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

Description of <i>NLRP7</i> mutations with methylation and expression profiling of imprinted loci.

<p>(A) Confirmation of recessive <i>NLRP7</i> mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect <i>NLRP7</i> mutations. (C) Confirmation of the methylation profile of the <i>NLRP7</i> mutated RHMs at the <i>NAP1L5</i>, <i>PEG10</i>, <i>RB1</i>, <i>L3MBTL1</i> and <i>H19</i> DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes <i>NAP1L5</i>, <i>HYMAI</i>, <i>PEG10</i> and <i>PEG3</i> in control placenta samples (PL) and <i>NLRP7</i>-mutated moles (RHM).</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

Methylation and expression analyses of placenta-specific DMRs in RHM samples.

<p>(A) Circular heatmap of the 153 Infinium array probes mapping to the 18 known placenta-specific imprinted DMRs. The inner circles represent the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect <i>NLRP7</i> mutations. (B) Confirmation of the methylation profile at the maternally methylated <i>GLIS3</i>, <i>DNMT1</i> and <i>MCCC1</i> DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (C) Allelic expression analysis of imprinted genes <i>MCCC1</i>, <i>LIN28B</i> and <i>GLIS3</i> in control placenta samples (PL) and <i>NLRP7</i>-mutated moles (RHM). (D) Quantitative RT-PCR for <i>H19</i>, <i>DNMT1</i> and <i>AGBL3</i> in RHM samples. The boxplot show the median expression (whiskers 5–95% percentile) determined for 15 control placenta samples with the values of RHMs highlighted.</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

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 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