Figure 1. Imprinted versus non-imprinted insulation at the H19/Igf2 locus by two distinct insulators.
(A) Imprinted insulation at the H19/Igf2 imprinted domain by the ICR. Maternal chromosome (M): unmethylated (white lollipops) ICR (shaded area) is inherited from the egg. CTCF (yellow ovals) imparts insulator activity (bracket) between the Igf2 promoters and the shared, downstream enhancers (orange oval). Initiation of H19 expression depends on an unmethylated ICR during embryogenesis. Paternal chromosome (P): methylated (black lollipops) ICR is inherited from the sperm, CTCF cannot bind, hence ICR has no insulator activity, Igf2 promoters and enhancers can interact. Early in postimplantation development, the H19 promoter is inactivated by an ICR-dependent mechanism (horizontal arrow). (B) Non-imprinted insulation at the H19/Igf2 locus by the chicken β-globin insulator duplex (ChβGI)2 [44]. The (ChβGI)2 is unmethylated and insulates the Igf2 promoter from the shared enhancers when substituted for the ICR and transmitted maternally (not shown) or paternally (P), with 10% Igf2 activity remaining. H19 is overactivated 1.5-fold by the (ChβGI)2 sequences in the paternal allele (bold arrow). (C) Structure of the (ChβGI)2 with the five in vitro footprints of the core insulator [8]: binding sites 1, 3 and 5 (blue circle): VEZF1 (BGP1); binding site 2: CTCF; and binding site 4 (pink oval): USF1. (D) Structure of the mutant chicken β-globin insulator duplex (mChβGI)2. Only the CTCF binding site (thick underlining) remains in each unit after deleting (x) binding sites 1, 3, 4 and 5 using site-directed mutagenesis. (E) Confirmation of the site-directed mutagenesis by DNA sequencing. Arrows indicate the positions of the deleted binding sites (deleted sequences shown underneath) and light underlining shows added nucleotides at footprint 1. Novel restriction sites, ScaI, StuI and NheI, marked above, were generated to aid the screening of mutant colonies. One out of two SmaI sites remained at the footprint 3 deletion.