Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.

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...

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...

The 5 region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.