Phenotype of +/<i>T</i>^int2 and +/<i>T</i>^ex1.

<p>Appearance of <i>+/T</i>^<i>int2</i> and +/<i>T</i>^<i>ex1</i> (A) Two +/<i>T</i>^<i>int2</i> and a wild-type sibling at postnatal day 2. The +/<i>T</i>^<i>int2</i> (labelled with an arrow) are smaller and leaner than wild-type. (B) A newborn +/<i>T</i>^<i>ex1</i> (labelled with an arrow) and a wild-type sibling. (C) Growth retardation. Shown is the growth curve of surviving and non-surviving +/<i>T</i>^<i>int2</i> and wild-type littermates from 1 to 29 days post birth. The mean weight of wild-type littermates at each time-point have been normalised to 1 and the weights of +/<i>T</i>^<i>int2</i> mice have been taken as a percentage of wild-type weights (n = 19–27 for surviving +/<i>T</i>^<i>int2</i>, 4–19 for non-surviving +/<i>T</i>^<i>int2</i> and 5–49 for +/+). Weights of both sexes have been combined as no significant differences in the weights of males and females were found when considered as a percentage of the weight of wild-type siblings. Error bars show standard error of the means.</p

Schematic of the <i>Gnas</i> cluster in (A) +/+ (B) <i>+/T</i>^ex1 and (C) <i>+/T</i>^int2 mice.

<p>Solid black fill boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> (XL) whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i> (1A). (B) Insertion of a poly-A cassette is shown as inverted ‘pA’ on the paternal <i>T</i>^<i>ex1</i> allele. On this allele, <i>Nespas</i> was truncated, and <i>Nesp</i> was de-repressed. (C) The poly-A cassette is inserted in the reverse orientation (shown as pA) on the paternal <i>T</i>^<i>int2</i> allele. On this allele, <i>Nespas</i> was not truncated but was expressed at a low level (low expression shown as grey arrow), and <i>Nesp</i> was de-repressed and truncated. (D) <i>Nesp</i> is expressed biallelically at 10.5 dpc in <i>SD2/T</i>^<i>ex1</i> embryos. <i>Nesp</i> expressed from an SD2 allele shows a band of 151bp, and <i>Nesp</i> from a <i>T</i>^<i>ex1</i> (129/<i>SvEv</i> inbred strain) allele band shows a band of 178bp upon digestion with <i>Bst</i>UI. Lanes 2,3 of <i>SD2/T</i>^<i>ex1</i> mutants show bands of both sizes, indicating that <i>Nesp</i> is expressed from both parental alleles at 10.5 dpc. Lane 1 shows 10.5 dpc <i>SD2</i>/+; Lane 4, 129<i>SvEv</i> wild-type neonatal brain; Lane 5, SD2 neonatal brain; Lane, 6,7, 10.5 dpc <i>T</i>^<i>ex1</i>/<i>SD2</i>; Lane 8, 10.5 dpc +/<i>SD2</i>; Lane 1 is a DNA ladder.</p

Expression of <i>Gnas</i>, <i>Gnasxl</i> and <i>Exon1A</i>, and methylation status of the <i>Nespas-Gnasxl</i> and <i>Exon1A</i> DMRs.

<p>Bar-charts (A, B, C) show relative quantification (RQ) of <i>Gnasxl</i>, <i>Gnas</i> and <i>Exon1A</i> transcripts in +/<i>T</i>^<i>int2</i> and +/<i>T</i>^<i>ex1</i> neonatal tissue compared to +/+ siblings (left). Error bars represent the range of possible RQ values defined by the standard error of the ΔCTs. * <i>p< 0</i>.<i>05</i>, ** <i>p<0</i>.<i>01</i>, as determined by a w-sample <i>t</i>-test. (D) Summary of methylation at the <i>Nespas-Gnasxl</i> DMR and the <i>Exon1A</i> DMR on paternal inheritance of a wild-type, <i>T</i>^<i>ex1</i> and <i>T</i>^<i>int2</i> allele. (E) Northern blot analysis of expression of <i>Exon1A</i> and <i>Gnas</i> in brown adipose tissue of +/<i>T</i>^<i>int2</i> and +/<i>T</i>^<i>ex1</i> neonates and their wild-type siblings. <i>Actb</i> is an endogenous loading control.</p

Methylation of the <i>Exon1A</i> DMR on paternal inheritance of the <i>T</i>^ex1 allele in (A) neonatal brain, n = 2 and (B) 10.5 dpc embryos, n = 2, both of the +/<i>T</i>^ex1; <i>ΔExon1A</i>/+ genotype.

<p>Each circle represents a CpG dinucleotide; filled when methylated and open when unmethylated. Each string of circles is a unique clone, and all clones from an individual are grouped into a block. (C) shows a summary of <i>Nespas</i> and <i>Nesp</i> expression and <i>Exon1A</i> DMR methylation on paternal inheritance of a <i>T</i>^<i>ex1</i> allele. The solid black filled boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i>. Hypomorphic <i>Nespas</i> expression is shown in grey. (D) Methylation of the paternally inherited <i>Exon1A</i> DMR in control littermates +/+; <i>ΔExon1A</i>/+ in neonatal brain, n = 1 and in (E) 10.5 dpc embryos, n = 2. (F) shows a summary of <i>Nespas</i> and <i>Nesp</i> expression and <i>Exon1A</i> DMR methylation on a wild-type paternal allele. (G) shows a methylation sensitive Southern blot performed on +/<i>T</i>^<i>ex1</i>; <i>ΔExon1A</i>/+ (lanes 4,5,6) and +/+; <i>ΔExon1A</i>/+ (lanes 1,2,3) neonatal brains. <i>Bam</i>HI digestion (-), <i>Bam</i>HI and <i>Hpa</i>II (H), and <i>Bam</i>HI and <i>Msp</i>I (M) digestions probed for the <i>Exon1A</i> DMR are shown for each sample. Sample +/<i>T</i>^<i>ex1</i>; <i>ΔExon1A</i>/+ resists complete digestion by the restriction sensitive <i>Hpa</i>II (lane 5) suggesting methylation at the <i>Exon1A</i> DMR.</p

Methylation of the <i>Exon1A</i> DMR on the <i>T</i>^int2<i>Exon1A</i>⁺ allele in neonatal brain.

<p>Upper panels show a schematic of the wildtype <i>Gnas</i> cluster or that of the <i>Gnas</i> cluster carrying a <i>T</i>^<i>int2</i> mutant allele. Lower panels show corresponding bilsulfite methylation profiles of the <i>Exon 1A</i> DMR. (A) Upper panel: Line drawing of the <i>Gnas</i> cluster on paternal inheritance of the <i>T</i>^<i>int2</i> allele. Lower panel: Bisulfite methylation profile of the paternal <i>Exon1A</i> DMR in neonatal brain of genotype <i>+/T</i>^<i>int2</i><i>; ΔExon1A</i>/ +, n = 3. A ‘-’ shows no result at that CpG. (B) Upper panel: Line drawing of a paternally inherited wild-type <i>Gnas</i> cluster. Lower panel: Bisulfite methylation profile of the paternal <i>Exon1A</i> DMR in neonatal brain of littermate controls, <i>+/+; ΔExon1A</i>/ +, n = 2. (C) Upper panel: Line drawing of the <i>Gnas</i> cluster on maternal inheritance of the <i>T</i>^<i>int2</i> allele. Lower panel: Bisulfite methylation profile of the maternal <i>Exon1A</i> DMR in neonatal brain of <i>T</i>^<i>int2</i>/+;+/<i>ΔExon1A</i>, n = 2. (D) Upper panel: Line drawing of a maternally inherited wild-type <i>Gnas</i> cluster. Lower panel: Bisulfite methylation profile of the maternal <i>Exon1A</i> DMR in neonatal brain of littermate controls, <i>+/+;+/ΔExon1A</i>, n = 2. (E) Methylation sensitive Southern blot performed on +/<i>T</i>^<i>int2</i>; Δ<i>Exon1A</i>/+ (lanes 4,5,6) and +/+; Δ<i>Exon1A</i>/+ (lanes 1,2,3) neonatal brains. <i>Bam</i>HI digestion (-), <i>Bam</i>HI and <i>Hpa</i>II (H), and <i>Bam</i>HI and <i>Msp</i>I (M) digestions probed for the <i>Exon1A</i> DMR are shown for each sample. Both samples are completely digested by the restriction sensitive <i>Hpa</i>II suggesting absence of methylation at <i>Hpa</i>II sites in the <i>Exon1A</i> DMR.</p

Composite of DMR methylation and transcript expression in (A) +/+, (B) +/<i>T</i>^ex1 and (C) +/<i>T</i>^int2.

<p>The solid black fill boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i>. A string of filled circles represents a methylated DMR, and a string of open circled represents an unmethylated DMR. ‘X’ shows that the corresponding transcript is repressed. Transcripts expressed at low levels are shown in grey.</p

Organisation of the mouse <i>Gnas</i> locus.

<p>Both the maternally and paternally inherited copies of the <i>Gnas</i> cluster are shown. Boxes represent exons. The solid black filled boxes represent first exons of the protein-coding transcripts <i>Nesp</i>, <i>Gnas</i> and <i>Gnasxl</i> (labelled XL) whereas shaded boxes are first exons of the non-coding transcripts <i>Nespas</i> and <i>Exon1A</i> (labelled 1A). Arrows show the direction of transcription. <i>Gnas</i> expression is shown as a dotted line as <i>Gnas</i> itself shows tissue-specific imprinted expression. The position of the differentially methylated regions (DMRs) is shown by a string of filled circles on the allele on which the DMR is methylated. <i>Nesp</i> transcription traverses the entire length of the cluster, including the <i>Nespas-Gnasxl</i> DMR and the <i>Exon1A</i> DMR, as shown by a long arrow. The figure is not to scale. Adapted from Williamson <i>et al</i> (2011).</p

Methylation of the <i>Exon1A</i> DMR in sperm.

<p>(A) <i>Exon1A</i> DMR methylation in sperm of mice carrying the <i>T</i>^<i>ex1</i> mutation (of genotype <i>T</i>^<i>ex1</i>/+; +/Δ<i>Exon1A</i>, n = 2). (B) <i>Exon1A</i> DMR methylation in sperm of littermate control males (of genotype +/+; +/Δ<i>Exon1A</i>, n = 2).</p

Representation of EEG frequencies in REM and NREM sleep.

<p>Here is represented the power density (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002706#s4" target="_blank">Methods</a>) for all frequencies in all 4-seconds REM (blue line) and NREM (red line) sleep epochs during a 48-hour baseline.</p

Exon 1A deletion: <i>Gnas</i> expression and body temperature.

<p>(a) Representation of <i>Gnas</i> expression when the <i>Exon1A-DMR</i> (Ex1A-DMR) is methylated on the maternal (M) allele in +/+ and +/<i>Ex1a</i> or deleted on the paternal (P) allele in +/<i>Ex1a</i> mice. (b) Expression levels, by q-PCR measurements, in BAT tissue of <i>Gnas</i> (left panel) and <i>Ucp1</i> (right panel) mRNA in <i>+/+</i> versus mutant <i>+/Ex1a</i> mice. (c) Grand-averages and shadowed ± s.e.m. of body temperature over a 24-hour period for +/+ and +/<i>Ex1a</i> mice. Statistics are reported as following: <i>t</i>-test; ** = <i>P</i><0.01.</p