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Fig. 5. Methylation level of CpG sites in the 5' region of Xist is reduced on X^pA. (A) The genomic structure of the 5' region of Xist on the wild-type X and X^pA is shown along with recognition sites of the methylation-sensitive restriction enzymes and positions of probes used in this assay. (B-D) The methylation level in the 5' region of Xist was examined by Southern blotting in the embryo proper and the visceral endoderm of E13.5 embryos recovered from X^pAX females crossed with wild-type males. Genomic DNA was digested with one of the four methylation-sensitive restriction enzymes shown in A in combination with BclI and probed with either HS0.7 or BE0.6. BclI digestion produced an 8.4 kb (black arrowhead shown on the right of the blot) and a 10.0-kb (white arrowhead shown on the right) band from the wild-type X and X^pA, respectively. Although a decrease in CpG methylation was observed on X^pA in both tissues of both male and female embryos, it was more prominent in the visceral endoderm. Since the restriction fragments derived from X^pA digested by HhaI and SacII, when probed with HS0.7, migrated together with those from the wild-type X, the reduction of CpG methylation on X^pA was less clear in the embryo proper of X^pAX on the blot probed by HS0.7 (B), the same blot was reprobed with BE0.6 to visualize the fragments derived from X^pA (indicated by asterisks), which appear only when the relevant restriction sites are unmethylated (C). Probing with HS0.7 was sufficient to show hypomethylation of the locus in the visceral endoderm (D). Hh, HhaI; S, SacII; Hp, HpaII; and P, PmaCI.

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