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Structure of the MLL CXXC domain–DNA complex and its functional role in MLL-AF9 leukemia

Nature Structural & Molecular Biology volume 17, pages 62–68 (2010)Cite this article

Abstract

The gene MLL (encoding the protein mixed-lineage leukemia) is the target of chromosomal translocations that cause leukemias with poor prognosis. All leukemogenic MLL fusion proteins retain the CXXC domain, which binds to nonmethylated CpG DNA sites. We present the solution structure of the MLL CXXC domain in complex with DNA, showing how the CXXC domain distinguishes nonmethylated from methylated CpG DNA. On the basis of the structure, we generated point mutations that disrupt DNA binding. Introduction of these mutations into the MLL-AF9 fusion protein resulted in increased DNA methylation of specific CpG nucleotides in Hoxa9, increased H3K9 methylation, decreased expression of Hoxa9-locus transcripts, loss of immortalization potential, and inability to induce leukemia in mice. These results establish that DNA binding by the CXXC domain and protection against DNA methylation is essential for MLL fusion leukemia. They also provide support for viewing this interaction as a potential target for therapeutic intervention.

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Figure 1: Structure of the CXXC–DNA complex and details of CpG recognition.
Figure 2: Design of mutations impairing interaction of the CXXC domain with DNA.
Figure 3: DNA-binding activity of the MLL CXXC domain is required for MLL-AF9 to protect Hoxa9 from DNA methylation and induce Hoxa9 and mir196b transcript expression, but not for binding to the locus.
Figure 4: DNA-binding activity of the MLL CXXC domain in MLL-AF9 is required for increased proliferative capacity and immortalization in a bone marrow progenitor serial re-plating assay.
Figure 5: DNA-binding activity of the MLL CXXC domain is required for MLL-AF9 to cause leukemia in vivo.
Figure 6: Model of the regulation of Hoxa9 locus transcription by the CXXC domain of MLL-AF9.

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References

  1. Tkachuk, D.C., Kohler, S. & Cleary, M.L. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 71, 691–700 (1992).

    Article  CAS  Google Scholar 

  2. Sedkov, Y., Tillib, S., Mizrokhi, L. & Mazo, A. The bithorax complex is regulated by trithorax earlier during Drosophila embryogenesis than is the Antennapedia complex, correlating with a bithorax-like expression pattern of distinct early trithorax transcripts. Development 120, 1907–1917 (1994).

    CAS  PubMed  Google Scholar 

  3. Yu, B.D., Hess, J.L., Horning, S.E., Brown, G.A. & Korsmeyer, S.J. Altered Hox expression and segmental identity in Mll-mutant mice. Nature 378, 505–508 (1995).

    Article  CAS  Google Scholar 

  4. Muller, J., Gaunt, S. & Lawrence, P.A. Function of the Polycomb protein is conserved in mice and flies. Development 121, 2847–2852 (1995).

    CAS  PubMed  Google Scholar 

  5. Orlando, V., Jane, E.P., Chinwalla, V., Harte, P.J. & Paro, R. Binding of trithorax and Polycomb proteins to the bithorax complex: dynamic changes during early Drosophila embryogenesis. EMBO J. 17, 5141–5150 (1998).

    Article  CAS  Google Scholar 

  6. Guenther, M.G. et al. Global and Hox-specific roles for the MLL1 methyltransferase. Proc. Natl. Acad. Sci. USA 102, 8603–8608 (2005).

    Article  CAS  Google Scholar 

  7. Rice, K.L. & Licht, J.D. HOX deregulation in acute myeloid leukemia. J. Clin. Invest. 117, 865–868 (2007).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Popovic, R. & Zeleznik-Le, N.J. MLL: how complex does it get? J. Cell. Biochem. 95, 234–242 (2005).

    Article  CAS  Google Scholar 

  10. Sorensen, P.H. et al. Molecular rearrangements of the MLL gene are present in most cases of infant acute myeloid leukemia and are strongly correlated with monocytic or myelomonocytic phenotypes. J. Clin. Invest. 93, 429–437 (1994).

    Article  CAS  Google Scholar 

  11. Cox, M.C. et al. Chromosomal aberration of the 11q23 locus in acute leukemia and frequency of MLL gene translocation: results in 378 adult patients. Am. J. Clin. Pathol. 122, 298–306 (2004).

    Article  CAS  Google Scholar 

  12. Dimartino, J.F. & Cleary, M.L. Mll rearrangements in haematological malignancies: lessons from clinical and biological studies. Br. J. Haematol. 106, 614–626 (1999).

    Article  CAS  Google Scholar 

  13. Meyer, C. et al. The MLL recombinome of acute leukemias. Leukemia 20, 777–784 (2006).

    Article  CAS  Google Scholar 

  14. Krivtsov, A.V. & Armstrong, S.A. MLL translocations, histone modifications and leukaemia stem-cell development. Nat. Rev. Cancer 7, 823–833 (2007).

    Article  CAS  Google Scholar 

  15. Yokoyama, A. et al. The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis. Cell 123, 207–218 (2005).

    Article  CAS  Google Scholar 

  16. Yokoyama, A. & Cleary, M.L. Menin critically links MLL proteins with LEDGF on cancer-associated target genes. Cancer Cell 14, 36–46 (2008).

    Article  CAS  Google Scholar 

  17. Birke, M. et al. The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation. Nucleic Acids Res. 30, 958–965 (2002).

    Article  CAS  Google Scholar 

  18. Ayton, P.M., Chen, E.H. & Cleary, M.L. Binding to nonmethylated CpG DNA is essential for target recognition, transactivation, and myeloid transformation by an MLL oncoprotein. Mol. Cell. Biol. 24, 10470–10478 (2004).

    Article  CAS  Google Scholar 

  19. Xia, Z.B., Anderson, M., Diaz, M.O. & Zeleznik-Le, N.J. MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein. Proc. Natl. Acad. Sci. USA 100, 8342–8347 (2003).

    Article  CAS  Google Scholar 

  20. Erfurth, F.E. et al. MLL protects CpG clusters from methylation within the Hoxa9 gene, maintaining transcript expression. Proc. Natl. Acad. Sci. USA 105, 7517–7522 (2008).

    Article  CAS  Google Scholar 

  21. Baylin, S.B. et al. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum. Mol. Genet. 10, 687–692 (2001).

    Article  CAS  Google Scholar 

  22. Ohki, I. et al. Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA. Cell 105, 487–497 (2001).

    Article  CAS  Google Scholar 

  23. Jorgensen, H.F., Ben-Porath, I. & Bird, A.P. Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains. Mol. Cell. Biol. 24, 3387–3395 (2004).

    Article  CAS  Google Scholar 

  24. Lee, J.H., Voo, K.S. & Skalnik, D.G. Identification and characterization of the DNA binding domain of CpG-binding protein. J. Biol. Chem. 276, 44669–44676 (2001).

    Article  CAS  Google Scholar 

  25. Inomata, K. et al. Kinetic and thermodynamic evidence for flipping of a methyl-CpG binding domain on methylated DNA. Biochemistry 47, 3266–3271 (2008).

    Article  CAS  Google Scholar 

  26. Allen, M.D. et al. Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukemia-associated MLL histone methyltransferase. EMBO J. 25, 4503–4512 (2006).

    Article  CAS  Google Scholar 

  27. Jones, S., van Heyningen, P., Berman, H.M. & Thornton, J.M. Protein-DNA interactions: a structural analysis. J. Mol. Biol. 287, 877–896 (1999).

    Article  CAS  Google Scholar 

  28. Popovic, R. et al. Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization. Blood 113, 3314–3322 (2009).

    Article  CAS  Google Scholar 

  29. Milne, T.A. et al. Menin and MLL cooperatively regulate expression of cyclin-dependent kinase inhibitors. Proc. Natl. Acad. Sci. USA 102, 749–754 (2005).

    Article  CAS  Google Scholar 

  30. Lavau, C., Du, C., Thirman, M. & Zeleznik-Le, N. Chromatin-related properties of CBP fused to MLL generate a myelodysplastic-like syndrome that evolves into myeloid leukemia. EMBO J. 19, 4655–4664 (2000).

    Article  CAS  Google Scholar 

  31. Krivtsov, A.V. et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature 442, 818–822 (2006).

    Article  CAS  Google Scholar 

  32. Somervaille, T.C. & Cleary, M.L. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell 10, 257–268 (2006).

    Article  CAS  Google Scholar 

  33. Bach, C., Mueller, D., Buhl, S., Garcia-Cuellar, M.P. & Slany, R.K. Alterations of the CxxC domain preclude oncogenic activation of mixed-lineage leukemia 2. Oncogene 28, 815–823 (2009).

    Article  CAS  Google Scholar 

  34. Milne, T.A., Martin, M.E., Brock, H.W., Slany, R.K. & Hess, J.L. Leukemogenic MLL fusion proteins bind across a broad region of the Hox a9 locus, promoting transcription and multiple histone modifications. Cancer Res. 65, 11367–11374 (2005).

    Article  CAS  Google Scholar 

  35. Slany, R.K. When epigenetics kills: MLL fusion proteins in leukemia. Hematol. Oncol. 23, 1–9 (2005).

    Article  CAS  Google Scholar 

  36. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

  37. Cierpicki, T. & Bushweller, J.H. Charged gels as orienting media for measurement of residual dipolar couplings in soluble and integral membrane proteins. J. Am. Chem. Soc. 126, 16259–16266 (2004).

    Article  CAS  Google Scholar 

  38. Tugarinov, V. & Kay, L.E. Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G. J. Mol. Biol. 327, 1121–1133 (2003).

    Article  CAS  Google Scholar 

  39. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  40. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods 25, 402–408 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants CA105049 and HL087188 and a grant from the Dr. Ralph and Marian Falk Medical Research Trust (to N.J.Z.-L.), NIH Experimental Immunology training grant T32 AI007508-11A1 (to L.E.R.) and by Leukemia and Lymphoma Society SCOR grant 7006-05 (to J.H.B.).

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Author notes

  1. Tomasz Cierpicki & Laurie E Risner

    Present address: These authors contributed equally to this work. Present address: Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA (T.C. and J.G.).,

Authors and Affiliations

  1. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA

    Tomasz Cierpicki, Jolanta Grembecka, Monika Omonkowska, David D Shultis & John H Bushweller

  2. Department of Medicine, Molecular Biology Program, Oncology Institute, Loyola University, Chicago, Maywood, Illinois, USA

    Laurie E Risner, Relja Popovic & Nancy J Zeleznik-Le

  3. Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA

    Stephen M Lukasik & John H Bushweller

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  1. Tomasz Cierpicki
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Contributions

T.C., L.E.R. and J.G. designed and performed experiments, analyzed data and wrote the manuscript; S.M.L., R.P., M.O. and D.S.S. designed and performed experiments and analyzed data. N.J.Z.-L. and J.H.B. designed experiments, analyzed data and wrote the manuscript.

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Correspondence to Nancy J Zeleznik-Le or John H Bushweller.

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Cierpicki, T., Risner, L., Grembecka, J. et al. Structure of the MLL CXXC domain–DNA complex and its functional role in MLL-AF9 leukemia. Nat Struct Mol Biol 17, 62–68 (2010). https://doi.org/10.1038/nsmb.1714

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