DNA methylation analysis.

<p>A. Structure of the <i>Kit</i> transcription unit on mouse chromosome 5. Regions analyzed by (h)meDIP are indicated as black boxes, the promoter-associated CpG island is shown as a green box and the transgene insertion site of the <i>Kit^tmlAlf1/+</i> allele is marked by an asterisk. B. (h)meDIP analysis of genomic DNA from mouse testes. Immunoprecipitated DNA was amplified by locus-specific qPCR and enrichments were calculated relative to the unmethylated actin control. 5mC values are shown as red bars and 5hmC values as blue bars. Diagrams show the results of at least three independent experiments, standard errors of the mean are indicated by error bars. C. Bisulfite sequencing analysis of genomic DNA from testes. Methylation maps show 454 sequencing reads (rows) and the methylation status of 9 CpGs (columns) within the Kit promoter and 4 CpGs from the Kit exon 14 region. Methylated CpGs are shown in red, unmethylated CpGs in cyan and gaps in white. Numbers in methylation maps indicate the number of sequencing reads.</p

Kit epigenetic variants are not generated in the <i>Dnmt2^−/−</i> genotype.

<p>Progenies of the indicated crosses were individually genotyped by PCR determination of the LacZ marker of the <i>Kit^tmlAlf1/+</i> allele. The epigenetic Kit variants maintain the white-tail phenotype of the mutant with the <i>Kit^+/+</i> homozygote genotype.</p><p>In Experiment #3, fertilized eggs were collected at day E 0.5 and implanted into wild type foster mothers. The fact that nearly all fertilized eggs (75 out of 77) developed into healthy progenies excludes any selective developmental arrest during development.</p

The epigenetic Kit modification is maintained in the <i>Dnmt2 ^−/−</i> progeny of heterozygote parents but not further transmitted.

<p>Analysis of 3 litters for each cross. In crosses #1 and 2, <i>Kit</i> genotypes are indicated in the <i>Dnmt2 ^−/−</i> fraction of the progeny. In cross #3, the male parent was a <i>Dnmt2 ^−/−</i> homozygote with a modified Kit allele born from cross #2. When mated with a wild type female, only non-modified <i>Kit⁺</i> alleles were transmitted to the progeny.</p

RNA of <i>Dnmt2 ^−/− Kit^tmlAlf1/+</i> heterozygotes does not induce paramutation in the wild-type one-cell embryo.

*<p><i>p</i><0.05 between RNAs of Dnmt2-positive and negative animals.</p

Induction of paramutation by a fragment of the <i>Kit</i> mRNA sequence is increased after methylation but requires Dnmt2 expression.

<p>Microinjection assays were performed comparatively on wild type fertilized eggs and eggs recovered after mating two <i>Dnmt2 ^−/−</i> parents. Controls received either buffer only. Sequence of the Kit oligoribonucleotide is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003498#pgen.1003498.s007" target="_blank">Table S2</a>. MetKit2 is identical to Kit2123–2150 but with all 8 cytosines methylated.</p>§<p><i>p</i><0.05 between the methylated and the non methylated RNAs.</p

RNA methylation analysis.

<p>A. Single-cell embryos were collected after mating of either two wild-type or two <i>Dnmt2^−/−</i> B6/D2 parents. After microinjection of the Kit2123–2150 oligoribonucleotide (representing 28 nt of the mRNA sequence) or buffer, the embryos were transferred to foster mothers (2 for each condition and 10 embryos per foster). At embryonic day E9.5, 6 to 8 embryos were obtained from each foster. Total RNA was prepared separately from each embryo and processed for <i>Kit</i> RNA methylation analysis. RNA bisulfite sequencing maps are shown for 45 cytosine residues from the Kit exon 14 region in microinjected wild-type and Dnmt2^−/− embryos. Each row represents one sequence read, each column a cytosine residue. Yellow boxes represent unmethylated cytosine residues, blue boxes indicate methylated cytosine residues, sequencing gaps are shown in white. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003498#s2" target="_blank">Results</a> are shown for three independent biological replicates, numbers in methylation maps indicate the total number of sequencing reads. Arrows mark two putative cytosine methylation sites, numbers below arrows indicate the site-specific methylation levels. B. Schematic drawing of the sequenced region of <i>Kit</i> mRNA. Position of the microinjected oligoribo-nucleotide is shown in red and underlined in the nucleotides sequence.</p

The Kit and Sox9 variant phenotypes are not generated in Dnmt2-negative embryos.

<p>A. Kit paramutants in heterozygote mating. In the progeny of crosses between <i>Kit^tmlAlf1/+</i> heterozygotes (either males or females) and <i>Kit^+/+</i> partners, a majority of the <i>Kit^+/+</i> offspring (red boxes) maintained the white-tail phenotype of the mutant shown in the insert photograph. In crosses performed in parallel between isogenic <i>Dnmt2^−/−</i> parents, all the <i>Kit^+/+</i> progeny exhibited the full-color tail phenotype (open boxes). Numeric values and results of <i>Kit^tmlAlf1/+</i> intercrosses are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003498#pgen-1003498-t001" target="_blank">Table 1</a>. B. The Sox9 paramutation induced by microinjection of miR-124 RNA. Fertilized B6D2 eggs were collected following crosses either between <i>Dnmt2^+/+</i> parents, or between a <i>Dnmt2^+/+</i> female and a <i>Dnmt2^−/−</i> male, or between two <i>Dnmt2^−/−</i> parents. Microinjection of single-stranded miR-124 RNA was performed as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003498#pgen.1003498-Grandjean1" target="_blank">[4]</a>. E7.5 embryos were collected. In the wild-type and heterozygote <i>Dnmt2</i> genotypes, but not in the negative homozygote, the characteristic “giant” phenotype was identified based on the increased size (insert, scaling bar 1 mm) and weight of the embryos. Bars represent the average weight and standard error of the mean (SEM) values for each series of 6 embryos. To minimize variations between foster mothers, controls (microinjected with unrelated RNAs) and miR-124-treated embryos were in each series separately implanted in the two uterine horns of the same mothers. <i>p</i><0.05 for <i>Dnmt2</i> negative <i>versus</i> wild-type and heterozygote embryos.</p