Bisulfite sequencing and dinucleotide content analysis of 15 imprinted mouse differentially methylated regions (DMRs): paternally methylated DMRs contain less CpGs than maternally methylated DMRs

Imprinted genes in mammals show monoallelic expression dependent on parental origin and are often associated with differentially methylated regions (DMRs). There are two classes of DMR: primary DMRs acquire gamete-specific methylation in either spermatogenesis or oogenesis and maintain the allelic methylation differences throughout development; secondary DMRs establish differential methylation patterns after fertilization. Targeted disruption of some primary DMRs showed that they dictate the allelic expression of nearby imprinted genes and the establishment of the allelic methylation of secondary DMRs. However, how primary DMRs are recognized by the imprinting machinery is unknown. As a step toward elucidating the sequence features of the primary DMRs, we have determined the extents and boundaries of 15 primary mouse DMRs (including 12 maternally methylated and three paternally methylated DMRs) in 12.5-dpc embryos by bisulfite sequencing. We found that the average size of the DMRs was 3.2 kb and that their average G+C content was 54%. Dinucleotide content analysis of the DMR sequences revealed that, although they are generally CpG rich, the paternally methylated DMRs contain less CpGs than the maternally methylated DMRs. Our findings provide a basis for the further characterization of DMRs.     

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.     

CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia

Genomic imprints-parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes-are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP-SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.     

Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base.     

Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.     

DNA methylation dynamics of a maternally methylated DMR in the mouse Dlk1–Dio3 domain

The mouse delta-like homolog 1 and type III iodothyronine deiodinase (Dlk1–Dio3) imprinted domain contains three known paternally methylated differentially methylated regions (DMRs): intergenic DMR (IG-DMR), maternally expressed 3-DMR (Gtl2-DMR), and Dlk1-DMR. Here, we report the first maternally methylated DMR, CpG island 2 (CGI-2), is located approximately 800 bp downstream of miR-1188. CGI-2 is highly methylated in sperm and oocytes, de-methylated in pre-implantation embryos, and differentially re-methylated during post-implantation development. CGI-2, similarly to Gtl2-DMR and Dlk1-DMR, acquires differential methylation prior to embryonic day 7.5 (E7.5). Both H3K4me3 and H3K9me3 histone modifications are enriched at CGI-2. Furthermore, CCCTC-binding factor (CTCF) binds to both alleles of CGI-2 in vivo. These results contribute to the investigation of imprinting regulation in this domain.     

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.     

Diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes

Epigenetic modifications are closely associated with embryo developmental potential. One of the epigenetic modifications thought to be involved in genomic imprinting is DNA methylation. Here we show that the maternally imprinted genes Snrpn and Peg1/Mest were nearly unmethylated or heavily methylated, respectively, in their differentially methylated regions (DMRs) at the two-cell stage in parthenogenetic embryos. However, both genes were gradually de novo methylated, with almost complete methylation of all CpG sites by the morula stage in parthenogenetic embryos. Unexpectedly, another maternally imprinted gene, Peg3, showed distinct dynamics of methylation during preimplantation development of diploid parthenogenetic embryos. Peg3 showed seemingly normal methylation patterns at the two-cell and morula stages, but was also strongly de novo methylated in parthenogenetic blastocysts. In contrast, the paternally imprinted genes H19 and Rasgrf1 showed complete unmethylation of their DMRs at the morula stage in parthenogenetic embryos. These results indicate that diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes and that the aberrantly homogeneous status of methylation imprints may partially account for developmental failure.     

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     

Identification of imprinting regulators at the Meg3 differentially methylated region

Genomic imprinting at the Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos.     

Chromosome-wide analysis of parental allele-specific chromatin and DNA methylation

To reveal the extent of domain-wide epigenetic features at imprinted gene clusters, we performed a high-resolution allele-specific chromatin analysis of over 100 megabases along the maternally or paternally duplicated distal chromosome 7 (Chr7) and Chr15 in mouse embryo fibroblasts (MEFs). We found that reciprocal allele-specific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of H3K27me3 (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H19-Igf2 domain. A paternal allele-specific gap was found along Kcnq1ot1, interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allele-specific chromatin marks at the Peg13 and Slc38a4 DMRs, Cdkn1c upstream region, and Inpp5f_v2 DMR and paternal allele-specific CTCF binding at the Peg13 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor H3K9ac and CTCF or H3K4me3 in one allele and H3K9me3 in the reciprocal allele, using a sliding-window approach, recognized with precision the parental allele specificity of known imprinted genes, H19, Igf2, Igf2as, Cdkn1c, Kcnq1ot1, and Inpp5f_v2 on Chr7 and Peg13 and Slc38a4 on Chr15. Chromatin features, therefore, can unequivocally identify genes with imprinted expression.     

Differential methylation of STOX1 in human placenta

The 10q22 chromosomal region with genomic linkage to pre-eclampsia in Dutch females shows a parent-of-origin effect with maternal transmission of the Y153H susceptibility allele of the STOX1 gene. Although the CpG island within the STOX1 promoter region shows no differential methylation, this study describes the identification of a differentially methylated region (DMR) in intron 1 of the STOX1 gene. Methylation coincides with STOX1 expression, where high methylation leads to reduced expression. In the SGHPL-5 extravillous trophoblast cell line allele-specific expression was observed in a subset of cells. Although no allele-specific expression could be detected in early placenta samples, these samples did show an increase in methylation when they were homozygous for the Y153H susceptibility allele. Allele-specific methylation was observed in column extravillous trophoblast samples with the methylated allele being paternal in origin. We conclude that STOX1 is paternally imprinted, maternally expressed, with the DMR identified in this study showing parental-specific methylation in specific cell-types, hypothesized to occur in villous cytotrophoblasts, and proven in column extravillous trophoblasts originating from the anchoring villus. In other (placental) cells methylation is independent of parental origin, but regulates STOX1 expression with the Y153H genotype directing the level of methylation.     

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.     

Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome

Differential methylation of the two parental genomes in placental mammals is essential for genomic imprinting and embryogenesis. To systematically study this epigenetic process, we have generated a base-resolution, allele-specific DNA methylation (ASM) map in the mouse genome. We find parent-of-origin dependent (imprinted) ASM at 1,952 CG dinucleotides. These imprinted CGs form 55 discrete clusters including virtually all known germline differentially methylated regions (DMRs) and 23 previously unknown DMRs, with some occurring at microRNA genes. We also identify sequence-dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Finally, we report a surprising presence of non-CG methylation in the adult mouse brain, with some showing evidence of imprinting. Our results provide a resource for understanding the mechanisms of imprinting and allele-specific gene expression in mammalian cells.