Allele-Specific RT-PCR Sequencing Assays for the Human <i>INPP5F_V2</i> (A), <i>NAP1L5</i> (B), and <i>MCTS2</i> (C) Transcripts

<p>The maternal and fetal genotype was determined for each family. Where mother and fetus were both heterozygous, the parental origin of the single expressing fetal allele could not be determined (“not informative”). For all three genes, the first family shows paternal-allele-specific expression in fetal spinal cord. In each case, the remaining two families exhibited monoallelic expression.</p

Identification of a Novel Imprinted Retrogene and gDMR

<div><p>(A) Allele-specific RT-PCR sequencing assays in inter-specific mouse hybrids. SNPs were identified between C57BL/6J (B6) and <i>Mus mus castaneus</i> (cast), such that the parental origin of the expressing allele(s) could be determined in F1 hybrids. The maternal allele is indicated first in the hybrid crosses.</p> <p>(B) Allele-specific RT-PCR sequencing assay for the <i>U2af1-rs1</i> and <i>Inpp5f_v2</i> genes in mouse testes. cDNA was prepared from whole testes.</p> <p>(C) Comparative analysis of the <i>H13</i> genomic sequence in multiple species, using mouse as the base genome. For clarity only the intron containing the imprinted murine retrogene is shown. Purple shading indicates coding exonic sequence, light-blue shading indicates noncoding exonic sequence, and pink shading indicates conserved nontranscribed sequence. Positions of mouse exons are shown as horizontal lines underneath the plot.</p> <p>(D) Methylation status of the <i>Mcts2</i> promoter region in germ cells and E13.5 embryo, determined by the sequencing of bisulphite-modified genomic DNA. Closed circles indicate methylated CpGs, open circles are unmethylated. E13.5 embryos were derived from B6 mothers and cast fathers, so the parental origin of each strand could be determined using a SNP within the PCR amplicon.</p></div

The Genomic Environment and Germ-Line Methylation Status of Three Imprinted Retrogenes

<p>The chromosome maps on the left-hand side show the position of each imprinted retrogene relative to other imprinted domains on the same chromosome. For each of the three loci, the top right-hand section shows the exonic structure and splicing pattern, the middle section shows the intron within which the retrogene is situated, and the bottom section shows the methylation status in oocytes and sperm, as determined by bisulphite sequencing. Circles on horizontal lines depict CpG dinucleotides on individual strands of genomic DNA. Filled circles represent methylated CpGs and open circles are unmethylated CpGs. The horizontal bar underneath each section marks the extent of the region below to depict scale. For <i>U2af1-rs1,</i> the methylation data is a summary of previously published results [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030020#pgen-0030020-b015" target="_blank">15</a>].</p

Evolutionary Tree for Placental Mammals, Using the Topology Determined in [54]

<div><p>(A) Based on the multi-species comparative sequence analysis in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030020#pgen-0030020-g002" target="_blank">Figures 2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030020#pgen-0030020-g003" target="_blank">3</a>, the approximate points in the mammalian radiation at which each of the four imprinted retrogenes originated are superimposed as grey boxes. The genome sequence of Dasypus novemcictus (armadillo) and Echinops telfari (tenrec) are currently only available in draft format and were therefore not used for the comparative analyses.</p> <p>(B) Allele-specific RT-PCR sequencing assays for <i>H13</i> in B6 × cast reciprocal F1 hybrids. The maternal allele is given first in the hybrid crosses.</p> <p>(C) Transcriptional overview of the <i>H13</i> locus. Protein-coding regions are shown as thick blocks, UTR regions as thin blocks, and introns as thin lines. Splice patterns are indicated. The paternally expressed <i>Mcts2</i> is shown in blue and the maternally expressed <i>H13</i> is in red. Arrows indicate the orientation of transcription.</p></div

Profiling of oxBS-450K 5-hydroxymethylcytosine in human placenta and brain reveals enrichment at imprinted loci

<p>DNA methylation (5-methylcytosine, 5 mC) is involved in many cellular processes and is an epigenetic mechanism primarily associated with transcriptional repression. The recent discovery that 5 mC can be oxidized to 5-hydromethylcytosine (5hmC) by TET proteins has revealed the “sixth base” of DNA and provides additional complexity to what was originally thought to be a stable repressive mark. However, our knowledge of the genome-wide distribution of 5hmC in different tissues is currently limited. Here, we sought to define loci enriched for 5hmC in the placenta genome by combining oxidative bisulphite (oxBS) treatment with high-density Illumina Infinium HumanMethylation450 methylation arrays and to compare our results with those obtained in brain. Despite identifying over 17,000 high-confidence CpG sites with consistent 5hmC enrichment, the distribution of this modification in placenta is relatively sparse when compared to cerebellum and frontal cortex. Supported by validation using allelic T4 β-glucosyltransferase assays we identify 5hmC at numerous imprinted loci, often overlapping regions associated with parent-of-origin allelic 5 mC in both placenta and brain samples. Furthermore, we observe tissue-specific monoallelic enrichment of 5hmC overlapping large clusters of imprinted snoRNAs-miRNAs processed from long noncoding RNAs (lncRNAs) within the <i>DLK1-DIO3</i> cluster on chromosome 14 and <i>SNRPN-UBE3A</i> domain on chromosome 15. Enrichment is observed solely on the transcribed alleles suggesting 5hmC is positively associated with transcription at these loci. Our study provides an extensive description of the 5hmC/5 mC landscape in placenta with our data available at <a href="http://www.humanimprints.net" target="_blank">www.humanimprints.net</a>, which represents the most comprehensive resource for exploring the epigenetic profiles associated with human imprinted genes.</p

Analysis of <i>Plagl1</i> region in <i>Dnmt3l −/+.</i>

<p>(A) The methylation status of the <i>Plagl1</i> promoter regions in wild type +/+ and <i>Dnmt3l</i> −/+ embryos examined by bisulphite PCR. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (•) or an unmethylated cytosine (○). (B) RT-PCRs on cDNA generated with (+) and without (−) reverse transcriptase show an increase in the expression of the imprinted transcripts in <i>Dnmt3l</i>−/+ embryos as a result of reactivation of the maternal allele. (C) The histone modification signature of the <i>Plagl1</i>-DMR in wild type B×C embryos, and after targeted deletion of the <i>Dnmt3l</i> gene. DNA extracted from antibody bound (B) and unbound (U) chromatin fractions were subject to either qPCR or PCR and SSCP analysis with primers that can discriminate parental alleles.</p

Cellular localization and RNA stability of the ncRNAs.

<p>(A) Distribution of the various transcripts in the nuclear (dark grey) and cytoplasmic (black) fractions, compared to total RNA (light grey). <i>U937 snoRNA</i> and <i>Airn</i> are nuclear-retained controls, whereas <i>Igf2</i> is cytoplasm-exported control. (B) Abundance of the various transcripts after exposure to Actinomycin D to determine RNA stability. The relative expression values of the control untreated samples are set to 1 (light grey bars) for each transcript. <i>C-Myc</i> and <i>Airn</i> are control transcripts for with short half-life; <i>Gapdh</i> and <i>Igf2r</i> are long half-life controls.</p

Schematic overview of the mouse chromosome 10 imprinted domain.

<p>(A) Map of the <i>Plagl1</i> locus, showing the location of the various imprinted transcripts and CpG islands (paternally expressed transcripts are in blue; biallelically expressed transcripts are in grey). Arrows represent direction of transcription. (B) The allelic expression of the various transcripts in embryonic tissues in reciprocal mouse crosses (for clarity only (B×C) F1 tissues are shown).</p

Identification of additional placenta-specific imprinted DMRs in RHM samples.

<p>(A) A heatmap for the β_mean of the Infinium probes with a methylation difference (>20%, minimum 3 consecutive probes) in RHMs associated with maternal effect <i>NLRP7</i> mutations compared to control placental biopsies. (B) Schematic representation of the methylation-sensitive <i>Hpa</i>II genotyping assay. (C) Methylation profiles as determined by methylation-sensitive genotyping and (D) bisulfite PCR and subcloning on placenta and somatic tissue DNA samples at the <i>SCIN</i>, <i>ST8AIA1</i> and <i>CABIN1</i> promoters. Note that the samples used for methylation-sensitive genotyping and bisulphite PCR maybe different to highlight that methylation is not associated with genotype but parental origin.</p

Additional file 2: of Genome-wide DNA methylation analysis of pseudohypoparathyroidism patients with GNAS imprinting defects

Detailed schematic representation of the human GNAS-AS region