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. Author manuscript; available in PMC: 2010 Mar 27.
Published in final edited form as: Genomics. 2008 Oct 2;92(6):464–473. doi: 10.1016/j.ygeno.2008.08.004

Fig. 5.

Fig. 5

The unmethylated human IGF2R intron 2 CpG island contains a DNase1 hypersensitive site. (A) Map of human IGF2R intron 2 (17.686 kb) showing the direction of IGF2R transcription. A map of the fragment used to generate the mouse transgenes is shown underneath. The length of the intronic CpG island (CpG 99) is indicated and the location of a cluster of tandem direct repeats (TDR) within this island is shown. The BstXI probe used in the Southern blots in B,C is shown. The transcription start sites T1, T2, mapped in Fig. 3A are indicated. The paternal-specific DHS site identified in B, is marked with a resolution of ±500 bp. (B) Identification of a paternal-specific DNase1 hypersensitive site. Top: Southern blot from HS27 human foreskin fibroblast cells treated with different concentrations of DNase1, digested with EcoRI and hybridized with probe BstXI. The last two lanes contain control nuclei treated either with incubation buffer at 37 °C or left on ice. Samples treated with DNase1 show a second 2.5 kb band in addition to the expected 5.7 kb EcoRI band. The position of this DNase1 hypersensitive site can be determined with a resolution of ±500 bp to be at the border between the 1440 bp fragment with promoter activity and the CpG island (indicated above in A). The middle and lower panel shows Southern blots from mouse A9 cells containing a full mouse genome plus a single human chromosome 6 from the maternal parent (HS6-MAT) or from the paternal parent (HS6-PAT) [25], treated as described for A. The 2.5 kb band produced by treatment with DNase1 is only present in somatic cell hybrids containing the paternal chromosome. (C) DNA methylation of the IGF2R intron 2 CpG island is maternal-specific. HS27 cells contain maternal and paternal chromosomes and produce 2 bands, one from the maternally-methylated allele (5.7 kb) and one from the unmethylated paternal allele (2.9 kb). HS6-MAT somatic cell hybrids retain the DNA-methylation imprint on the maternal copy of the IGF2R intron 2 CpG island (5.7 kb), while HS6-PAT have a methylation-free island (2.9 kb). The HS6-PAT somatic cell hybrid lacks AIR expression (data not shown and [25]).