Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30?017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.     

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30 017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.     

Breast cancer family history and allele-specific DNA methylation in the legacy girls study

Family history, a well-established risk factor for breast cancer, can have both genetic and environmental contributions. Shared environment in families as well as epigenetic changes that also may be influenced by shared genetics and environment may also explain familial clustering of cancers. Epigenetic regulation, such as DNA methylation, can change the activity of a DNA segment without a change in the sequence; environmental exposures experienced across the life course can induce such changes. However, genetic-epigenetic interactions, detected as methylation quantitative trait loci (mQTLs; a.k.a. meQTLs) and haplotype-dependent allele-specific methylation (hap-ASM), can also contribute to inter-individual differences in DNA methylation patterns. To identify differentially methylated regions (DMRs) associated with breast cancer susceptibility, we examined differences in white blood cell DNA methylation in 29 candidate genes in 426 girls (ages 6–13 years) from the LEGACY Girls Study, 239 with and 187 without a breast cancer family history (BCFH). We measured methylation by targeted massively parallel bisulfite sequencing (bis-seq) and observed BCFH DMRs in two genes: ESR1 (Δ4.9%, P = 0.003) and SEC16B (Δ3.6%, P = 0.026), each of which has been previously implicated in breast cancer susceptibility and pubertal development. These DMRs showed high inter-individual variability in methylation, suggesting the presence of mQTLs/hap-ASM. Using single nucleotide polymorphisms data in the bis-seq amplicon, we found strong hap-ASM in SEC16B (with allele specific-differences ranging from 42% to 74%). These findings suggest that differential methylation in genes relevant to breast cancer susceptibility may be present early in life, and that inherited genetic factors underlie some of these epigenetic differences.     

Germline-derived DNA methylation and early embryo epigenetic reprogramming: The selected survival of imprints

DNA methylation is an essential epigenetic mechanism involved in many essential cellular processes. During development epigenetic reprograming takes place during gametogenesis and then again in the pre-implantation embryo. These two reprograming windows ensure genome-wide removal of methylation in the primordial germ cells so that sex-specific signatures can be acquired in the sperm and oocyte. Following fertilization the majority of this epigenetic information is erased to give the developing embryo an epigenetic profile coherent with pluripotency. It is estimated that ∼65% of the genome is differentially methylated between the gametes, however following embryonic reprogramming only parent-of-origin methylation at known imprinted loci remains. This suggests that trans-acting factors such as Zfp57 can discriminate imprinted differentially methylated regions (DMRs) from the thousands of CpG rich regions that are differentially marked in the gametes. Recently transient imprinted DMRs have been identified suggesting that these loci are also protected from pre-implantation reprograming but succumb to de novo remethylation at the implantation stage. This highlights that “ubiquitous” imprinted loci are also resilient to gaining methylation by protecting their unmethylated alleles. In this review I examine the processes involved in epigenetic reprograming and the mechanisms that ensure allelic methylation at imprinted loci is retained throughout the life of the organism, discussing the critical differences between mouse and humans.This article is part of a Directed Issue entitled: Epigenetics Dynamics in development and disease.     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Familial molar tissues due to mutations in the inflammatory gene, NALP7, have normal postzygotic DNA methylation

An imprinting disorder has been believed to underlie the etiology of familial biparental hydatidiform moles (HMs) based on the abnormal methylation or expression of imprinted genes in molar tissues. However, the extent of the epigenetic defect in these tissues and the developmental stage at which the disorder begins have been poorly defined. In this study, we assessed the extent of abnormal DNA methylation in two HMs caused by mutations in the recently identified 19q13.4 gene, NALP7. We demonstrate normal postzygotic DNA methylation patterns at major repetitive and long interspersed nuclear elements (LINEs), genes on the inactive X-chromosome, three-cancer related genes, and CpG rich regions surrounding the PEG3 differentially methylated region (DMR). Our data provide a comprehensive assessment of DNA methylation in familial molar tissues and indicate that abnormal DNA methylation in these tissues is restricted to imprinted DMRs. The known role of NALP7 in apoptosis and inflammation pinpoints previously unrecognized pathways that could directly or indirectly underlie the abnormal methylation of imprinted genes in molar tissues.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ～3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ～2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ～750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Autonomous silencing of the imprinted Cdkn1c gene in stem cells

Parent-of-origin specific expression of imprinted genes relies on the differential DNA methylation of specific genomic regions. Differentially methylated regions (DMRs) acquire DNA methylation either during gametogenesis (primary DMR) or after fertilisation when allele-specific expression is established (secondary DMR). Little is known about the function of these secondary DMRs. We investigated the DMR spanning Cdkn1c in mouse embryonic stem cells, androgenetic stem cells and embryonic germ stem cells. In all cases, expression of Cdkn1c was appropriately repressed in in vitro differentiated cells. However, stem cells failed to de novo methylate the silenced gene even after sustained differentiation. In the absence of maintained DNA methylation (Dnmt1^-/-), Cdkn1c escapes silencing demonstrating the requirement for DNA methylation in long term silencing in vivo. We propose that postfertilisation differential methylation reflects the importance of retaining single gene dosage of a subset of imprinted loci in the adult.     

Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α^−/−;Gsk-3β^−/− ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α^−/−;Gsk-3β^−/− ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3–dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3–mediated signal transduction in the epigenome.     

Bovine DNA methylation imprints are established in an oocyte size-specific manner, which are coordinated with the expression of the DNMT3 family proteins

A subset of genes, known as imprinted genes, is present in the mammalian genome. Genomic imprinting governs the monoallelic expression of these genes, depending on whether the gene was inherited from the sperm or the egg. This parent-of-origin specific gene expression is generally dependent on the epigenetic modification, DNA methylation, and the DNA methylation status of CpG dinucleotides residing in loci known as differentially methylated regions (DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3)) groups to the cytosine residue in the CpG dinucleotides are known as DNA methyltransferases (DNMTs). Correct establishment and maintenance of methylation patterns at imprinted genes has been associated with placental function and regulation of embryonic/fetal development. Much work has been carried out on imprinted genes in mouse and human; however, little is known about the methylation dynamics in the bovine oocyte. The primary objective of the present study was to characterize the establishment of methylation at maternally imprinted genes in bovine growing oocytes and to determine if the expression of the bovine DNMTs-DNMT3A, DNMT3B, and DNMT3L-was coordinated with DNA methylation during oocyte development. To this end, a panel of maternally imprinted genes was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1) and putative DMRs for MEST, IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene; the SNRPN DMR has been reported previously. Conventional bisulfite sequencing revealed that methylation marks were acquired at all five DMRs investigated in an oocyte size-dependent fashion. This was confirmed for a selection of genes using pyrosequencing analysis. Furthermore, mRNA expression and protein analysis revealed that DNMT3A, DNMT3B, and DNMT3L are also present in the bovine oocyte during its growth phase. This study demonstrates for the first time that an increase in bovine imprinted gene DMR methylation occurs during oocyte growth, as is observed in mouse.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.Key words: genomic imprinting, DNA methylation, Gtl2, secondary DMR, epigenetics     

Differentially methylated regions of imprinted genes in prenatal, perinatal and postnatal human tissues

Epigenetic plasticity in relation to in utero exposures may mechanistically explain observed differences in the likelihood of developing common complex diseases including hypertension, diabetes and cardiovascular disease through the cumulative effects of subtle alterations in gene expression. Imprinted genes are essential mediators of growth and development and are characterized by differentially methylated regulatory regions (DMRs) that carry parental allele-specific methylation profiles. This theoretical 50% level of methylation provides a baseline from which endogenously- or exogenously-induced deviations in methylation can be detected. We quantified DNA methylation at imprinted gene DMRs in a large panel of human conceptal tissues, in matched buccal cell specimens collected at birth and at one year of age, and in the major cell fractions of umbilical cord blood to assess the stability of methylation at these regions. DNA methylation was measured using validated pyrosequencing assays at seven DMRs regulating the IGF2/H19, DLK1/MEG3, MEST, NNAT and SGCE/PEG10 imprinted domains. DMR methylation did not significantly differ for the H19, MEST and SGCE/PEG10 DMRs across all conceptal tissues analyzed (ANOVA p>0.10). Methylation differences at several DMRs were observed in tissues from brain (IGF2 and MEG3-IG DMRs), liver (IGF2 and MEG3 DMRs) and placenta (both DLK1/MEG3 DMRs and NNAT DMR). In most infants, methylation profiles in buccal cells at birth and at one year of age were comparable, as was methylation in the major cell fractions of umbilical cord blood. Several infants showed temporal deviations in methylation at multiple DMRs. Similarity of inter-individual and intra-individual methylation at some, but not all of the DMRs analyzed supports the possibility that methylation of these regions can serve as useful biosensors of exposure.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

Regulation of imprinted DNA methylation

DNA methylation is an essential enzymatic modification in mammals. This common epigenetic mark occurs predominantly at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3'. The majority of methylated CpGs are located within repetitive elements including centromeric repeats, satellite sequences and gene repeats encoding ribosomal RNAs. CpG islands, frequently located at the 5' end of genes, are typically unmethylated. DNA methylation also occurs at imprinted genes which exhibit parent-of-origin-specific patterns of methylation and expression. Imprinted methylation at differentially methylated domains (DMDs) is one of the regulatory mechanisms controlling the allele-specific expression of imprinted genes. Proper control of DNA methylation is needed for normal development and loss of methylation control can contribute to initiation and progression of tumorigenesis (reviewed in Plass and Soloway, 2002). Because patterns of imprinted DNA methylation are highly reproducible, imprinted loci make useful models for studying regulation of DNA methylation and may provide insights into how this regulation goes awry in cancer. Here, we review what is currently known about the mechanisms regulating imprinted DNA methylation. We will focus on cis-acting DNA sequences, trans-acting protein factors and the possible involvement of RNAs in control of imprinted DNA methylation.     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.     

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.