An evolutionarily conserved putative insulator element near the 3′ boundary of the imprinted Igf2/H19 domain

Igf2 and H19 are closely linked imprinted genes lying at the centromeric end of a 1 Mb imprinted domain on mouse chromosome 7. L23mrp and other genes located 3' (more centromeric) to H19 are not imprinted and do not interact with the enhancers shared by Igf2 and H19. It is therefore suggested that the intergenic region between H19 and L23mrp contains a boundary or an insulator element. We have identified a binding site for CTCF, a nuclear factor that mediates insulator activity in vertebrates, in the intergenic region. This site is conserved between human and mouse, associated with a major DNase I-hypersensitive site, and bound by CTCF in vivo. Functional assays using reporter constructs demonstrated that this element functions as an insulator in transfected cells. The findings suggest that this CTCF site contributes to the 3' boundary of this imprinted domain. Together with the findings on the differentially methylated CTCF sites 5' to H19, CTCF-dependent insulators may not only regulate but also delimit the imprinted domain.