Allele-specific expression of H19, Snurf/Snrpn and Peg3 imprinted genes were analyzed in individual wild-type and DNMT1o-deficient blastocysts using real time RT-PCR. Expression of maternal allele is shown in red and expression of paternal allele is shown in blue. Percentages of expression were determined as described in materials and methods.

The 12 autosomes known to contain imprinted genes are depicted as colored bars in 8-cell stage blastomeres (left). Methylated DNA single strands are chromatids indicated by filled areas and unmethylated DNA single strands and chromatids indicated by outlined areas. The fates of chromatids following replication, cell division and cell distribution within embryo are shown.

Bisulfite genomic sequencing was used to establish allele-specific methylation of H19 and Snurf/Snrpn DMDs in E7.5 embryos. A representative wild-type and 4 different DNMT1o-deficient embryos are shown. M=maternal allele; P=paternal allele. Position of methylated CpG dinucleotides are indicated by the filled circles. The percentages of methylated CpGs are indicated. * denotes significantly different levels of CpGs methylation (p< 0.05). ** denotes highly significant differences (p< 0.01). ns = not statistically significant differences (p>0.05).

Methylation patterns of a representative wild-type and three DNMT1o-deficient embryos were determined by the method of bisulfite genomic sequencing. Allele-specific expression for H19 gene was analyzed using RT-PCR followed by restriction endonuclease digestion to distinguish parental alleles. M = maternal allele; P = paternal allele. Position of methylated CpG dinucleotides are indicated as filled circles. The percentages of methylated CpGs are indicated. * denotes significantly different levels of CpGs methylation (p< 0.05). ** denotes highly significant differences (p< 0.01). ns = not statistically significant differences (p>0.05).

Top: Schematics of the mouse Snurf/Snrpn and H19 DMDs (grey boxes). The arrows represent the start of transcription and arrowheads indicate the tandem repeats within the Snurf/Snrpn DMD. Bottom: Patterns of DNA methylation on DMD sequences of the endogenous Snurf/Snrpn and H19 genes were analyzed with the bisulfite genomic sequencing technique in pools of wild-type and DNMT1o-deficient morulae. One pool of wild-type and two pools of DNMT1o-deficient morulae were analyzed for each DMD. M = maternal allele; P = paternal allele. Each line represents one sequenced allele. Filled circles indicate the position of methylated CpG dinucleotides. The percentages of methylated CpGs are indicated. * denotes significantly different levels of CpGs methylation (p< 0.05). ** denotes highly significant differences (p< 0.01). ns = not statistically significant differences (p>0.05).

(A) Schematic of the TR2+3Igmyc transgene. The black box indicates Igf2r DMD sequences. The Cα and Sα regions of the mouse IgA locus (Ig) and the 3’ UTR and the c-myc exons (grey boxes) are shown. The arrows indicate the three primers used for semi-nested PCR. (B) Analysis of DNA methylation of the maternal TR2+3Igmyc transgene in wild-type and DNMT1o-deficient oocytes and preimplantation embryos. The top line shows the position of the CpG dinucleotides analyzed (open circles). The filled circles represent the position of methylated CpG dinucleotides. (C) Summary of CpG methylation levels of the TR2+3Igmyc transgene on DNMT1o-deficient MII oocytes (MII) and preimplantation embryos. 4 = 4-cell embryos; 8 = 8-cell embryos; M = morulae; Bl = blastocysts. The percentages of methylated CpGs are indicated. Error bars represent standard error of the mean (SEM). * denotes significantly different levels of CpGs methylation (p< 0.05). ** denotes highly significant differences (p< 0.01). ns = not statistically significant differences (p>0.05).

(A) Methylation analysis of the Snurf/Snrpn DMD on ES cells (ESC) derived from DNMT1o-deficient (Δ1o) blastocysts. Left: A restriction map of the 5’-end of Snrpn gene is shown. Hybridization probe is indicated by the dark bar. Right: DNAs were digested with PstI (P) and the methylation sensitive HhaI (Hh) restriction enzymes. Lanes 1 to 6 are ESC clones from DNMT1o-deficient line 1 (L1). Lane 7 is DNA from DNMT1o-deficient ESC line 2 (L2 Δ1o) and lanes 8 to 12 are ESC clones from DNMT1o-deficient line 2. RI = RI ES cells, c/c = Dnmt1^c/c ES cells. Lanes 15 and 16 are ESC clones from a wild-type line. (B) Methylation analyses of the H19 DMD on ES cells derivated from DNMT1o-deficient (Δ1o) blastocysts. Left: A restriction map of the 5’ non-transcribed region of H19 gene is shown. Hybridization probe is indicated by the dark bar. Right: DNAs were digested with MspI (M) and HhaI (Hh) restriction enzymes. Lanes 1 to 16 same as in A. Molecular weights are expressed in kb and the expected methylated and unmethylated bands are shown. (C) Prediction of four chromosome 7 epigenotypes following loss of DNMT1o maintenance methylation at the fourth embryonic S phase; these first appear in cells of 32-cell embryos after random assortment of chromatids. Each pair of vertical lines (M = maternal, P = paternal) represents the two homologues of chromosome 7. m indicates a parent-specific imprinted methylation mark on the normally methylated chromosome.