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CTCF maintains differential methylation at the Igf2/H19 locus

Nature Genetics volume 33, pages 66–69 (2003)Cite this article

Abstract

Genomic imprinting relies on establishing and maintaining the parental-specific methylation of DNA elements that control the differential expression of maternal and paternal alleles. Although the essential DNA methyltransferases have been discovered, proteins that regulate the sequence-specific establishment and maintenance of allelic methylation have not been identified. One candidate regulator of methylation, the zinc-finger protein CTCF, binds to the imprinting control region (ICR) of the genes Igf2 (encoding insulin-like growth factor 2) and H19 (fetal liver mRNA; refs. 1,2). The unmethylated maternal ICR is a chromatin boundary that prevents distant enhancers from activating Igf2 (refs. 3–6). In vitro experiments have suggested that CTCF mediates boundary activity of the maternal ICR, and that methylation of the paternal ICR abolishes this activity by preventing CTCF binding3,4,6. Using mice with point mutations in all four CTCF sites in the ICR, we show that maternally transmitted mutant ICRs in neonatal mice acquire a substantial but heterogeneous degree of methylation. Mutant ICRs in oocytes and blastocysts are not methylated, however, indicating that binding of CTCF is not required to establish the unmethylated ICR during oogenesis. We also show that the mutant ICR lacks enhancer-blocking activity, as the expression of Igf2 is activated on mutant maternal chromosomes. Conversely, maternal H19 expression is reduced, suggesting a positive role for CTCF in the transcription of that gene. This study constitutes the first in vivo demonstration of the multiple functions of CTCF in an ICR.

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Figure 1: Targeting of the mutant and wild-type ICR and the relative binding affinity of mutant CTCF sites.
Figure 2: Methylation analysis of the mutant ICR.
Figure 3: Bisulfite sequencing of regions of maternally transmitted mutant ICRs.
Figure 4: Allelic Igf2 mRNA expression in neonatal mice.
Figure 5: Effect of the mutant ICR on maternal H19 expression and promoter methylation.

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Acknowledgements

We thank R. Ingram for DNA sequencing; B. Jones for RNA samples; D. Mancini-DiNardo for advice on bisulphite analysis; M. Capecchi for the pACN plasmid; and members of S.M.T.'s laboratory for comments. This work was supported by a grant from the US National Institute for General Medical Sciences. S.M.T. was an investigator of the Howard Hughes Medical Institute.

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  1. Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign, Urbana, 61801, Illinois, USA

    Christopher J. Schoenherr

  2. Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, 08544, New Jersey, USA

    John M. Levorse & Shirley M. Tilghman

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Correspondence to Christopher J. Schoenherr or Shirley M. Tilghman.

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Schoenherr, C., Levorse, J. & Tilghman, S. CTCF maintains differential methylation at the Igf2/H19 locus. Nat Genet 33, 66–69 (2003). https://doi.org/10.1038/ng1057

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