Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive

in mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1] and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles [1,2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the HIS enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4,5], A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated [6,7], We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8,9] and references therein) to the His DMD is methylation-sensitive and parent of origin-dependent, Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the His DMD enhancer blocking property. These observations suggest that molecular mechanisms of genomic Imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.