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

Abstract

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.

We are grateful to Wolf Reik for generously providing both SD7 mice and sequence information of the M. musculus spretus H19 ICR, to Gail Martin for providing the Cre-transgenic mouse line, and to Ragnar Mattsson at the Lund University transgenic core facility for help with blastocyst injections.

This work was supported in part by the Swedish Science Research Council (VR), the Swedish Cancer Research Foundation (CF), the Swedish Pediatric Cancer Foundation, and the Wallenberg and Lundberg Foundations, as well as Stiftelsen Wenner-Grenska Samfundet, and the intramural research funding from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (to V.L.) and the Association for International Cancer Research (IC, EK).