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. 2007 Mar 5;27(9):3499–3510. doi: 10.1128/MCB.02170-06

FIG. 1.

FIG. 1.

Long-range interactions at the Igf2 locus on wild-type (WT) and ΔICR chromosomes. (A) Schematic depiction of the 100-kb Igf2-H19 locus includes the three Igf2 promoters (P1 at kb −78, P2 at kb −76, and P3 at kb −74), the shared ICR (at kb −4.4 to −2), the H19 promoter (H19P at bp 0), and the shared endodermal (open circle at kb +8) and mesodermal (filled circle at kb +25) enhancers. DMR1 and DMR2, flanking the Igf2 promoters, become methylated on the paternal chromosome in the postimplantation embryo and play a role in the activation of paternal Igf2 in liver cells and in the repression of maternal Igf2 in muscle cells, respectively. The ΔICR chromosome carries a 5-kb deletion from kb −6 to −1 that removes the ICR. The vertical bars above and below the map indicate BamHI and BglII restriction sites, respectively. Arrowheads depict the orientations and locations of PCR primers used for 3C analysis. Asterisks indicate RFLPs that distinguish between wild-type M. castaneus alleles and M. domesticus alleles. (B to K) 3C analysis of long-range interactions at the Igf2 locus was carried out on using the primers indicated. Animal genotypes C/D, D/C, C/ΔICR, and ΔICR/C are indicated (maternal allele listed first). The top panels for each experiment represent the 3C PCR product. The bottom panels, when included, depict the banding patterns after digestion with enzymes distinguishing between the M. castaneus (C-labeled arrowheads)- and M. domesticus (D-labeled arrowheads)-derived DNAs. Note that the ΔICR mutation is on an M. domesticus chromosome. (B and C) In wild-type muscle cells (B) and liver cells (C), the Igf2 promoters associate with the mesodermal and endodermal enhancers, respectively, only on the active paternal chromosome. That is, the C/D and D/C extracts yield only PCR products that are all M. domesticus (D-labeled arrowheads) and all M. castaneus (C-labeled arrowheads) alleles, respectively. (D and E) The maternally inherited ICR insulator is necessary to prevent maternal promoter-enhancer interactions in both muscle (D) and liver (E) cells. Maternal inheritance of the ICR deletion mutant results in biallelic interactions between the Igf2 promoters and the enhancers, as indicated by the presence of both M. domesticus and M. castaneus bands in the 3C products of ΔICR/C extracts. (F and G) Inactive maternal Igf2 promoters associate with the ICR insulator in both muscle (F) and liver (G) cells. (H and I) Igf2 promoters interact only with a maternally inherited ICR in both muscle (H) and liver (I) cells. (J) The maternal ICR interacts with blocked downstream enhancers. (K) Only the unmethylated maternal ICR interacts with downstream mesodermal and endodermal enhancers. NS, nonspecific PCR product; C+D, digestion products indicative of both M. castaneus and M. domesticus DNAs comigrate.