Model: chromosome conformation guides <i>Airn</i> lncRNA to target silencing of distant imprinted genes in extra-embryonic tissues.

Left: In early development where Airn is not expressed, and the genes that it regulates Igf2r, Slc22a2 and Slc22a3 are expressed at low levels from both alleles (dashed arrows). Both parental alleles have a chromosomal conformation that brings the Airn gene body and the promoters of Slc22a2 and Slc22a3 in close proximity. These chromosome interactions may be mediated by interaction factors (IF) at regions of open chromatin marked by H3K27ac histone modification (triangles). The imprint control element (ICE) or Airn promoter is methylated on the maternal allele (filled diamond) and unmethylated on the paternal allele (unfilled diamond). Middle: As development progresses, methylation on the maternal allele prevents expression of Airn, the chromosome conformation is maintained and Igf2r, Slc22a2 and Slc22a3 are upregulated (solid arrows). On the unmethylated paternal allele, Airn (wavy line) starts to be expressed and recruits repressive histone modifiers (HM) and targets them to the promoters of Slc22a2 and Slc22a3, preventing these genes from being upregulated. Airn prevents Igf2r from being upregulated by transcriptional interference with its promoter [7]. Right: Later in development, on the maternal allele the chromosome conformation is maintained and Igf2r, Slc22a2 and Slc22a3 are expressed. On the paternal allele, the loss of H3K27ac and the establishment of a compact repressive chromatin domain including H3K27me3, maintains silencing of Slc22a2 and Slc22a3, and leads to the loss of chromosome interactions.</p

Allele-specific expression analysis shows imprinted expression is unaffected by maternal deletion of the <i>Airn</i> gene, but lost upon paternal deletion.

(A) VYS endoderm and placenta was isolated from E12.5 F1 embryos from RSDel x CAST reciprocal crosses and subject to RNA-seq (3 wildtype and 3 RSDel from each cross and tissue). The data was subject to allelic expression analysis using the Allelome.PRO pipeline [33] as detailed in the methods. (B) The maternal RSDel deletion does not affect imprinted expression of Slc22a3 and Slc22a2 in VYS endoderm. Within the deletion Airn is unaffected as it is exclusively paternally expressed, while the non-imprinted gene Tcp1 becomes paternally expressed, and Mllt4 lying 750kb outside of the deletion is unaffected. (C) The maternal RSDel deletion does not affect imprinted expression of Slc22a3 and Pde10a in placenta. Within the deletion Airn and Tcp1, show paternal expression, while Mllt4 is unaffected. (D) The paternal RSDel deletion leads to loss of Slc22a3 and Slc22a2 imprinted expression in VYS endoderm. Within the deletion Airn expression is lost, as it is shows exclusive paternal expression, while Tcp1 shows maternal expression, and Mllt4 outside the deletion is unaffected. (E) The paternal RSDel deletion leads to loss of Slc22a3 and Pde10a imprinted expression in placenta. Airn expression is lost, Tcp1 becomes maternally expressed, and Mllt4 is unaffected.</p

Broad enrichments of H3K27me3 cover the silenced allele of imprinted genes in VYS endoderm.

(A) Maternal enrichment of H3K27ac and paternal enrichment of H3K27me3 is present at sites across the entire 10Mb Igf2r cluster in VYS endoderm, despite imprinted expression being limited to the 450Kb from Slc22a3 to Airn in this tissue. In this region showing imprinted expression, broad enrichment of H3K27me3 covers the silenced paternal alleles of Slc22a3 and Slc22a2, while more focal maternal enrichment of H3K27ac is seen within these genes. (B) Genome-wide 97.2% of H3K27me3 parental allele enriched 20kb windows in VYS endoderm lie within imprinted clusters, with the Igf2r cluster showing the highest number, followed by the Kcnq1 cluster and then the Sfmbt2 cluster. (C) H3K27me3 is enriched over the silenced maternal allele of Sfmbt2 and Blustr in the Sfmbt2 cluster.</p

Deletion of the <i>Airn</i> gene indicates that it contains no essential enhancers for <i>Slc22a3</i>.

(A) The enhancer interference hypothesis. The Airn gene body contains an essential enhancer (E) that interacts with the Slc22a3 promoter (solid arrow) and potentially also with the Slc22a2 promoter (dashed arrow) on the maternal allele (red) activating gene expression (arrows). On the paternal allele (blue) Airn (wavy line) prevents this interaction causing silencing (blocked arrows). (B) The RSDel deletion spans 270 kb from the Airn promoter to the third intron of the Sod2 gene, and includes the entire 118 kb Airn gene and 6 addition genes. The deletion was constructed by Cre-mediated trans recombination between loxP sites in the R2Δ Airn promoter deletion and the Sod2Δ alleles as detailed in the text. (C) Predicted expression patterns in the RSDel deletion that includes the entire Airn gene. Left: Prediction if enhancer interference hypothesis is correct: The maternal deletion removes the essential enhancer for Slc22a3 and Slc22a2 preventing their upregulation, and leading to a loss of expression. The paternal deletion removes Airn, but also the essential enhancer, so Slc22a3 and Slc22a2 remain silenced on the paternal allele, leading to normal levels of expression. Right: Prediction if enhancer interference hypothesis is false: The maternal deletion has no effect on expression of Slc22a3 and Slc22a2, leading to normal levels of expression. The paternal deletion removes Airn leading to a loss of imprinted silencing on the paternal allele, and a doubling of Slc22a3 and Slc22a2 expression. (D) The RSDel maternal deletion does not affect Slc22a3 expression, whereas the paternal deletion leads to a doubling of Slc22a3 expression. RT-qPCR expression analysis of the RSDel maternal deletion (red, RSDel/+) and paternal deletion (blue, +/RSDel) in E9.5 VYS endoderm (left) and E12.5 placenta (right). Expression levels are normalized to wildtype for each cross (set to 100). Bars show the mean and triangles indicate all data points (biological replicates). Note that Airn and Tcp1 (non-imprinted gene) are within the deletion while Slc22a3 is outside.</p

Chromosome Conformation Capture (3C) indicates that the <i>Airn</i> gene body may contain multiple enhancers for <i>Slc22a3</i>.

(A) Chromosome interactions between the Slc22a3 promoter and the Airn gene body are enriched on the maternal allele. Top: In the Igf2r imprinted cluster in visceral yolk sac (VYS), Slc22a3, Slc22a2 and Igf2r are expressed from the maternal (red) and repressed on the paternal (blue) allele. Long arrows indicate active expression, blocked arrows indicate repression. The Slc22a3 promoter region (3C bait fragment) and Airn gene body (3C prey fragments) are indicated by grey boxes. Multiple lines indicate the interactions assayed by 3C. Bottom: The relative level of 3C interactions identified on the maternal (red, +/Thp) and paternal (blue, Thp/+) alleles. (B) Paternal allele chromosome interactions between the Slc22a3 promoter and the Airn gene body are increased following truncation of Airn. Top: Imprinted silencing in the Igf2r cluster in the VYS is lost following truncation of Airn (AirnT, colors and arrows as in A). Middle: The relative level of 3C interactions identified in the wildtype (black, +/+) and AirnT (grey, +/AirnT) mice (both parental alleles present). Bottom: The relative level of 3C interactions detected on the wildtype (dark blue, Thp/+) and the AirnT (cyan, Thp/AirnT) paternal alleles. 3C interactions were determined using Taqman qPCR, normalized to the mean of 2 interactions in the Igf2 cluster, and then the highest interaction for A and B were set to 1, as detailed in the methods. Positions and size of prey fragments investigated in the 3C assay are indicated at the bottom. Points and error bars are mean and standard deviation of 3 technical replicates.</p

Tandem direct repeats regulate the length of <i>Airn</i>.

<p>(A) qPCR of total (spliced+unspliced) <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g002" target="_blank">Figure 2</a> map for location of qPCR assays). Relative <i>Airn</i> levels were set to 100% in <i>S12/+</i> cells at d14. Bars and error bars: mean and standard deviation of three differentiation sets. <i>S12/+</i> and <i>S12/TDRΔ</i> were compared using an unpaired t-test (*P = 0.1–0.5, **P = 0.001–0.01, ***P<0.001). The data show that <i>Airn</i> steady-state levels are unchanged up to 53 kb but are greatly reduced and lost at the 3′ end. (B) qPCR of spliced <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells. Details as in (A). These data show that the TDR deletion does not affect <i>Airn</i> splicing suppression but leads to a shortening at the 3′ end. (C) qPCR of unspliced <i>Airn</i> in 12.5–13.5 dpc mouse embryos confirms the significant loss of <i>Airn</i> steady-state levels at the 3′ end as seen in differentiated ES cells (A,B). Embryos from 3 litters were assayed carrying wildtype (<i>+/+</i>, <i>Thp/+</i>) or <i>TDRΔ</i> (<i>+</i>/<i>TDRΔ</i>, <i>Thp/TDRΔ</i>) paternal alleles. The <i>Thp</i> allele carries a deletion of the entire <i>Igf2r</i> cluster thus only the paternal allele is present. Samples of the same genotype were averaged and the horizontal lines and error bars show mean and standard deviation. Values for individual embryos are plotted as single data points. The number of samples is given below the genotype (n). Relative <i>Airn</i> levels were set to 100% for <i>+/+</i>, all others are displayed relative to it. Samples were compared to <i>+/+</i> using an unpaired t-test. Details as (A).</p

The methylation-free state of the paternal ICE depends on the CGI.

<p>(A) DNA blot assaying methylation of the <i>Airn</i> promoter MluI site as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a>, in undifferentiated ES cells carrying a paternal <i>CGIΔ</i> or wildtype (<i>+</i>) allele. The 5.0 kb band identified by probe MEi (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map) indicates a gain of methylation on the <i>CGIΔ</i> paternal allele. This band is weaker in cells with lower passage numbers that still retain the selection cassette (<i>S12/CGIΔ+cas</i>-1,-2) compared to cells that have been in culture for 8 more passages (<i>S12/CGIΔ</i>-1A,-1B,-2A,-2B) with a deleted selection cassette. The lower panel confirms this by showing a matching loss of the unmethylated 1.1 kb fragment specific to the paternal allele in cells with a higher passage number. Both panels were from the same blot and the intervening area lacking any hybridisation signal removed. (B) DNA blot as in (A) assaying <i>Airn</i> promoter MluI methylation during ES cell differentiation showing that the level of paternal methylation on the <i>CGIΔ</i> allele in undifferentiated ES (d0) cells (5 kb band) does not change in differentiated d5 and d14 cells. Probe MEi is a 1 kb EcoRI-MluI fragment shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map. (C) Bisulfite sequencing of two undifferentiated <i>S12/CGIΔ</i> ES cell clones using primers spanning the deletion that specifically amplify the paternal <i>CGIΔ</i> allele, confirms the strong gain of DNA methylation, but also shows that some alleles are more methylated than others (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5B</a>).</p

TDR absence has a minor effect on paternal <i>Igf2r</i> repression.

<p>(A) Genomic DNA digested with EcoRI (E) or EcoRI+methyl-sensitive NotI (E/N) hybridised with probe EEi. wt:wildtype, met:methylated, unmet:unmethylated, <i>Thp</i>:deletes the entire <i>Igf2r</i> cluster. Quantification of the methylated/unmethylated hybridisation signal shown below for d14, shows equal gain of DNA methylation on the paternal <i>Igf2r</i> promoter in <i>S12/+</i> and <i>S12/TDRΔ</i> cells. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540.s004" target="_blank">Figure S4A</a> shows two further differentiation sets. (B) RT-PCR followed by digestion of a paternal-specific PstI site to assay allelic <i>Igf2r</i> expression in ES cells carrying a paternal wild type (<i>S12/+</i>) or mutated (<i>S12/TDRΔ</i>) allele in four targeted clones. Two further differentiation sets are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540.s004" target="_blank">Figure S4B</a>. -: minus RT, u: undigested, P: PstI digested, Mat: maternal, Pat: paternal. Impaired paternal <i>Igf2r</i> repression indicated by the clear presence of two paternal bands at d5 and faint presence at d14 (*) was seen in all four <i>S12/TDRΔ</i> cell lines. (C) Allele-specific qPCR quantifying <i>Igf2r</i> expression using the same SNP as in (B). The mean maternal∶paternal <i>Igf2r</i> expression ratio and standard deviation of three differentiation sets is displayed. As undifferentiated ES cells show biallelic <i>Igf2r</i> expression the ratio was set to 1 in <i>S12/+</i> d0 cells. Also the <i>S12/TDRΔ</i> cells show biallelic expression in undifferentiated ES cells, as the ratio mat/pat is around 1. During differentiation, the ratio in <i>S12/+</i> cells increases twofold more compared to <i>S12/TDRΔ</i> cells, indicating a compromised although not statistically significantly impaired imprinted expression of <i>Igf2r</i> in <i>S12/TDRΔ</i> cells. <i>S12/+</i> and <i>S12/TDRΔ</i> were compared using an unpaired t-test. (D) qPCR of total <i>Igf2r</i> steady-state levels in 12.5–13.5 dpc mouse embryos shows a minor loss of paternal <i>Igf2r</i> repression. Embryos from 3 litters carrying wildtype (<i>+/+</i>, <i>Thp/+</i>) or <i>TDRΔ</i> (<i>+/TDRΔ</i>, <i>Thp/TDRΔ</i>) paternal alleles were assayed and compared using an unpaired t-test. The <i>Thp</i> allele carries a deletion of the entire <i>Igf2r</i> cluster thus only the paternal allele is present. Details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3C</a>.</p

Tandem direct repeats play a role in <i>Airn</i> processivity.

<p><i>Airn</i> expression by genome tiling array in day 5 differentiated ES cells carrying a paternal wildtype (<i>S12/+</i>) or mutated (<i>S12/TDRΔ</i>-2A and <i>+/TDRΔ</i>) allele. Note the maternal allele is always written on the left side (Mat/Pat). x-axis: basepairs, y-axis: averaged relative signal intensities with standard deviation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#s4" target="_blank">Materials and Methods</a>). Single and double dashed arrows: position after which consistent differences between wildtype and two <i>TDRΔ</i> cell lines are seen. Grey arrow: <i>Airn</i> hybridisation signals are lost after 90 kb in two <i>TDRΔ</i> cell lines. Below: <i>Airn</i> (wavy arrow) and <i>Airn</i> splice variants (black boxes: exons). Grey font: <i>Airn</i> qPCR assays with their distance from <i>Airn</i>-TSS. RP11, RP6, RP21, RP5, RP4 were combined with FP1+TQ-AS. This analysis shows that <i>Airn</i> in <i>TDRΔ</i> cells is reduced after 68 kb and lost after 90 kb.</p

The <i>Airn</i> CGI plays a major role in <i>Airn</i> transcription and function.

<p>(A) <i>Airn</i> expression by genome-tiling array (left axis) and strand-specific expression analysis by RNA-Seq (right axis) for differentiated <i>S12/+</i> and <i>S12/CGIΔ</i>-1A cells. Dashed arrows: sharp drop of <i>Airn</i> hybridisation signals in the <i>Airn</i>-specific region (single) and absence after 73 kb (doublet). Below: qPCR assays relative to <i>Airn</i>-TSS with colour code as (B,C). Striped box: overlapping START+RP11 assays. (B) qPCR of total+unspliced <i>Airn</i> in d0/d5/d14 differentiated <i>S12/+</i> and four <i>S12/CGIΔ</i> clones shows unspliced <i>Airn</i> is reduced by ∼40% at the 5′ end (RP11/154 bp), but when assayed downstream (<i>Airn</i>-middle/53 kb, <i>Airn</i>-end/99 kb) or at positions which include splice variants (START), is reduced by >70% in <i>S12/CGIΔ</i> cells. Shown are mean and standard deviation of three differentiation sets (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3A</a>). (C) <i>Airn</i> qPCR in <i>S12/+</i> and four <i>S12/CGIΔ</i> d14 clones shows that unspliced <i>Airn</i> is reduced by 79–83% at 0.57 kb and ∼85% at 7.3 kb, while spliced <i>Airn</i> reduced by >85%. Shown are mean and standard deviation of three differentiation sets (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3A</a>). (D) ChIP for Ser5P/Ser2P RNAPII in <i>S12/+</i>, <i>S12/TDRΔ</i>-1A and <i>S12/CGIΔ</i>-1A d11 cells shows unaffected <i>Airn</i> initiation and elongation (except at <i>Airn</i>-end) in <i>TDRΔ</i> and a sharp RNAPII decrease in the <i>CGIΔ</i> allele. The mean and standard deviation of three technical replicates is shown. Assay <i>Airn</i>-132 controls for background from the overlapping <i>Igf2r</i> transcript, which is 2-fold higher in <i>CGIΔ</i> that fails to repress the paternal <i>Igf2r</i> promoter. Map for qPCR assays as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g002" target="_blank">Figure 2</a>. (E) DNA blot analysing methylation of the <i>Igf2r</i> promoter NotI site (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g004" target="_blank">Figure 4A</a>). *methylated fragment in d0 cells originating from feeder-cells. This blot shows that cells carrying a paternal <i>CGIΔ</i> allele contrary to wildtype cells do not gain the methylated 5 kb band on the paternal <i>Igf2r</i> promoter. White lines: indicate the order of samples run on the same gel was changed electronically. (F) qPCR quantifying allelic expression shows absence of <i>Igf2r</i> imprinted expression (Mat∶Pat ratio is close to 1), in four <i>CGIΔ</i> (<i>S12/CGIΔ</i>) cell lines compared to wildtype (<i>S12/+</i>). Three differentiation sets are shown separately due to variability in Mat∶Pat ratios in wildtype controls for each set. Bars represent the mean, error bars the standard deviation of 3 technical replicates (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g004" target="_blank">Figure 4C</a>).</p