DNA methylation analysis in the modified Rxrα locus of Dnmt1^−/− mice.

<p>Bisulfite sequencing analysis at the <i>LoxP</i> site in 8.5 <i>dpc</i> embryos either heterozygous or homozygous for the <i>Dnmt1</i> null mutation, including representative examples of partially methylated molecules. Genomic DNA from three embryos of each genotype have been independently subjected to bisulfite treatment.</p

Heritable methylation profiles require the Dnmt1 but neither the Dnmt2 nor the Dnmt3a methyltransferase.

*<p>CpG/non-CpG methylation profiles as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001136#pone-0001136-g001" target="_blank">Fig. 1</a>.</p><p>n and x correspond to the number of clones sequenced and the number of mice analyzed, respectively.</p>$<p>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001136#pone-0001136-g003" target="_blank">Fig. 3</a>.</p

Epigenetic marks in sperm nuclei.

<p>A. Distribution in sperm DNA of the methylation profiles shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001136#pone-0001136-g001" target="_blank">Fig. 1</a>. Numbers of molecules of each profile are indicated and correspond to the analysis of two independent PCR reactions where two different preparations of sperm bisulfite-treated DNA were used as template. B. The <i>Rxr</i>α locus is included in the protamine-associated fraction of the sperm chromatin. Wild type (+/+) and heterozygote (<i>AF1</i>/+) sperm chromatin preparations were partitioned into histone-enriched (H) or protamine-enriched (P) DNA fractions after <i>EcoR</i>I and <i>BamH</i>1 restriction cleavage. PCR analysis of the methylated <i>LoxP</i> region and of <i>Line I</i> elements, known to be present in the nucleosomal fraction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001136#pone.0001136-Bird2" target="_blank">[18]</a>, was performed on the fractions using the primers and conditions indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001136#pone-0001136-t001" target="_blank">Table 1</a>.</p

Allele-specific maintenance of a complex methylation pattern.

<p>A. Upper part: schematic representation of the <i>AF1</i> mutant and wild type <i>Rxr</i>α alleles. Boxes indicate exons. The nucleotide sequence surrounding the <i>LoxP</i> site in <i>AF1</i> (arrow head) and the methylation state of the cytosine residues are shown below. Open stars correspond to unmethyled cytosines, filled stars to methylated non-CpG and filled circles to methylated CpG sites. The three classes of methylated molecules are shown and their frequency (per cent) in a total of 287 clones sequenced after amplification. B. Distribution of the three classes of methylated molecules amplified from the <i>AF1</i> allele. Mice indicated as “Family 1” were derived from the same unmethylated <i>Rxr</i>α^AF1 male carrying the Sycp1-Cre transgene and that indicated “Family 2”, from a distinct ancestor. Genomic DNA from organs (liver, kidney and testis) dissected from 2 to 3 independent mice and genomic tail DNA from 2 to 4 different mice of each generation were subjected independently to bisulfite treatment. Each genomic DNA were then PCR amplified separately and classified in three different classes of methylated molecules. Number of mice used in each experiment are indicated in parentheses. From left to right: Family 1 liver, kidney, testis and tail DNA after 15 generations, tail DNA after 18 and 23 generations in the B6D2 F1 genetic background; tail DNA after 8 generations of crosses into the indicated backgrounds; Family 2 tail DNA after 15 generations in B6D2 F1. n correspond to the numbers of molecules analysed.</p

Oligonucleotides used in this study.

<p>For the oligonucleotide, RTQ primer, V represents the nucleotides A, C, or G; N indicates that the nucleotide is equivalent to A, C, G, or T.</p

Quantitative PCR determination of spR-12 in somatic and germ line cells.

*<p>Expression relative to that of miR-16.</p>**<p>Expression relative to actual number of cells. Serial dilutions of synthetic spR-12 RNA were used to generate a standard curve of the log of template quantity versus threshold cycle (C_(t)). The concentration of RNA ranged from 10–10⁶ initial copies. A minimum of eight oocytes and/or embryos was used in each experiment. Each value corresponds to three or four independent experiments, performed in duplicate. The analysis for spR-13 was not done as it was not possible to synthesize the spR-13 RT-primer.</p

Illumina GA sequencing reads of spR-12 sequences and variants.

<p>Bold type letters show nucleotide sequences which are not mapped on the genome.</p

Size distribution of testis and sperm RNAs.

<p>Electropherograms were obtained using the RNA 6000 Nano Chip. <b>A.</b> RNA prepared from mouse sperm contained no or very low levels of long RNAs, including ribosomal RNAs (18S- and 28S-rRNA) compared with the total RNA from mouse testis <b>(B)</b>. <b>C.</b> Classification of all sequence reads in the mouse sperm total RNA library.</p

Sperm/testis ratio of read counts of spR-12 and flanking-spR-12.

<p>Read counts of each sequence were normalized to reads per million (RPM).</p

Search for double-stranded piRNA precursors of spR-12 and -13 by strand-specific RT-PCR.

<p>Total RNA from adult mouse testes and epididymis was reverse-transcribed with a primer with an antisense sequence, with a primer with a sense sequence, and with no primer. PCR was conducted using gene-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone-0044542-t005" target="_blank">Table 5</a>). The product was 301 bp for the spR-12 locus, and 351 bp for the spR-13 locus, whose nucleotide sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone-0044542-g007" target="_blank">Figure 7</a>. LINE 1 and β-actin were used as positive and negative controls, respectively.</p