CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2

Abstract

It is thought that the H19 imprinting control region (ICR) directs the silencing of the maternally inherited Igf2 allele through a CTCFdependent chromatin insulator. The ICR has been shown to interact physically with a silencer region in Igf2, differentially methylated region (DMR)1, but the role of CTCF in this chromatin loop and whether it restricts the physical access of distal enhancers to Igf2 is not known. We performed systematic chromosome conformation capture analyses in the Igf2͞H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF binding sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF target site within Igf2 DMR1, showing that CTCF can coordinate regional epigenetic marks. This systematic chromosome conformation capture analysis of an imprinting cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome.genomic imprinting ͉ insulators ͉ chromosome biology A variety of control elements, such as enhancers, silencers, and chromatin insulators, are thought to establish and maintain domains of gene expression or repression in the genome. Insulators, for example, are needed to keep genes in silent domains, away from the influence of neighboring enhancers. The prototypical insulator from the chicken ␤-globin locus contains binding sites for the 11-zinc-finger protein CTCF, and binding of CTCF is necessary for insulator function, not only in the chicken but also in many mammalian insulators (1-4). How CTCF confers chromatin insulation is not clear, but recent work shows that DNA-bound CTCF molecules can dimerize in a binding site-specific fashion (5) and that CTCF binds nucleophosmin and may be tethered to the nuclear matrix (6). CTCF emerges, therefore, as a potential mediator of long-range interactions that are involved in establishing repressed domains.Regional coordination of gene expression and repression is found in many imprinted gene clusters in the mammalian genome and is regulated by imprinting centers or imprinting control regions (ICRs) (7-10). A well characterized cluster is that containing the paternally expressed Igf2 and maternally expressed H19. Both genes share enhancers, and the ICR is located in the 5Ј flank of the H19 gene (11). The deletion of this ICR results in biallelic expression of both Igf2 and H19, demonstrating a role of the ICR to repress the maternal Igf2 allele, which is located Ͼ80 kb away (12). The mechanism underlying this function has been proposed to involve a CTCF-dependent...

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

“…A mouse fibroblast cell line (MBW2) derived by breeding M. spretus males with C57BL/c females (6) was used to study the allelic interactions with CTCF. We first examined whether CTCF interacts directly with the Igf2 promoters by 3C methodology (17). CTCFbound DNA in the ICR (EcoRI sites 5 and 6) interacted with the DNA near the imprinted promoters primarily on the maternal allele (EcoRI sites 2 and 3, lanes 3 to 6, Fig.…”

“…When this ICR is deleted (35) or mutated (32, 34), the maternal Igf2 allele is expressed, leading to biallelic expression. In addition, CTCF has recently been shown to act as a tethering protein, serving as a molecular glue to secure long-range intrachromosomal (17) and interchromosomal (18) interactions.By chromosome configuration capture (3C) methodology, it has been shown that CTCF participates in the formation of a long-range chromosomal loop to the upstream Igf2 DMRs when it is bound to the maternal ICR (17,42,21). This model suggests that CTCF may not only function as a physical insulator but also actively participate in the regulation of the imprinted Igf2 allele.…”

“…By chromosome configuration capture (3C) methodology, it has been shown that CTCF participates in the formation of a long-range chromosomal loop to the upstream Igf2 DMRs when it is bound to the maternal ICR (17,42,21). This model suggests that CTCF may not only function as a physical insulator but also actively participate in the regulation of the imprinted Igf2 allele.…”

“…When this ICR is deleted (35) or mutated (32,34), the maternal Igf2 allele is expressed, leading to biallelic expression. In addition, CTCF has recently been shown to act as a tethering protein, serving as a molecular glue to secure long-range intrachromosomal (17) and interchromosomal (18) interactions.…”

CTCF terminal segments are unstructured

“…15,16 In the mouse genome, the imprinted H19/Igf2 locus in particular has yielded several insights into chromosomal organization mediated by CTCF. Not only has intrachromosomal association of elements within the locus been detected, 17,18 but interchromosomal association between the locus and elements on different chromosomes has also been observed. 19,20 Loss of a CTCFmediated loop is sometimes also correlated with reduced transcription.…”