The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and theH19imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains

Abstract

The repression of the maternally inherited Igf2 allele has been proposed to depend on a methylation-sensitive chromatin insulator organized by the 11 zinc finger protein CTCF at the H19 imprinting control region (ICR). Here we document that point mutations of the nucleotides in physical contact with CTCF within the endogenous H19 ICR lead to loss of CTCF binding and Igf2 imprinting only when passaged through the female germline. This effect is accompanied by a significant loss of methylation protection of the maternally derived H19 ICR. Because CTCF interacts with other imprinting control regions, it emerges as a central factor responsible for interpreting and propagating gamete-derived epigenetic marks and for organizing epigenetically controlled expression domains. The manifestation of genomic imprinting involves the translation of gametic marks into parent of origin-dependent gene expression patterns (Bartolomei and Tilghman 1997;Horsthemke et al. 1999). The neighboring IGF2 and H19 genes emerge as paradigms of genomic imprinting, because their expression is monoallelic from opposite parental alleles and governed by shared enhancers (Bartolomei and Tilghman 1997;Horsthemke et al. 1999). The repression of the maternal IGF2 and paternal H19 alleles depends on a differentially methylated imprinting control region (ICR) in the 5Ј region of the H19 gene (Olek and Walter 1997;Kaffer et al. 2000). The proposal that the complex between the H19 ICR and the 11 zinc finger protein CTCF organizes a CpG methylationsensitive insulation of the maternal Igf2 allele (Bell and Felsenfeld 2000;Hark et al. 2000; Kanduri et al. 2000a,b;Bell et al. 2001;Ohlsson et al. 2001) is supported by the observations that CTCF interacts with only the maternal H19 ICR allele (Kanduri et al. 2000b) and that the chromatin insulator function is regulated by CpG methylation (Holmgren et al. 2001).To directly prove this proposal, we mutated sequences within the H19 ICR, which we have previously shown to be in direct physical contact with CTCF. Our results document that the CTCF-H19 ICR complex is vital in the manifestation and propagation of gametic marks. Results and DiscussionTo directly demonstrate the function of the CTCF target sites within the H19 ICR in association with the establishment and manifestation of the imprinting phenomenon, we changed the sequence GTGG to ATAT in three of the four CTCF target sites within the CGCG(T/ G)GGTGGCAG-core motif. This sequence change deletes essential contact points for the CTCF while preserving the CpGs responsible for the methylation-sensitive portion of the CTCF target sites (Kanduri et al. 2000b). The remaining second CTCF target site was ignored, because it does not, in contrast to the other target sites, display any marked in vivo footprint or nuclease hypersensitivity (Kanduri et al. 2000a;Szabó et al. 2000). Following electroporation of the targeting construct into ES cells and exploiting the fact that the mutations introduced an EcoRV site in each of the three mutated target sites (Kanduri et a...

H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas: A pilot case-control analysis

“…A novel finding in our study was the C/T polymorphism identified by the endonuclease, HhaI, in CTCF binding site 6. It is thought that the number of functional CTCF binding sites in the DMR correlates with their insulator activity and this C/T SNP could influence the stability of the binding to the unmethylated allele in this critical imprinting control region (45). The comparison of the genotypes, CC and CT + TT, in the 95 patients with cancer and in the 157 volunteers revealed a statistical trend towards a lower frequency of the homozygous wild-type CC genotype in the cancer group.…”

Role of CTCF Binding Sites in the Igf2/H19 Imprinting Control Region

“…We can therefore conclude that the CTCF insulator binding sites are dispensable for the maintenance of ICR hypomethylation in female germ cells and for the acquisition and maintenance of ICR hypermethylation in male germ cells. Thus, the binding of CTCF, or another protein which recognizes the site, e.g., the "brother of the regulator of imprinted sites" (BORIS) protein (20,26), is not required in either of these processes. With respect to what other ICR sites might be involved, we have identified a set of conserved nuclear hormone receptor-like sites in the ICR which show in vivo footprints in somatic cells and which can bind proteins specifically in male fetal germ cell nuclear extracts in EMSAs (41a.…”

“…1B to D). We would also expect this mutation to fully eliminate binding of CTCF to the ICR in vivo; it was shown that changing the mouse CTCF insulator binding site core sequence from CCGC(G/A)(T/C)GG(T/C)GGCAG to CCGCG(G/A)(T/C)AT ATGCAG was sufficient to eliminate all CTCF binding to the ICR in vivo at the level of detection of chromatin immunoprecipitation assays (26). In comparison, our mutation involved changing the three invariant nucleotides of these same four nucleotides: three G3T, four other invariant nucleotides, two C3A and two G3T, and two other nucleotides, (G/A)3T and (T/C)3G, to give CATA TGTTTTTCAG (Fig.…”

“…This result was interpreted to mean that CTCF binding at the ICR-even at low levels, in the case of the previously described mutant ICR-functions to inhibit methylation of the region in oocytes (7). After fertilization, maternal ICRs with mutated insulator sites have been observed to acquire methylation, suggesting that CTCF binding is normally required to maintain the hypomethylation of this region in somatic cells (26,32). Overall, the results point to a crucial role for the CTCF insulator binding sites, and CTCF binding, in establishing and maintaining imprinting of the Igf2/H19 region.…”

Epigenetic deregulation of imprinting in congenital diseases of aberrant growth

“…For the H19 ICR, CTCF binding might prevent methylation in the female germline. (37,38) However, it is not known whether, in the male germline, methylation is put onto the ICR by default, or via an active recruitment process. It remains unknown also, which mechanism(s) dictates the establishment of methylation at the KvDMR1 specifically in the female germline.…”