TDRs are required for the regulation of ICE DNA methylation.

<p>(A) Genomic DNA digested with EcoRI (E) or EcoRI+methyl-sensitive MluI (E/M) and hybridised with probe <i>Airn</i>T. Cells carrying a paternal <i>TDRΔ</i> (<i>S12/TDRΔ</i>) or wildtype (<i>S12/+</i>) allele have a 6.2 kb band from the maternally-methylated plus 4.3 kb or 5.0 kb from the paternally-unmethylated allele. <i>S12/TDRΔ</i> cells additionally show at d0, a faint 5.5 kb band representing a paternal partially methylated allele (*) that is lost by d14 in differentiated cells. (B) Bisulfite sequencing of undifferentiated <i>S12/TDRΔ</i>-1A ES cells. Each row represents one allele, single columns: one CpG, double columns: two neighbouring CpGs. White/black circles: unmethylated/methylated CpGs. Arrows: position of MluI site and <i>TDRΔ</i> deletion. Primers DMR2-4F and -9R specifically amplify the paternal allele in <i>S12/TDRΔ</i> ES cells (details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540.s005" target="_blank">Figure S5B</a>). Bisulfite sequencing confirms a low level of DNA methylation on the paternal <i>TDRΔ</i> allele at d0 only. (C) Percent methylation levels for two <i>S12/TDRΔ</i> and control <i>R2Δ/+</i> ES cells. The <i>R2</i>Δ deletion removes the maternal ICE, thus only the wildtype paternal ICE is detected. Each point represents one sequenced clone and shows that low-level DNA methylation is a general feature of the paternal ICE in d0 ES cells and not a consequence of the TDR deletion. (D) DNA blot analysing the MluI methylation status as in (A) for 12.5–13.5 dpc embryos carrying a maternal <i>TDRΔ</i> (<i>TDRΔ/+, TDRΔ/Thp</i>) or wildtype (<i>+/+, +/Thp</i>) allele. The 5.0 kb methylated band is faint or absent in <i>TDRΔ/+</i> embryos, but present in <i>+/+</i> embryos (6.2 kb), showing that maternal inheritance of the <i>TDRΔ</i> allele leads to loss of DNA methylation. (E) Bisulfite sequencing of embryonic genomic DNA (details as in B) shows that maternal transmission of the TDR deletion leads to a major loss in DNA methylation. For <i>TDRΔ/Thp</i> only the maternal allele, for <i>+/+</i> both parental alleles were analysed. (F) Percent methylation level for <i>TDRΔ/Thp</i> and <i>+/+</i> embryos as in (C).