Human Oocyte-derived Methylation Differences Persist In The Placenta Revealing Widespread Transient Imprinting

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation

Single-cell multi-omic analysis profiles defective genome activation and epigenetic reprogramming associated with human pre-implantation embryo arrest

During pre-implantation stages of mammalian development, maternally stored material promotes both the erasure of the sperm and oocyte epigenetic profiles and is responsible for concomitant genome activation. Here, we have utilized single-cell methylome and transcriptome sequencing (scM&T-seq) to quantify both mRNA expression and DNA methylation in oocytes and a developmental series of human embryos at single-cell resolution. We fully characterize embryonic genome activation and maternal transcript degradation and map key epigenetic reprogramming events in developmentally high-quality embryos. By comparing these signatures with early embryos that have undergone spontaneous cleavage-stage arrest, as determined by time-lapse imaging, we identify embryos that fail to appropriately activate their genomes or undergo epigenetic reprogramming. Our results indicate that a failure to successfully accomplish these essential milestones impedes the developmental potential of pre-implantation embryos and is likely to have important implications, similar to aneuploidy, for the success of assisted reproductive cycles

Human oocyte-derived methylation differences persist in the placenta revealing widespread transient imprinting

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation

Human Oocyte-derived Methylation Differences Persist In The Placenta Revealing Widespread Transient Imprinting

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation

Human Oocyte-Derived Methylation Differences Persist in the Placenta Revealing Widespread Transient Imprinting

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation.This work was supported by Spanish Ministerio de Educación y Competitividad (MINECO) (BFU2014-53093 to DM) co-funded with the European Union Regional Development Fund (FEDER). AMS is a recipient of a FPI PhD studentship from MINECO. TMB is supported by ICREA, EMBO YIP 2013, MINECO BFU2014-55090-P (FEDER), BFU2015-7116-ERC and BFU2015-6215-ERCU01 MH106874 grant, Fundacio Zoo Barcelona and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya

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 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

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 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