysine methyltransferase selectivity screening; O. Fedorov for support with the bromodomain selectivity screening; A. Tumber for support with the lysine demethylase selectivity screening; E. Hull-Ryde for support with the CellTiter-Glo cell viability assay; K. Hahn (University of North Carolina (UNC)) for providing mero76; B. Roth for helpful discussion regarding the GPCR selectivity studies; and G. Wang (UNC) for providing PHF23 and JARID1 proteins. Results shown in this report are derived from work performed at the Structural Biology Center at the Advanced Photon Source at Argonne National Laboratory. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. The research described here was supported by the US National Institute of General Medical Sciences; US National Institutes of Health (grant RC1GM090732 and R01GM100919); the Carolina Partnership and the University Cancer Research Fund; University of North Carolina at Chapel Hill; the Center for Environmental and Molecular Carcinogenesis at the M.D. Anderson Cancer Center; the National Institute of Mental Health Psychoactive Drug Screening Program; the Ontario Research Fund (grant ORF-GL2); the Natural Sciences and Engineering Research Council of Canada; the Ontario Ministry of Health and Long-Term Care; the American Cancer Society (C.J.M.; 119169-PF-10-183-01-TBE); and the Structural Genomics Consortium, which is a registered charity (number 1097737) that receives funds from Canadian Institutes of Health Research; Eli Lilly Canada; Genome Canada; GlaxoSmithKline; the Ontario Ministry of Economic Development and Innovation; the Novartis Research Foundation; Pfizer; Abbott; Takeda; and the Wellcome Trust. M.T.B. is supported by an institutional grant from the US National Institute of Environmental Health Sciences (ES007784) and Cancer Prevention Research Institute of Texas funding (RP110471). C.H.A. holds a Canada Research Chair in Structural Genomics.

Author information

Authors and Affiliations

Authors

Contributions

L.I.J. synthesized all compounds and related analogs and performed ITC studies; D.B.-L. and L.K. performed immunofluorescence FRAP, affinity purification and coimmunoprecipitation studies; N.Z. and W.T. solved and analyzed the X-ray crystal structure of the UNC1215–L3MBTL3 complex; V.K.K. and W.P.J. performed and analyzed AlphaScreen studies; C.J.M. synthesized the mero76-UNC1215 conjugate; J.L.N. purified proteins and performed mutagenesis; C.A.S. and M.T.B. performed protein array and protein pull-down experiments; E.M., H.G., A.E. and J.F.G. performed MS-based studies; S.D. cloned mammalian expression vectors for all cellular studies; X.-P.H. performed and analyzed GPCR selectivity studies; L.I.J., D.B.-L., N.Z., L.K., J.M.H., V.K.K., C.G., D.B.K., J.J., W.P.J., P.J.B., M.T.B, C.H.A. and S.V.F. designed studies and discussed results; L.I.J., C.H.A. and S.V.F. wrote the paper.

Corresponding authors

Correspondence to Cheryl H Arrowsmith or Stephen V Frye.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 4334 kb)

Supplementary Note 1

SuppNote_final.pdf (PDF 795 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

James, L., Barsyte-Lovejoy, D., Zhong, N. et al. Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain. Nat Chem Biol 9, 184–191 (2013). https://doi.org/10.1038/nchembio.1157

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1157

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing