Tissue-specific and mosaic imprinting defects underlie opposite congenital growth disorders in mice

<div><p>Differential DNA methylation defects of <i>H19/IGF2</i> are associated with congenital growth disorders characterized by opposite clinical pictures. Due to structural differences between human and mouse, the mechanisms by which mutations of the <i>H19/IGF2</i> Imprinting Control region (IC1) result in these diseases are undefined. To address this issue, we previously generated a mouse line carrying a humanized IC1 (hIC1) and now replaced the wildtype with a mutant IC1 identified in the overgrowth-associated Beckwith-Wiedemann syndrome. The new humanized mouse line shows pre/post-natal overgrowth on maternal transmission and pre/post-natal undergrowth on paternal transmission of the mutation. The mutant hIC1 acquires abnormal methylation during development causing opposite <i>H19/Igf2</i> imprinting defects on maternal and paternal chromosomes. Differential and possibly mosaic <i>Igf2</i> expression and imprinting is associated with asymmetric growth of bilateral organs. Furthermore, tissue-specific imprinting defects result in deficient liver- and placenta-derived <i>Igf2</i> on paternal transmission and excessive <i>Igf2</i> in peripheral tissues on maternal transmission, providing a possible molecular explanation for imprinting-associated and phenotypically contrasting growth disorders.</p></div

Strategy for generating the <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2</i>} allele.

(A)Schematics of the endogenous mouse H19 locus, targeting vector for mutant hIC1 (pH19hIC1Δ2.2-neo), targeted mutant allele with the neomycin resistance cassette (H19hIC1Δ2.2-neo), targeted mutant allele after neoR excision (H19hIC1Δ2.2) and targeted wildtype hIC1 allele (H19hIC1). Depicted are the IC1 (white rectangle) with CTCF binding sites (vertical lilac bars within IC1), ZFP57 binding sites (purple circles), H19 exons (gray rectangles); pBluescriptIIKS vector with the human insert flanked by the mouse sequences (outlined with dashed blue lines) used as arms for the homologous recombination, neoR cassette (yellow rectangle), loxP sites (black arrowheads). Restriction sites and their relative positions (in kb) to the 5’ end of H19 are also indicated. Probes (A and B) used for Southern blot analysis are indicated as thick green lines below the endogenous locus. Positions of the primers for testing the neo cassette excision are indicated by green arrows. Dashed green lines define the region of hIC1 deleted in the hIC1Δ2.2 allele. (B) Southern blot analysis to confirm targeting of mutant hIC1 allele. DNA from wildtype (+/+) and targeted (+/hIC1Δ2.2-neo) ES cells was digested with EcoRV and hybridized to probe A (lying 5’ of the targeting arm) or digested with StuI and hybridized to probe B (lying 3’ of the targeting arm). (C) Confirmation of Cre-mediated excision of neoR cassette. Excision was confirmed by PCR with primers (green arrows in fig 1A) flanking neoR. A PCR product of 550 bp confirming excision was obtained from genomic DNA of F1 (Cre/hIC1Δ2.2) mice, and F2 (+/hIC1Δ2.2) derived from F1 crossed to wildtype mice. No PCR product was obtained from KI mice (+/hIC1Δ2.2-neo) with the NeoR cassette.</p

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

<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

Somatic overgrowth on maternal transmission of the <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2</i>} allele.

Box plot of birth weights (A), growth charts (B), and box plots of organ/whole animal weights at 14 weeks of age (C) of H19hIC1Δ2.2/+ and H19+/+ littermates. Box plots display the full range of variation (from minimum to maximum values), the likely range of variation (first and third quartiles) and the median (horizontal bar within box). Values in brackets indicate the number of animals derived from three litters that was used for this study. P = P-value calculated by two-tailed Student’s T-test. In (B) the growth chart is expressed as ratio between the weights of KI and weights of +/+ animals in three litters. Bars represent the mean ± SEM. In (C) the box plots represent the relative values of organ weight/whole animal weight ratios, obtained by arbitrarily setting the median value of H19+/+ as 1.</p

Somatic undergrowth on paternal transmission of the <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2</i>} allele.

<p>(A-C) Box plot of birth weights (A), growth charts (B), and box plots of organ weights at 14 weeks of age (C) of <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} and <i>H19</i>^<i>+/+</i> littermates. Box plots in (A) and (C) and growth chart in (B) are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007243#pgen.1007243.g002" target="_blank">Fig 2</a>. (D-E) Box plot of embryo body and placenta weights of <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} (D) and <i>H19</i>^{<i>+/hIC1</i>} (E) mice at E15.5 compared with <i>H19</i>^<i>+/+</i> littermates. The animals used for this study derived from three (A-C) or two litters (D-E).</p

Analysis of <i>H19</i> and <i>Igf2</i> expression in <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>} newborn mice.

<p>(A) Histograms of total <i>H19</i> and <i>Igf2</i> expression in three different neonatal organs of <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>} and <i>H19</i>^<i>+/+</i> littermates analysed by RT-qPCR. The mean value of <i>H19</i>^<i>+/+</i> is set arbitrarily as 1. NS, Not Significant. Bars represent the mean ± SEM. (B) Allele-specific expression of <i>H19</i> and <i>Igf2</i>. Dots indicate the percent expression of the maternal allele in each individual sample. The animals used for this study derived from three litters.</p

Kidney asymmetry and <i>H19/Igf2</i> expression and imprinting on maternal and paternal transmission of the <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} allele.

<p>(A-F) Analysis of maternal transmission. Weight difference of the kidneys at 12 weeks of age (A), kidney weights at birth (B), DNA methylation level of CTS1 (C), total <i>H19</i> expression (D), allele-specific <i>Igf2</i> expression (E), and total <i>Igf2</i> expression (F) in heavier and lighter kidneys of <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2/+</i>} and <i>H19</i>^<i>+/+</i> mice. (G-L) Analysis of paternal transmission. Weight difference of the kidneys at 12 weeks of age (G), kidney weights at birth (H), DNA methylation level of CTS1 (I), total <i>H19</i> expression (J), allele-specific <i>H19</i> expression (K), and total <i>Igf2</i> expression (L) analysed in heavier and lighter kidneys of <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} and <i>H19</i>^<i>+/+</i> mice. Bars in A and G represent the mean ± SEM. Bars in C-F and I-L represent the mean ± SEM of three technical replicates of the same sample. P-value calculated by two-tailed Student’s T-test on lighter vs heavier kidneys of littermates with the same genotype. The animals used for this study derived from two litters. Note that differences in weight, <i>Igf2</i> expression and <i>H19/Igf2</i> imprinting were present only in mice carrying the <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2</i>} allele.</p

Analysis of <i>H19</i> and <i>Igf2</i> expression in <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} newborn mice.

<p>(A) Histograms of total <i>H19</i> and <i>Igf2</i> expression in three different neonatal organs of <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} and <i>H19</i>^<i>+/+</i> littermates analysed by RT-qPCR. The mean value of <i>H19</i>^<i>+/+</i> is set arbitrarily as 1. NS, Not Significant. Bars represent the mean ± SEM. (B) Dots indicate the percent expression of the paternal allele in each individual sample. The animals used for this study derived from three litters.</p

IC1 methylation in <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} and <i>H19</i>^{<i>+/hIC1</i>} mice at E15.5.

<p>(A) See legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007243#pgen.1007243.g004" target="_blank">Fig 4A</a>. (B) Percent methylation measured by pyrosequencing at two CTCF target sites (CTS1 and CTS6) from embryo body and placenta of <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} and <i>H19</i>^{<i>+/hIC1</i>} mice at E15.5 (left panel). The endogenous mIC1 was analysed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007243#pgen.1007243.g004" target="_blank">Fig 4</a>. Each histogram represents the methylation mean value of 5 (CTS1 and mIC1) or 6 (CTS6) CpGs, tested in two litters of each KI: +/hIC1Δ2.2 (n = 6), <i>+/+</i> (n = 5); +/hIC1 (n = 6) and <i>+/+</i> (n = 4) embryos. Bars represent the mean ± SEM. (C) IC1 methylation analysed by bisulphite treatment followed by cloning and sequencing in embryo body and placenta of <i>H19</i>^{<i>+/hIC1</i>Δ<i>2</i>.<i>2</i>} and <i>H19</i>^{<i>+/hIC1</i>} mice at E15.5. See legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007243#pgen.1007243.g004" target="_blank">Fig 4</a> for more details.</p

Diagram summarizing the tissue-specific imprinting defects of the <i>H19</i>^{<i>hIC1</i>Δ<i>2</i>.<i>2</i>} mice.

<p>White rectangles represent the <i>Igf2</i> and <i>H19</i> genes, grey rectangle mIC1 and green rectangles hIC1Δ2.2. Enhancers are indicated by green ovals. Transcription direction is indicated by arrows and transcription intensity by arrow thickness. Lollipops indicate CTS methylation status: filled lollipops, methylated CTSs; open lollipops: non-methylated CTSs; half-filled lollipops: partially methylated CTSs; dashed lollipops: missing CTSs in the deleted region of hIC1Δ2.2. Curved arrows indicate <i>Igf2</i> activation by downstream enhancers: arrow thickness is inversely proportional to the insulator strength inferred from allele-specific <i>Igf2</i> expression in each specific tissue. P: paternal chromosome; M: maternal chromosome.</p