Gain of IC1 methylation in <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>} mice.

Abstract

<p>(A) Graph representing the humanized and endogenous <i>H19/Igf2</i> locus with the regions (indicated by black bars) whose methylation was analysed by pyrosequencing. The region deleted in <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2</i>} is represented by a shaded light blue box. The coordinates of the sequenced CpGs are: CTS1 in hg39—chr11:2,024,193–2,024,261; CTS6 in hg39—chr11:2,021,096–2,021,153; mIC1 in mm9—chr7:149,767,625–149,767,705; mIC2 in mm9—chr7:150,482,365–150,482,486. Other details are described in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007243#pgen.1007243.g001" target="_blank">Fig 1</a>. (B) Percent methylation measured by pyrosequencing at two CTCF target sites (CTS1 and CTS6) in three different organs collected from <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>} and <i>H19</i>^{<i>hIC1/+</i>} mice at birth (left panel). Endogenous mIC1 analysed as control of methylation: the paternal allele (pat) was assayed in <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>}, the maternal allele (mat) in <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>}, and both alleles (pat + mat) in <i>H19</i>^<i>+/+</i> mice (right panel). Each histogram represents the methylation mean value of 5 (CTS1 and mIC1) or 6 (CTS6) CpGs, tested in <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>} (n = 10), <i>H19</i>^{<i>hIC1/+</i>} (n = 9) and <i>H19</i>^<i>+/+</i> (n = 7) mice derived from three litters. Bars represent the mean ± SEM. Note that methylation (ranging from 55% to 75%) was observed at the <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2</i>} and not at the <i>H19</i>^<i>hIC1</i> allele. (C) IC1 methylation analysed by bisulphite treatment followed by cloning and sequencing in tongue of <i>H19</i>^{<i>hIC1/+</i>} and <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>} mice. Each line corresponds to a single template DNA molecule cloned; each circle corresponds to a CpG dinucleotide. Filled circles designate methylated cytosines; open circles, unmethylated cytosines. CpGs measured by pyrosequencing are framed.</p