Fig. 5. Disruption of methylation and expression of maternally imprinted genes in Dnmt3L^mat–/– embryos. (A-D) Methylation of four maternally imprinted genes (Igf2r, Peg3, Snrpn and Peg1) were analyzed by either bisulfite sequencing or southern blot hybridization with genomic DNA isolated from E9.5 wild-type (+/+), Dnmt3L^+/– (+/–), Dnmt3L^–/– (–/–) and Dnmt3L^mat–/– (mat–/–) embryos. (A,B) Bisulfite sequencing analysis (methylation shown as black circles) shows that the Igf2r region 2 and Peg3 DMR are almost completely unmethylated in Dnmt3L^mat–/– embryos, whereas they are partially methylated in wild-type and Dnmt3L^–/– embryos. (C,D) Southern blot analysis of the Snrpn DMR1 and the Peg1 DMR shows that the maternal alleles (m) of both genes are methylated in wild-type and Dnmt3L^–/– embryos, but unmethylated in Dnmt3L^mat–/– embryos. (E,F) No change in methylation of paternally imprinted genes, Rasgrf1 and H19, was observed in Dnmt3L^mat–/– embryos. (G,H) Expression of four maternally imprinted genes (Igf2r, p57^kip2, Peg1 and Snrpn) in E9.5 embryos was analyzed by either RT-PCR (G) or northern blot hybridization (H). RT-PCR analysis of expression of Igf2r and p57^kip2 in two wild-type, two Dnmt3L^–/–, and four Dnmt3L^mat–/– embryos showed that expression of Igf2r and p57^kip2 was drastically reduced in all Dnmt3L^mat–/– embryos. Northern analysis of Peg1 and Snrpn expression with total RNA isolated from a pool of 5-6 E9.5 embryos for each genotype showed an increased expression (approximately twice the wild-type level) of Peg1 and Snrpn in Dnmt3L^mat–/– embryos as compared to the normal embryos. m, maternal allele; p, paternal allele.

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