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CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus

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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Figure 1: Testing boundary activity in transgenic mice.
Figure 2: Boundary activity in stable transfection of Hep3B cells.
Figure 3: Structure and functional analysis of the human ICR.
Figure 4: Methylation-sensitive binding of CTCF to multiple sites within the ICR.

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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

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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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