CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus (original) (raw)

Nature volume 405, pages 486–489 (2000)Cite this article

Abstract

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2,3,4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream2. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3′ of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function7, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.

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References

  1. Leighton, P. A., Ingram, R. S., Eggenschwiler, J., Efstratiadis, A. & Tilghman, S. M. Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature 375, 34–39 (1995).
    Article ADS CAS PubMed Google Scholar
  2. Thorvaldson, J. L., Duran, K. L. & Bartolomei, M. S. Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H10 and Igf2. Genes Dev. 12, 3693–3702 (1998).
    Article Google Scholar
  3. Bartolomei, M. S., Webber, A. L., Brunkow, M. E. & Tilghman, S. M. Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev. 7, 1663–1673 (1993).
    Article CAS PubMed Google Scholar
  4. Ferguson-Smith, A. C., Sasaki, H., Cattanach, B. M. & Surani, M. A. Parental-origin-specific epigenetic modifications of the mouse H19 gene. Nature 362, 751–755 (1993).
    Article ADS CAS PubMed Google Scholar
  5. Webber, A., Ingram, R. I., Levorse, J. & Tilghman, S. M. Location of enhancers is essential for imprinting of H19 and Igf2 . Nature 391, 711–715 (1998).
    Article ADS CAS PubMed Google Scholar
  6. Hark, A. T. & Tilghman, S. M. Chromatin conformation of the H19 epigenetic mark. Hum. Mol. Genet. 7, 1979–1985 (1998).
    Article CAS PubMed Google Scholar
  7. Bell, A. C., West, A. G. & Felsenfeld, G. The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98, 387–396 (1999).
    Article CAS PubMed Google Scholar
  8. Kellum, R. & Schedl, P. A position-effect assay for boundaries of higher order chromosomal domains. Cell 64, 941–950 (1991).
    Article CAS PubMed Google Scholar
  9. Kellum, R. & Schedl, P. A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol. Cell. Biol. 12, 2424–2431 ( 1992).
    Article CAS PubMed PubMed Central Google Scholar
  10. Kellum, R. & Elgin, S. C. R. Chromatin boundaries: punctuating the genome. Curr. Biol. 8, R521– R524 (1998).
    Article CAS PubMed Google Scholar
  11. Khosla, S., Aitchison, A., Gregory, R., Allen, N. D. & Fell, R. Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene. Mol. Cell. Biol. 19, 2556– 2566 (1999).
    Article CAS PubMed PubMed Central Google Scholar
  12. Elson, D. A. & Bartolomei, M. S. A 5′ differentially methylated sequence and the 3′ flanking region are necessary for H19 transgene imprinting. Mol. Cell. Biol. 17, 309–317 (1997).
    Article CAS PubMed PubMed Central Google Scholar
  13. Pfeifer, K., Leighton, P. A. & Tilghman, S. M. The structural H19 gene is required for its own imprinting. Proc. Natl Acad. Sci. USA 93, 13876–13883 (1996).
    Article ADS CAS PubMed PubMed Central Google Scholar
  14. Frevel, M. A., Hornberg, J. J. & Reeve, A. E. A potential imprint control element: identification of a conserved 42 bp sequence upstream of H19. Trends Genet. 15, 216–218 ( 1999).
    Article CAS PubMed Google Scholar
  15. Stadnick, M. P. et al. Role of a 461-bp G-rich repetitive element in H19 transgene imprinting. Dev. Genes Evol. 209, 239– 248 (1999).
    Article CAS PubMed Google Scholar
  16. Filippova, G. N. et al. An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol. Cell. Biol. 16, 2802–2813 (1996).
    Article CAS PubMed PubMed Central Google Scholar
  17. Vostrov, A. A. & Quitschke, W. W. The zinc finger protein CTCF binds to the APBβ domain of the amyloid β-protein precursor promoter. Evidence for a role in transcriptional activation. J. Biol. Chem. 272, 33353–33359 (1997).
    Article CAS PubMed Google Scholar
  18. Burcin, M. et al. Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone-receptors, is identical to the multivalent zinc finger repressor CTCF. Mol. Cell. Biol. 17, 1281–1288 ( 1997).
    Article CAS PubMed PubMed Central Google Scholar
  19. Awad, T. A. et al. Negative transcriptional regulation mediated by thyroid hormone response element 144 requires binding of the multivalent factor CTCF to a novel target DNA sequence. J. Biol. Chem. 274, 27092–27098 (1999).
    Article CAS PubMed Google Scholar
  20. Bird, A. P. & Wolffe, A. P. Methylation-induced repression—belts, braces, and chromatin. Cell 99, 451– 454 (1999).
    Article CAS PubMed Google Scholar
  21. Macleod, D., Charlton, J., Mullins, J. & Bird, A. P. Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 8, 2282– 2292 (1994).
    Article CAS PubMed Google Scholar
  22. Brandeis, M. et al. Sp1 elements protect a CpG island from de novo methylation. Nature 371, 435–438 (1994).
    Article ADS CAS PubMed Google Scholar
  23. Bell, A. C. & Felsenfeld, G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482–485 ( 2000).
    Article ADS CAS PubMed Google Scholar
  24. Szabo, P. E., Pfeifer, G. P. & Mann, J. R. Characterization of novel parent-specific epigenetic modifications upstream of the imprinted mouse H19 gene. Mol. Cell. Biol. 18, 6767–6776 (1998).
    Article CAS PubMed PubMed Central Google Scholar
  25. Feil, R. & Khosla, S. Genomic imprinting in mammals: an interplay between chromatin and DNA methylation? Trends Genet. 15, 431–435 ( 1999).
    Article CAS PubMed Google Scholar
  26. Auffray, C. & Rougeon, F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107, 303–314 ( 1980).
    Article CAS PubMed Google Scholar
  27. Leighton, P. A., Saam, J. R., Ingram, R. S., Stewart, C. L. & Tilghman, S. M. An enhancer deletion affects both H19 and Ifg2 expression. Genes Dev. 9, 2079–2089 (1995).
    Article CAS PubMed Google Scholar
  28. Hagenbuchle, O. & Wellauer, P. K. A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture. Nucleic Acids Res. 20, 3555–2559 (1992).
    Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank A. Bell and G. Felsenfeld for communicating results before publication. We also thank J. Shen for her assistance with the sequence alignment analysis and members of our laboratory for helpful discussions. This work was supported by a grant from the National Institute of General Medical Sciences. C.J.S. was a recipient of an NRSA Postdoctoral Fellowship from the NIH and S.M.T. is an Investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Amy T. Hark and Christopher J. Schoenherr: These authors contributed equally to this work
  2. Shirley M. Tilghman: Correspondence and requests for materials should be address to S.M.T.

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Molecular Biology Princeton University, Princeton, 08544, New Jersey, USA
    Amy T. Hark, Christopher J. Schoenherr, David J. Katz, Robert S. Ingram, John M. Levorse & Shirley M. Tilghman

Authors

  1. Amy T. Hark
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  2. Christopher J. Schoenherr
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  3. David J. Katz
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  4. Robert S. Ingram
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  5. John M. Levorse
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  6. Shirley M. Tilghman
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Hark, A., Schoenherr, C., Katz, D. et al. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.Nature 405, 486–489 (2000). https://doi.org/10.1038/35013106

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