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

in mammals, a subset of genes inherit gametic marks that establish parent o f origin-dependent expression patterns in the soma ([1] and references ther ein). The currently most extensively studied examples of this phenomenon, t ermed genomic imprinting, are the physically linked Igf2 (insulin like grow th factor II) and H19 genes, which are expressed mono-allelically from oppo site parental alleles [1,2]. The repressed status of the maternal Igf2 alle le is due to cis elements that prevent the HIS enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4,5], A differentially meth ylated domain (DMD) in the 5' flank of H19 is maintained paternally methyla ted and maternally unmethylated [6,7], We show here by gel-shift and chroma tin immunopurification analyses that binding of the highly conserved multiv alent factor CTCF ([8,9] and references therein) to the His DMD is methylat ion-sensitive and parent of origin-dependent, Selectively mutating CTCF-con tacting nucleotides, which were identified by methylation interference with in the extended binding sites initially revealed by nuclease footprinting, abrogated the His DMD enhancer blocking property. These observations sugges t that molecular mechanisms of genomic Imprinting may use an unusual abilit y 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.