Fig. 2

Suppression of Dnmt1 or Dnmt7 activity affects ntl methylation. A: Methylation status of the ntl CpG island was analyzed 48 hr after the injection of morpholino oligonucleotide (MO) against each dnmt. mm-dnmt1 and mm-dnmt7 are negative-control MOs carrying mismatch nucleotides in their original dnmt1-MO and dnmt7-MO sequences, respectively. Part of the ntl CpG island containing 16 CpG dinucleotides was PCR amplified and 10 independent clones were sequenced. Each column represents the mean ± standard error of methylated cytosines. B:ntl methylation level reduced by dnmt7 52UTR-MO was partially restored by coinjection of in vitro-synthesized dnmt7 mRNA (approximately 500 pg), which has no complementary sequence to the dnmt7 5′UTR-MO. Also, 200 pg of the same mRNA was unable to rescue the de novo methylation (data not shown). Because dnmt7 52 UTR-MO caused a neural death phenotype that at low doses is transient, we used less of the 52 UTR-MO (1.5 ng) than that of the original dnmt7-MO (2.0 ng). C: Methylation status of an ntl 32 exon region. An ntl 3′ region harboring 15 CpGs was amplified, and 10 independent clones were sequenced.