Letter | Published:

Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX

Nature volume 497, pages 122126 (02 May 2013) | Download Citation

Subjects

Abstract

TET (ten-eleven-translocation) proteins are Fe(ii)- and α-ketoglutarate-dependent dioxygenases1,2,3 that modify the methylation status of DNA by successively oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine1,3,4,5, potential intermediates in the active erasure of DNA-methylation marks5,6. Here we show that IDAX (also known as CXXC4), a reported inhibitor of Wnt signalling7 that has been implicated in malignant renal cell carcinoma8 and colonic villous adenoma9, regulates TET2 protein expression. IDAX was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The IDAX CXXC domain binds DNA sequences containing unmethylated CpG dinucleotides, localizes to promoters and CpG islands in genomic DNA and interacts directly with the catalytic domain of TET2. Unexpectedly, IDAX expression results in caspase activation and TET2 protein downregulation, in a manner that depends on DNA binding through the IDAX CXXC domain, suggesting that IDAX recruits TET2 to DNA before degradation. IDAX depletion prevents TET2 downregulation in differentiating mouse embryonic stem cells, and short hairpin RNA against IDAX increases TET2 protein expression in the human monocytic cell line U937. Notably, we find that the expression and activity of TET3 is also regulated through its CXXC domain. Taken together, these results establish the separate and linked CXXC domains of TET2 and TET3, respectively, as previously unknown regulators of caspase activation and TET enzymatic activity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Gene Expression Omnibus

Data deposits

The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE42958.

References

  1. 1.

    et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009)

  2. 2.

    , , & Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids. Cell Cycle 8, 1698–1710 (2009)

  3. 3.

    et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468, 839–843 (2010)

  4. 4.

    et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011)

  5. 5.

    et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011)

  6. 6.

    & Dependence of substrate binding and catalysis on pH, ionic strength, and temperature for thymine DNA glycosylase: Insights into recognition and processing of G.T mispairs. DNA Repair 10, 545–553 (2011)

  7. 7.

    et al. Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein. Mol. Cell. Biol. 21, 330–342 (2001)

  8. 8.

    et al. Decreased expression of CXXC4 promotes a malignant phenotype in renal cell carcinoma by activating Wnt signaling. Oncogene 28, 297–305 (2009)

  9. 9.

    , & Differentiation of tubular and villous adenomas based on Wnt pathway-related gene expression profiles. Int. J. Mol. Med. 26, 121–125 (2010)

  10. 10.

    , & Natural history of eukaryotic DNA methylation systems. Prog. Mol. Biol. Transl. Sci. 101, 25–104 (2011)

  11. 11.

    , & Identification and characterization of the DNA binding domain of CpG-binding protein. J. Biol. Chem. 276, 44669–44676 (2001)

  12. 12.

    et al. Structure of the MLL CXXC domain–DNA complex and its functional role in MLL-AF9 leukemia. Nature Struct. Mol. Biol. 17, 62–68 (2010)

  13. 13.

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

  14. 14.

    , , , & The structural basis for selective binding of non-methylated CpG islands by the CFP1 CXXC domain. Nature Commun. 2, 227 (2011)

  15. 15.

    , , & Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–1040 (2011)

  16. 16.

    et al. CpG islands recruit a histone H3 lysine 36 demethylase. Mol. Cell 38, 179–190 (2010)

  17. 17.

    et al. Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development. Cell 151, 1200–1213 (2012)

  18. 18.

    et al. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200–213 (2011)

  19. 19.

    et al. Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice. Proc. Natl Acad. Sci. USA 108, 14566–14571 (2011)

  20. 20.

    et al. RINF (CXXC5) is overexpressed in solid tumors and is an unfavorable prognostic factor in breast cancer. Ann. Oncol. 22, 2208–2215 (2011)

  21. 21.

    et al. A novel Wilms tumor 1 (WT1) target gene negatively regulates the WNT signaling pathway. J. Biol. Chem. 285, 14585–14593 (2010)

  22. 22.

    et al. Functional involvement of RINF, retinoid-inducible nuclear factor (CXXC5), in normal and tumoral human myelopoiesis. Blood 113, 3172–3181 (2009)

  23. 23.

    et al. Caspase activity mediates the differentiation of embryonic stem cells. Cell Stem Cell 2, 595–601 (2008)

  24. 24.

    , & Ubiquitin and proteasomes in transcription. Annu. Rev. Biochem. 81, 177–201 (2012)

  25. 25.

    et al. Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res. 22, 467–477 (2012)

  26. 26.

    et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS ONE 5, e15367 (2010)

  27. 27.

    et al. Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers. Oncotarget 2, 627–637 (2011)

  28. 28.

    Maintaining embryonic stem cell pluripotency with Wnt signaling. Development 138, 4341–4350 (2011)

  29. 29.

    et al. Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation. Oncogene 32, 663–669 (2013)

  30. 30.

    et al. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell 150, 1135–1146 (2012)

  31. 31.

    et al. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  32. 32.

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

  33. 33.

    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)

  34. 34.

    et al. CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282 (1987)

  35. 35.

    et al. The UCSC Genome Browser database: extensions and updates 2011. Nucleic Acids Res. 40, D918–D923 (2012)

  36. 36.

    et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004)

  37. 37.

    et al. The protein model portal. J. Struct. Funct. Genomics 10, 1–8 (2009)

  38. 38.

    et al. MODBASE, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res. 32, D217–D222 (2004)

  39. 39.

    The SWISS-MODEL Repository.

  40. 40.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  41. 41.

    , , & Chimera: an Extensible Molecular Modeling Application Constructed Using Standard Components Vol. 1 724 (Pacific Symposium on Biocomputing, 1996)

Download references

Acknowledgements

We thank G. Seumois, M. Ku and J. Day for help with library preparation, B. Ren for use of his Illumina Hi-Seq 2000, J. A. Zepeda-Martínez for the recombinant Flag–TET2CD, and members of the Rao laboratory for discussions. This work was supported by National Institutes of Health (NIH) R01 grants HD065812 and CA151535, grant RM-01729 from the California Institute of Regenerative Medicine and Translational Research, grant TRP 6187-12 from the Leukemia and Lymphoma Society (to A.R.) and NIH R01 grant AI40127 (to P.G.H. and A.R). We also gratefully acknowledge a Special Fellow Award from the Leukemia and Lymphoma Society (to M.K.), postdoctoral fellowships from the Lady Tata Memorial Trust and from the GlaxoSmithKline-Immune Disease Institute Alliance (to H.S.B.) and a predoctoral graduate research fellowship from the National Science Foundation (to W.A.P.).

Author information

Author notes

    • Myunggon Ko
    •  & Jungeun An

    These authors contributed equally to this work.

    • William A. Pastor
    • , Huiming Li
    •  & Kian Peng Koh

    Present addresses: Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Terasaki Life Sciences Building, 610 Charles Young Drive East, Los Angeles, California 90095-723905, USA (W.A.P.); Bristol-Myers Squibb, 700 Bay Road, Redwood City, California 94063, USA (H.Li); KU Leuven Department of Development and Regeneration & Stem Cell Institute Leuven, Herestraat 49, 3000 Leuven, Belgium (K.P.K.).

Affiliations

  1. Division of Signaling and Gene Expression, La Jolla Institute for Allergy & Immunology, La Jolla, California 92037, USA

    • Myunggon Ko
    • , Jungeun An
    • , Hozefa S. Bandukwala
    • , Lukas Chavez
    • , Tarmo Äijö
    • , William A. Pastor
    • , Matthew F. Segal
    • , Patrick G. Hogan
    •  & Anjana Rao
  2. Department of Information and Computer Science, Aalto University School of Science, FI-00076 Aalto, Finland

    • Tarmo Äijö
    •  & Harri Lähdesmäki
  3. Harvard Medical School and Program in Cellular and Molecular Medicine, Children’s Hospital, Boston, Massachusetts 02115, USA

    • Huiming Li
    • , Kian Peng Koh
    •  & Anjana Rao
  4. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA

    • L. Aravind
  5. Sanford Consortium for Regenerative Medicine, La Jolla, California 92037, USA

    • Anjana Rao

Authors

  1. Search for Myunggon Ko in:

  2. Search for Jungeun An in:

  3. Search for Hozefa S. Bandukwala in:

  4. Search for Lukas Chavez in:

  5. Search for Tarmo Äijö in:

  6. Search for William A. Pastor in:

  7. Search for Matthew F. Segal in:

  8. Search for Huiming Li in:

  9. Search for Kian Peng Koh in:

  10. Search for Harri Lähdesmäki in:

  11. Search for Patrick G. Hogan in:

  12. Search for L. Aravind in:

  13. Search for Anjana Rao in:

Contributions

L.A., P.G.H. and A.R. conceived the project and supervised project planning and execution. M.K. and J.A. performed cellular and molecular experiments including ChIP-seq, gene knockdown, establishment of stable cell lines, site-directed mutagenesis, dot blot, immunocytochemistry, in vitro caspase and TET assays, and in vitro differentiation studies. J.A. performed the in-cell western blots. H.S.B. obtained the initial data showing downregulation of TET2 protein by IDAX. M.K. conducted the electrophoretic mobility shift assays with help from W.A.P. and M.F.S. H.Li and P.G.H. generated the homology model of the IDAX CXXC domain. K.P.K. provided mRNAs from ESC samples. L.C., T.A. and H.Lähdesmäki performed the bioinformatic analyses of ChIP-seq data. M.K. and A.R. wrote the manuscript with input from other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Anjana Rao.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1-16, Supplementary Methods and Supplementary Tables 1, 3 and 4.

Excel files

  1. 1.

    Supplementary Data

    This file contains Supplementary Table 2.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature12052

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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