Hirasawa et al. 1
Supplemental Figure S1. Conditional deletion of the Dnmt genes in oocytes
Schematic representation of the wildtype, 2lox and 1lox alleles of Dnmt1, Dnmt3a and Dnmt3b. In the Dnmt12lox allele, exons 4 and 5 were flanked by the loxP sequence (green rectangle). In the Dnmt3a2lox allele, exon 19, which contains the conserved catalytic motif PC, and in the Dnmt3b2lox allele, exons 17-20, which contain the conserved catalytic motifs PC and ENV, were flanked by the loxP sequence. In the presence of Cre-recombinase, the two loxP sites at each locus are recombined, resulting in a deletion of the exon(s) and creation of the 1lox null allele.
Supplemental Figure S2. Expression of Dnmt3b in early and late 2-cell-stage embryos
Dnmt3b was not detectable at the early 2-cell stage (left panel) and became detectable in the cell nuclei at the late 2-cell stage (right panel).
Supplemental Figure S3. Morphology of Dnmt3a/Dnmt3b mutant embryos at E9.5
All embryos obtained from [Dnmt3a2lox/2lox, Dnmt3b2lox/2lox, Zp3-Cre] females crossed with [Dnmt3a1lox/+, Dnmt3b1lox/+] males showed growth retardation and various developmental defects at E9.5 and died before E10.5, partly due to the lack of the methylation imprint in the oocyte (Kaneda et al. 2004; Kaneda M, Hirasawa, R., Chiba, H., Okano, M., Li, E. and Sasaki, H., in preparation). Embryos lacking any Dnmt3a or Dnmt3b (either in the maternal or the zygotic form; [Dnmt3a1lox/1lox, Dnmt3b1lox/1lox]), showed the severest phenotype. Most of the embryos of this genotype did not undergo embryonic turning and the observed defects were very similar to those of the Dnmt1-null mutants, which showed global loss of genomic DNA methylation (Lei et al., 1996). Scale bar: 2 mm.
Supplemental Figure S4. Maternal-specific mono-allelic expression of H19 in the double mutants
H19 transcript was detected by RT-PCR and the amplified cDNA was digested with BglI. The JF1-strain-specific BglI SNP confirmed the normal maternal-specific expression of H19 in embryos without any (maternal or zygotic) Dnmt3a or Dnmt3b proteins ([Dnmt3a1lox/1lox, Dnmt3b1lox/1lox]). dom, Mus musculus domesticus.
Supplemental Figure S5. Methylation status of the Rasgrf1 DMRs in Dnmt3a/Dnmt3b double mutants
Methylation status of the Rasgrf1 DMR was analyzed by bisulfite sequencing in E9.5 [Dnmt3a1lox/1lox, Dnmt3b1lox/1lox] (A), [Dnmt3a1lox/+, Dnmt3b1lox/+] (B), [Dnmt3a1lox/1lox, Dnmt3b1lox/+] (C), and [Dnmt3a1lox/+, Dnmt3b1lox/1lox] embryos (D), all derived from [Dnmt3a2lox/2lox, Dnmt3b2lox/2lox, Zp3-Cre] females crossed with [Dnmt3a1lox/+, Dnmt3b1lox/+] males (Fig. 2A). While embryos lacking zygotic Dnmt3b showed a partial reduction in methylation at the paternal allele (A,D), the other embryos maintained the normal methylation patterns (B,C). Open circles and filled circles indicate unmethylated and methylated cytosines, respectively. JF1, JF1-derived allele; dom, domesticus-derived allele.
Supplemental Figure S6. Efficient deletion of Dnmt1 by Zp3-Cre in growing oocytes
(A) Genotype analysis of growing oocytes at P3, P5, P10 and P20 and FG oocytes, all derived from [Dnmt12lox/2lox, Zp3-Cre] females. The genotyping was carried out by PCR with primers that simultaneously amplify the 2lox and 1lox alleles, but not the wildtype allele. Dnmt1 gene was efficiently deleted before P10 at early stages of oocyte growth. (B) Detection of Dnmt1 isoforms by immunoblotting. Dnmt1o is efficiently disrupted in ovary (left panel) and FG oocytes (right panel) from [Dnmt12lox/2lox, Zp3-Cre] females. Extracts from sixty pooled oocytes were loaded in each lane (right panel). Extracts from ES cells were used as controls.