Analysis of allelic expression for genes associated with novel placenta-specific maternally methylated gDMRs.

<p>(<b>A</b>) Allelic RT-PCR analysis for nine transcripts originating from placenta-specific DMRs in control placenta samples. Monoallelic paternal expression was observed in heterozygous placenta biopsies. (<b>B</b>) The identification of a ~10 kb ncRNA overlapping a placenta-specific gDMR ~12 kb downstream of the <i>TET3</i> gene. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. The green box highlights the position of the gDMRs. (<b>C</b>) The 2.7 kb maternally methylated placenta-specific DMR identified by methyl-seq and confirmed with allelic-specific bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. For clarity only the first 10 CpG dinucleotides are shown. (<b>D</b>) Paternal expression of the RNA-seq peak was determined by RT-PCR, whilst allele-specific RT-PCR revealed that <i>TET3</i> is biallelically expressed in term placenta samples.</p

Methylation profiling of opposing gDMRs using bisulphite PCR in human gametes and blastocysts.

<p>(<b>A</b>) The confirmation that the <i>H19</i> DMR acquires methylation from the sperm and maintains in preimplantation embryos (separated into ICM and TE) and in somatic tissues. The <i>MSCT2</i> DMR shows the opposite profile with sperm devoid of methylation. (<b>B</b>) The bisulphite PCR profiles for the novel ubiquitous <i>FANCC</i> and <i>SVOPL</i> DMRs in human sperm, blastocysts and placenta. (<b>C</b>) Methyl-seq datasets reveal that the <i>R3HCC1</i> gene has two adjacent gDMRs, an upstream paternal gDMR (region 1) that subsequently gains methylation on both alleles during the blastocyst stage and a placenta-specific maternally methylated promoter region (region 2). The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. Green boxes highlight the position of the gDMRs. (<b>D</b>) Confirmation of the methylation profile by bisulfite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. If informative, the parental-origin of methylation is indicated. For clarity only the first 10 CpG dinucleotides are shown.</p

Methylation profiling of human gametes, embryos and tissues.

<p>(<b>A</b>) Heatmap of all 25 CpG dinucleotide tiles with maternal (left) and paternal (right) germline-derived methylation maintaining an intermediate state in blastocysts and arranged in descending order according to their placenta methylation profile. Tiles were partitioned according to their hypo- (<20%), hyper- (>80%) or intermediate (>20%, <80%) methylation. Genomic features are included as separate heatmaps. (<b>B</b>) Violin plots classified as the % repetitive sequences per 20 CpG dinucleotide tile for the paternally methylated, maternally methylated and known imprinted DMRs. The numbers in red represent the tiles with unique sequences (defined as >25% repeats). (<b>C</b>) Two novel maternally methylated ubiquitous DMRs associated with the <i>SVOPL</i> and <i>FANCC</i> genes exhibit promoters that are unmethylated in sperm, hypermethylated in oocytes and intermediately methylated in blastocysts, placenta and somatic tissue in methyl-seq datasets. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. Green boxes highlight the position of the gDMRs. (<b>D</b>) Bisulphite PCR and subcloning was used for confirmation. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. If heterozygous for a SNP, the parental-origin of methylation is indicated. For clarity only the first 10 CpG dinucleotides are shown. (E) Allelic RT-PCR for <i>SVOPL</i> reveals transcription from the maternal allele in placenta and monoallelic expression in brain and leukocytes.</p

Allele-specific expression and methylation analysis of genes with variable maternal placenta-specific gDMRs.

<p>(<b>A</b>) Allelic RT-PCR analysis for <i>SH3BP2</i> and <i>MOCS1</i> in placenta samples with bisulphite PCR and subcloning of the associated gDMR in the same biopsy. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (•) or an unmethylated cytosine (o). For clarity only the first 10 CpG dinucleotides are shown. (<b>B</b>) Pyrosequencing quantification of 29 placenta-specific DMRs reveals hypomethylation indicative of a stochastic trait. The average methylation of 55 controls placenta samples from uncomplicated pregnancies reveals profiles consistent with one methylated and unmethylated allele. The controls represented as Tukey box-and-whisker plots with whiskers spanning from 25th to 75th percentiles +/- 1.5IQR to highlight outliers. Individual hypomethylated samples are highlighted. (<b>C</b>) Allelic specific RT-PCR and strand-specific bisulphite PCR and subcloning of placenta samples lacking maternal methylation at <i>LIN28B</i> identified in (<b>B</b>) compared to a normal imprinted control sample.</p

Analysis of tissue-specific maintenance of germline methylation in different tissues.

<p>(<b>A</b>) A bar graph showing the fate of gDMRs in tissues. The bars represent the profiles of known ubiquitous (black) and placenta-specific (orange) gDMRs with numbers corresponding to the left y-axis. The superimposed line graph represent the profile of all remaining germline difference that are maintained to the blastocyst stage and correspond to the right y-axis. * placenta-specific DMRs identified by Court (2014) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006427#pgen.1006427.ref010" target="_blank">10</a>] and Sanchez-Delgado (2015) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006427#pgen.1006427.ref015" target="_blank">15</a>]. (<b>B</b>) A pie graph showing the distribution of individual tissues maintaining a partially methylated profile. (<b>C</b>) The <i>GRID2</i> gene exhibits high inter- and intragenic methylation and several regions with oocyte-derived methylation. The methyl-seq data reveals that a 1.9 kb region overlapping the promoter remains an imprinted gDMR in placenta while it is demethylated in all other tissues. A second oocyte-derived gDMR, consisting largely of an Alu/SINE repeat, becomes fully methylated in all tissues analysed. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. A green box highlights the position of the gDMR. (<b>D</b>) Bisulfite PCR and subcloning on heterozygous placenta DNA samples for the <i>GRID2</i> promoter and intragenic regions. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. If informative for a SNP, the parental-origin of methylation is indicated. For clarity only the first 10 CpG dinucleotides are shown.</p

Identification of novel imprinted genes in human embryos using allele-specific RNA-seq datasets.

<p>(<b>A</b>) Schematic drawing of the sequential transcriptome switching from oocyte-derived transcripts to the embryonic genome in human preimplantation embryos. (<b>B</b>) The expression pattern of the <i>ZHX3</i> gene during human preimplantation development. High expression was observed from the zygote to the 8-cell stage, declining in the morula. Paternal expression as observed from the 4-cell stage onwards. (<b>C</b>) Allele-specific RT-PCR was performed on term placenta samples heterozygous for the exonic SNP rs17265513. (<b>D</b>) Methyl-seq traces reveal the location of the placenta-specific maternal gDMR overlapping the <i>ZHX3</i> promoter. The vertical black lines in the methyl-seq tracks represent the mean methylation value for individual CpG dinucleotides. The green box highlights the position of the gDMRs. (<b>E</b>) The methylation profile confirmed using bisulphite PCR and cloning in sperm, blastocysts and placenta. Each circle represents a single CpG dinucleotide on a DNA strand. (•) Methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. For clarity only the first 10 CpG dinucleotides are shown.</p