CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, result ing in silencing of the maternal and paternal alleles, respectively. This e vent is dependent upon an imprinted-control region two kilobases upstream o f H19 (refs 1, 2). On the paternal chromosome this element is methylated an d required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 k ilobases upstream(2). We have proposed that the unmethylated imprinted-cont rol region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking acti vity would then be lost when the region was methylated, thereby allowing ex pression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and hu man H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function(7), binds to several sites in the unmethylated imprinted-control region that are ess ential for enhancer blocking. Consistent with our model, CTCF binding is ab olished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.