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Molecular and Cellular Biology, April 2004, p. 3497-3504, Vol. 24, No. 8
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.8.3497-3504.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mutation of a Single CTCF Target Site within the H19 Imprinting Control Region Leads to Loss of Igf2 Imprinting and Complex Patterns of De Novo Methylation upon Maternal Inheritance

Department of Development and Genetics, Evolution Biology Centre, Uppsala University, S-752 36 Uppsala, Sweden,¹ Molecular Pathology Section, Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,² Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CQ4 3SQ, United Kingdom,³ Genetisches Institut der Justus-Liebig-Universität, D-35392 Giessen, Germany⁴

Received 4 September 2003/ Returned for modification 20 November 2003/ Accepted 14 January 2004

The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.

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