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.

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

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 options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: IDAX preferentially binds CpG-rich DNA.
Figure 2: IDAX downregulates TET2 protein through caspase activation.
Figure 3: Reciprocal relationship between TET2 and IDAX in mouse ESCs and human U937 cells.
Figure 4: Negative regulation of TET3 by its CXXC domain.

Accession codes

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

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

    CAS  ADS  Article  Google Scholar 

  2. 2

    Iyer, L. M., Tahiliani, M., Rao, A. & Aravind, L. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids. Cell Cycle 8, 1698–1710 (2009)

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  ADS  Article  Google Scholar 

  4. 4

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

    CAS  ADS  Article  Google Scholar 

  5. 5

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

    CAS  ADS  Article  Google Scholar 

  6. 6

    Maiti, A. & Drohat, A. C. 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)

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

    Nguyen, A. V., Albers, C. G. & Holcombe, R. F. Differentiation of tubular and villous adenomas based on Wnt pathway-related gene expression profiles. Int. J. Mol. Med. 26, 121–125 (2010)

    PubMed  Google Scholar 

  10. 10

    Iyer, L. M., Abhiman, S. & Aravind, L. Natural history of eukaryotic DNA methylation systems. Prog. Mol. Biol. Transl. Sci. 101, 25–104 (2011)

    CAS  Article  Google Scholar 

  11. 11

    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)

    CAS  Article  Google Scholar 

  12. 12

    Cierpicki, T. 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)

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    Xu, C., Bian, C., Lam, R., Dong, A. & Min, J. The structural basis for selective binding of non-methylated CpG islands by the CFP1 CXXC domain. Nature Commun. 2, 227 (2011)

    ADS  Article  Google Scholar 

  15. 15

    Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–1040 (2011)

    CAS  ADS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

    Koh, K. P. 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)

    CAS  Article  Google Scholar 

  19. 19

    Ko, M. 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)

    CAS  ADS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

    Geng, F., Wenzel, S. & Tansey, W. P. Ubiquitin and proteasomes in transcription. Annu. Rev. Biochem. 81, 177–201 (2012)

    CAS  Article  Google Scholar 

  25. 25

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

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  ADS  Article  Google Scholar 

  27. 27

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

    Article  Google Scholar 

  28. 28

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

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

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

    Article  Google Scholar 

  32. 32

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

    Article  Google Scholar 

  33. 33

    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)

    CAS  Article  Google Scholar 

  34. 34

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

    CAS  Article  Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

  36. 36

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

    Article  Google Scholar 

  37. 37

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

    CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

    The SWISS-MODEL Repository. http://swissmodel.expasy.org/repository/

  40. 40

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

    Article  Google Scholar 

  41. 41

    Huang, C. C., Couch, G. S., Pettersen, E. F. & Ferrin, T. E. Chimera: an Extensible Molecular Modeling Application Constructed Using Standard Components Vol. 1 724 (Pacific Symposium on Biocomputing, 1996)

    Google Scholar 

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

Affiliations

Authors

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.

Corresponding author

Correspondence to Anjana Rao.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16, Supplementary Methods and Supplementary Tables 1, 3 and 4. (PDF 1836 kb)

Supplementary Data

This file contains Supplementary Table 2. (XLSX 3065 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ko, M., An, J., Bandukwala, H. et al. Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX. Nature 497, 122–126 (2013). https://doi.org/10.1038/nature12052

Download citation

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.

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