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.The neighboring Igf2 and H19 genes are expressed monoallelically from opposite chromosomes and are generally recognized as the paradigm of mammalian genomic imprinting (1, 11). The cis elements underlying their parent-of-origin-dependent expression patterns were traced to a differentially methylated region (imprinting control region [ICR]) located 2 kb upstream of the H19 transcriptional unit (5, 26-28). Subsequently, it was documented that repression of the paternal H19 allele was initiated, but not maintained by the H19 ICR, while the continuous presence of this region was required to suppress the maternal Igf2 allele (25). Arguments that this region contained a methylation-sensitive chromatin insulator to block communication between downstream enhancers and Igf2 promoters (3, 9, 12, 13) were later confirmed by experiments (10).Despite similar functions, the mouse and human H19 ICRs display little sequence similarity with the exception of a CpGrich element repeated four times in the mouse H19 ICR and seven times in the human H19 ICR (9). We and others have noted that the 11-zinc finger protein CTCF, which associates with all known vertebrate chromatin insulator cis elements (2, 19), interacted with these repeat elements in a methylationsensitive manner (3, 9, 13). Importantly, CTCF interacts in vivo with only the maternal H19 ICR allele (13), which is consistent with an involvement with the in vivo insulation of the maternal Igf2 from downstream lineage-specific enhancers. To confirm an in vivo role of the CTCF target sites at the Igf2/H19 locus, we previously cr...

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

“…In human tumors where IGF2 is biallelically expressed (24,32,35), the CTCF model predicts that both parental alleles are methylated and no CTCF insulation occurs at either allele, leading to loss of IGF2 imprinting. Indeed, maternally transmitted microdeletion of two CTCF sites in the ICR results in biallelic IGF2 expression and H19 silencing in BeckwithWiedemann syndrome (33).…”

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

“…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)(2)(3)(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.…”

“…However, the Igf2 gene itself also contains differentially methylated regions (DMRs), with DMR1 being a methylation-sensitive silencer (18, 19) and DMR2 being a methylation sensitive activator (20). Additional enhancer sequences located 5Ј of the ICR (21, 22) suggest that a simple insulator function of the ICR is not sufficient to explain all aspects of maternal silencing of Igf2.In addition to their insulator function, the CTCF target sites within the H19 ICR confer protection against de novo methylation in somatic cells (5,23,24), preimplantation conceptuses, embryonic stem cells, and oocytes (25,26). Furthermore, maternal deletion of the H19 ICR leads to de novo methylation at DMR1 and DMR2 in Igf2, showing regional coordination of epigenetic modifications by the ICR, which was proposed to occur by higher-order chromatin conformations (27).…”

“…In addition to their insulator function, the CTCF target sites within the H19 ICR confer protection against de novo methylation in somatic cells (5,23,24), preimplantation conceptuses, embryonic stem cells, and oocytes (25,26). Furthermore, maternal deletion of the H19 ICR leads to de novo methylation at DMR1 and DMR2 in Igf2, showing regional coordination of epigenetic modifications by the ICR, which was proposed to occur by higher-order chromatin conformations (27).…”

Genomic Imprinting and Epigenetic Control of Development

“…As the expression of the de novo methyltransferases is not limited to gametogenesis, and can occur in somatic tissues of both the embryo and the adult, the protective role of CTCF in maintaining a hypomethylated maternal H19 DMD extends beyond the female germline. Genetically engineered mutations in the DMD that alter or completely delete CTCF-binding sites,acquire DNA methylation during development (Schoenherr et al 2003;Pant et al 2004;Engel et al 2006). These results demonstrate a role for CTCF in establishing and maintaining maternal identity of the H19 DMD.…”