CTCF Regulates Allelic Expression of Igf2 by Orchestrating a Promoter-Polycomb Repressive Complex 2 Intrachromosomal Loop

Abstract

CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.

ACKNOWLEDGMENTS

We are indebted to M. S. Bartolomei, Piroska E. Szabo, and Jeffrey R. Mann for providing us with tissues and cell lines that are mutated at CTCF-binding sites of the ICR.

This study was supported by a Department of Defense grant (W81XWH-04-1-0597) and an NIH SBIR grant (R43 CA86664-01) to J.F.H., an NIH grant (DK36054) to A.R.H., and the Research Service of the Department of Veterans Affairs.