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.

Imprinting control regions (ICRs) are marked by mono-allelic bivalent chromatin when transcriptionally inactive.

Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire life-span. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover, they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters, regardless of the developmental stage, thereby contributing to protect cell identity.

Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting.

Familial recurrent hydatidiform mole (RHM) is a maternal-effect autosomal recessive disorder usually associated with mutations of the NLRP7 gene. It is characterized by HM with excessive trophoblastic proliferation, which mimics the appearance of androgenetic molar conceptuses despite their diploid biparental constitution. It has been proposed that the phenotypes of both types of mole are associated with aberrant genomic imprinting. However no systematic analyses for imprinting defects have been reported. Here, we present the genome-wide methylation profiles of both spontaneous androgenetic and biparental NLRP7 defective molar tissues. We observe total paternalization of all ubiquitous and placenta-specific differentially methylated regions (DMRs) in four androgenetic moles; namely gain of methylation at paternally methylated loci and absence of methylation at maternally methylated regions. The methylation defects observed in five RHM biopsies from NLRP7 defective patients are restricted to lack-of-methylation at maternal DMRs. Surprisingly RHMs from two sisters with the same missense mutations, as well as consecutive RHMs from one affected female show subtle allelic methylation differences, suggesting inter-RHM variation. These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte. In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci. These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment.

Differential methylation between the two alleles of a gene has been observed in imprinted regions, where the methylation of one allele occurs on a parent-of-origin basis, the inactive X-chromosome in females, and at those loci whose methylation is driven by genetic variants. We have extensively characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies, and hydatidiform moles, using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as to identify 21 novel loci harboring parent-of-origin methylation, 15 of which are restricted to the placenta. We observe that the extent of imprinted differentially methylated regions (DMRs) is extremely similar between tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Further, we profiled all imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically activated oocytes, individual blastomeres, and blastocysts, in order to identify primary DMRs and reveal the extent of reprogramming during preimplantation development. Intriguingly, we find that in contrast to ubiquitous imprints, the majority of placenta-specific imprinted DMRs are unmethylated in sperm and all human embryonic stem cells. Therefore, placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci.

HBx induces hypomethylation of distal intragenic CpG islands required for active expression of developmental regulators.

Epigenetic alterations caused by viral oncoproteins are strong initiation factors for cancer development, but their mechanisms are largely unknown. To identify the epigenetic effects of viral hepatitis B virus X (HBx) that lead to hepatocellular carcinoma (HCC), we profiled the DNA methylomes of normal and HBx transgenic mouse liver. Intriguingly, severe hypomethylation of intragenic CpG islands (CGIs) was observed in HBx liver before the full development of HCC. Normally, these CGIs were highly methylated (mCGIs) by the DNMT3L complex and marked with epigenetic signatures associated with active expression, such as H3K36me3. Hypomethylation of mCGI was caused by the downregulation of Dnmt3L and Dnmt3a due to HBx bound to their promoters, along with HDAC1. These events lead to the downregulation of many developmental regulators that could facilitate tumorigenesis. Here we provide an intriguing epigenetic regulation mediated by mCGI that is required for cell differentiation and describe a previously unidentified epigenetic role for HBx in promoting HCC development.

Methylation screening of reciprocal genome-wide UPDs identifies novel human-specific imprinted genes.

Nuclear transfer experiments undertaken in the mid-80's revealed that both maternal and paternal genomes are necessary for normal development. This is due to genomic imprinting, an epigenetic mechanism that results in parent-of-origin monoallelic expression of genes regulated by germline-derived allelic methylation. To date, ∼100 imprinted transcripts have been identified in mouse, with approximately two-thirds showing conservation in humans. It is currently unknown how many imprinted genes are present in humans, and to what extent these transcripts exhibit human-specific imprinted expression. This is mainly due to the fact that the majority of screens for imprinted genes have been undertaken in mouse, with subsequent analysis of the human orthologues. Utilizing extremely rare reciprocal genome-wide uniparental disomy samples presenting with Beckwith-Wiedemann and Silver-Russell syndrome-like phenotypes, we analyzed ∼0.1% of CpG dinculeotides present in the human genome for imprinted differentially methylated regions (DMRs) using the Illumina Infinium methylation27 BeadChip microarray. This approach identified 15 imprinted DMRs associated with characterized imprinted domains, and confirmed the maternal methylation of the RB1 DMR. In addition, we discovered two novel DMRs, first, one maternally methylated region overlapping the FAM50B promoter CpG island, which results in paternal expression of this retrotransposon. Secondly, we found a paternally methylated, bidirectional repressor located between maternally expressed ZNF597 and NAT15 genes. These three genes are biallelically expressed in mice due to lack of differential methylation, suggesting that these genes have become imprinted after the divergence of mouse and humans.

Placenta-specific epimutation at H19-DMR among common pregnancy complications: its frequency and effect on the expression patterns of H19 and IGF2.

BACKGROUND: H19 and IGF2 genes are imprinted and involved in regulating fetal and placental growth. The H19 differentially methylated region (DMR) is paternally methylated and maternally unmethylated and regulates the imprinted expression of H19 and IGF2. Epimutation at the H19-DMR in humans results in congenital growth disorders, Beckwith-Wiedemann and Silver-Russell syndromes, when erroneously its maternal allele becomes methylated and its paternal allele becomes unmethylated, respectively. Although H19 and IGF2 have been assessed for their involvement in pregnancy complications including fetal growth restriction (FGR) and pregnancy-induced hypertension (PIH)/hypertensive disorder of pregnancy (HDP) intensively in the last decade, it is still not established whether epimutation at the H19-DMR in the placenta results in pathogenic conditions in pregnancy. We aimed to assess the frequency of H19-DMR epimutation and its effects on the allelic expression patterns of H19 and IGF2 genes among normal and abnormal pregnancy cases. RESULTS: We enrolled two independently collected sets of placenta samples from normal pregnancies as controls and common pregnancy complications, FGR and PIH (HDP). The first set consisted of 39 controls and 140 FGR and/or PIH cases, and the second set consisted of 29 controls and 62 cases. For these samples, we initially screened for DNA methylation changes at H19-DMR and IGF2-DMRs by combined bisulfite restriction analysis, and further analyzed cases with methylation changes for their allelic methylation and expression patterns. We identified one case each of FGR and PIH showing hypomethylation of H19-DMR and IGF2-DMRs only in the placenta, but not in cord blood, from the first case/control set. For the PIH case, we were able to determine the allelic expression pattern of H19 to be biallelically expressed and the H19/IGF2 expression ratio to be highly elevated compared to controls. We also identified a PIH case with hypomethylation at H19-DMR and IGF2-DMRs in the placenta from the second case/control set. CONCLUSIONS: Placental epimutation at H19-DMR was observed among common pregnancy complication cases at the frequency of 1.5% (3 out of 202 cases examined), but not in 68 normal pregnancy cases examined. Alteration of H19/IGF2 expression patterns due to hypomethylation of H19-DMR may have been involved in the pathogenesis of pregnancy complications in these cases.

Characterization of parent-of-origin methylation using the Illumina Infinium MethylationEPIC array platform.

AIM: This study aimed to establish a catalog of probes corresponding to imprinted differentially methylated regions (DMRs) on the Infinium HumanMethylationEPIC BeadChip. MATERIALS & METHODS: Reciprocal uniparental diploidies with low normal biparental mosaic contribution, together with normal diploid controls, were subjected to EPIC BeadChip hybridization. The methylation profiles were assessed for imprinted differential methylation. Top candidates were validated using locus-specific PCR-based assays. RESULTS: Seven hundred and eighty-nine CpG probes coincided with 50 known imprinted DMRs and 467 CpG probes corresponding to 124 novel imprinted DMR candidates were identified. Validation led to identification of several subtle DMRs within known imprinted domains as well as novel maternally methylated regions associated with PTCHD3 and JAKMIP1. CONCLUSION: Our comprehensive list of bona fide-imprinted DMR probes will simplify and facilitate methylation profiling of individuals with imprinting disorders and is applicable to other diseases in which aberrant imprinting has been implicated, such as cancer and fetal growth.

Genomic, Epigenomic, and Transcriptomic Profiling towards Identifying Omics Features and Specific Biomarkers That Distinguish Uterine Leiomyosarcoma and Leiomyoma at Molecular Levels.

Uterine leiomyosarcoma (LMS) is the worst malignancy among the gynecologic cancers. Uterine leiomyoma (LM), a benign tumor of myometrial origin, is the most common among women of childbearing age. Because of their similar symptoms, it is difficult to preoperatively distinguish the two conditions only by ultrasound and pelvic MRI. While histopathological diagnosis is currently the main approach used to distinguish them postoperatively, unusual histologic variants of LM tend to be misdiagnosed as LMS. Therefore, development of molecular diagnosis as an alternative or confirmatory means will help to diagnose LMS more accurately. We adopted omics-based technologies to identify genome-wide features to distinguish LMS from LM and revealed that copy number, gene expression, and DNA methylation profiles successfully distinguished these tumors. LMS was found to possess features typically observed in malignant solid tumors, such as extensive chromosomal abnormalities, overexpression of cell cycle-related genes, hypomethylation spreading through large genomic regions, and frequent hypermethylation at the polycomb group target genes and protocadherin genes. We also identified candidate expression and DNA methylation markers, which will facilitate establishing postoperative molecular diagnostic tests based on conventional quantitative assays. Our results demonstrate the feasibility of establishing such tests and the possibility of developing preoperative and noninvasive methods.

Integration of transcriptome and methylome analysis of aldosterone-producing adenomas.

OBJECTIVE: The pathophysiology of aldosterone-producing adenomas (APA) has been investigated intensively through genetic and genomic approaches. However, the role of epigenetics in APA is not fully understood. In the present study, we explored the relationship between gene expression and DNA methylation status in APA. METHODS: We conducted an integrated analysis of transcriptome and methylome data of paired APA-adjacent adrenal gland (AAG) samples from the same patient. The adrenal specimens were obtained from seven Japanese patients with APA who underwent adrenalectomy. Gene expression and genome-wide CpG methylation profiles were obtained from RNA and DNA samples that were extracted from those seven paired tissues. RESULTS: Methylome analysis showed global CpG hypomethylation in APA relative to AAG. The integration of gene expression and methylation status showed that 34 genes were up-regulated with CpG hypomethylation in APA. Of these, three genes (CYP11B2, MC2R, and HPX) may be related to aldosterone production, and five genes (PRRX1, RAB38, FAP, GCNT2, and ASB4) are potentially involved in tumorigenesis. CONCLUSION: The present study is the first methylome analysis to compare APA with AAG in the same patients. Our integrated analysis of transcriptome and methylome revealed DNA hypomethylation in APA and identified several up-regulated genes with DNA hypomethylation that may be involved in aldosterone production and tumorigenesis.

Molecular and clinical studies in 138 Japanese patients with Silver-Russell syndrome.

BACKGROUND: Recent studies have revealed relative frequency and characteristic phenotype of two major causative factors for Silver-Russell syndrome (SRS), i.e. epimutation of the H19-differentially methylated region (DMR) and uniparental maternal disomy 7 (upd(7)mat), as well as multilocus methylation abnormalities and positive correlation between methylation index and body and placental sizes in H19-DMR epimutation. Furthermore, rare genomic alterations have been found in a few of patients with idiopathic SRS. Here, we performed molecular and clinical findings in 138 Japanese SRS patients, and examined these matters. METHODOLOGY/PRINCIPAL FINDINGS: We identified H19-DMR epimutation in cases 1-43 (group 1), upd(7)mat in cases 44-52 (group 2), and neither H19-DMR epimutation nor upd(7)mat in cases 53-138 (group 3). Multilocus analysis revealed hyper- or hypomethylated DMRs in 2.4% of examined DMRs in group 1; in particular, an extremely hypomethylated ARHI-DMR was identified in case 13. Oligonucleotide array comparative genomic hybridization identified a ∼3.86 Mb deletion at chromosome 17q24 in case 73. Epigenotype-phenotype analysis revealed that group 1 had more reduced birth length and weight, more preserved birth occipitofrontal circumference (OFC), more frequent body asymmetry and brachydactyly, and less frequent speech delay than group 2. The degree of placental hypoplasia was similar between the two groups. In group 1, the methylation index for the H19-DMR was positively correlated with birth length and weight, present height and weight, and placental weight, but with neither birth nor present OFC. CONCLUSIONS/SIGNIFICANCE: The results are grossly consistent with the previously reported data, although the frequency of epimutations is lower in the Japanese SRS patients than in the Western European SRS patients. Furthermore, the results provide useful information regarding placental hypoplasia in SRS, clinical phenotypes of the hypomethylated ARHI-DMR, and underlying causative factors for idiopathic SRS.