Differentially Methylated Domain Research Articles

Loss of inherited genomic imprints in mice leads to severe disruption in placental lipid metabolism

IntroductionMonoallelic expression of imprinted genes is necessary for placental development and normal fetal growth. Differentially methylated domains (DMDs) largely determine the parental-specific monoallelic expression of imprinted genes. Maternally derived DNA (cytosine-5-) -methyltransferase 1o (DNMT1o) maintains DMDs during the eight-cell stage of development. DNMT1o-deficient mouse placentas have a generalized disruption of genomic imprints. Previous studies have demonstrated that DNMT1o deficiency alters placental morphology and broadens the embryonic weight distribution in late gestation. Lipids are critical for fetal growth. Thus, we assessed the impact of disrupted imprinting on placental lipids. MethodsLipids were quantified from DNMT1o-deficient mouse placentas and embryos at E17.5 using a modified Folch method. Expression of select genes critical for lipid metabolism was quantified with RT-qPCR. Mitochondrial morphology was assessed by TEM and mitochondrial aconitase and cytoplasmic citrate concentrations quantified. DMD methylation was determined by EpiTYPER. ResultsWe found that DNMT1o deficiency is associated with increased placental triacylglycerol levels. Neither fetal triacylglycerol concentrations nor expression of select genes that mediate placental lipid transport were different from wild type. Placental triacylglycerol accumulation was associated with impaired beta-oxidation and abnormal citrate metabolism with decreased mitochondrial aconitase activity and increased cytoplasmic citrate concentrations. Loss of methylation at the MEST DMD was strongly associated with placental triacylglycerol accumulation. DiscussionA generalized disruption of genomic imprints leads to triacylglycerol accumulation and abnormal mitochondrial function. This could stem directly from a loss of methylation at a given DMD, such as MEST, or represent a consequence of abnormal placental development.

English

Usually, most of the genes are biallelically expressed but imprinted gene exhibit monoallelic expression based on their parental origin. Genomic imprinting exhibit differences in control between flowering plants and mammals, for instance, imprinted gene are specifically activated by demethylation, rather than targeted for silencing in plants and imprinted gene expression in plant which occur in endosperm. It also displays sexual dimorphism like differential timing in imprint establishment and RNA based silencing mechanism in paternally repressed imprinted gene. Within imprinted regions, the unusual occurrence and distribution of various types of repetitive elements may act as genomic imprinting signatures. Imprinting regulation probably at many loci involves insulator protein dependent and higher-order chromatin interaction, and/or non-coding RNAs mediated mechanisms. However, placenta-specific imprinting involves repressive histone modifications and non-coding RNAs. The higher-order chromatin interaction involves differentially methylated domains (DMDs) exhibiting sex-specific methylation that act as scaffold for imprinting, regulate allelic-specific imprinted gene expression. The paternally methylated differentially methylated regions (DMRs) contain less CpGs than the maternally methylated DMRs. The non-coding RNAs mediated mechanisms include C/D RNA and microRNA, which are invovled in RNA-guided post-transcriptional RNA modifications and RNA-mediated gene silencing, respectively. The maintenance and reprogramming of imprinting are not significantly affected by reduced expression of Dicer1 and the evolution of imprinting might be related to acquisition of DNMT3L (de novo methyltransferase 3L) by a common ancestor of eutherians and marsupials. The common feature among diverse imprinting control elements and evolutionary significance of imprinting need to be identified.   Key words: Genomic imprinting, differentially methylated regions (DMRs), non-coding RNA, imprinting evolution.

Gene therapy for cancer through adenovirus vector-mediated expression of the Ad5 early region gene 1A based on loss of IGF2 imprinting

Loss of (genomic) imprinting (LOI) of the insulin-like growth factor 2 gene (IGF2) is a common epigenetic abnormality in many human cancers. IGF2 imprinting is regulated by differentially methylated domains (DMD) in the imprinting control region that is located between IGF2 and H19 on human chromosome 11. In the present study, combined expression of adenoviral vectors (Ad-EGFP and Ad-E1A) driven by H19 enhancer-DMD-H19 promoter complex was investigated and their effects on the tumor growth were assessed in vitro and in vivo. When infected with Ad-EGFP, the cancer cell lines with the LOI, such as HRT-18 and HT-29 cells, had the expression of the EGFP protein, whereas three cancer cell lines with the maintenance of imprinting (MOI) (HCT-116, MCF-7 and GES-1) had weak expression of EGFP. Furthermore, the expressed Ad-E1A significantly decreased cell viability and induced cell apoptosis only in HRT-18 and HT-29 cells in vitro, and effectively suppressed tumor development in HRT-18 and HT-29 xenograft in nude mice. It is concluded that this gene therapy vector is effective in the suppression of the growth of human colon cancer cells in vitro and in vivo, and that cancer gene therapy based on loss of IGF2 imprinting may prove to be a novel therapeutic option.

Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia

DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.

Generalized disruption of inherited genomic imprints leads to wide-ranging placental defects and dysregulated fetal growth

Monoallelic expression of imprinted genes, including ones solely expressed in the placenta, is essential for normal placental development and fetal growth. To better understand the role of placental imprinting in placental development and fetal growth, we examined conceptuses developing in the absence of maternally derived DNA (cytosine-5-)-methyltransferase 1o (DNMT1o). Absence of DNMT1o results in the partial loss of methylation at imprinted differentially methylated domain (DMD) sequences in the embryo and the placenta. Mid-gestation E9.5 DNMT1o-deficient placentas exhibited structural abnormalities of all tissue layers. At E17.5, all examined placentas had aberrant placental morphology, most notably in the spongiotrophoblast and labyrinth layers. Abnormalities included an expanded volume fraction of spongiotrophoblast tissue with extension of the spongiotrophoblast layer into the labyrinth. Many mutant placentas also demonstrated migration abnormalities of glycogen cells. Additionally, the volume fraction of the labyrinth was reduced, as was the surface area for maternal fetal gas exchange. Despite these placental morphologic abnormalities, approximately one-half of DNMT1o-deficient fetuses survived to late gestation (E17.5). Furthermore, DNMT1o-deficient placentas supported a broad range of fetal growth. The ability of some DNMT1o-deficient and morphologically abnormal placentas to support fetal growth in excess of wild type demonstrates the importance of differential methylation of DMDs and proper imprinting of discrete gene clusters to placental morphogenesis and fetal growth.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer1

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2

BackgroundPorcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD) controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2).ResultsBisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele.ConclusionsOur results indicate that the IGF2 imprinting status is transcript-specific. Biallelic IGF2 expression in adult porcine liver and relaxation of IGF2 imprinting in porcine muscle were a common feature. These results were consistent with the IGF2 promoter P1 usage in adult liver and IGF2 promoter P2, P3 and P4 usages in muscle. The results showed further that bialellic IGF2 expression in liver and relaxation of imprinting in muscle and kidney were not associated with DNA methylation variation at and around at least one CTCF binding site in H19 DMD. The imprinting status in adult liver, muscle and kidney tissues were also not reflected in the methylation patterns of IGF2 DMRs 1 and 2.

Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.

A Non-Coding RNA Within the Rasgrf1 Locus in Mouse Is Imprinted and Regulated by Its Homologous Chromosome in Trans

Background Rasgrf1 is imprinted in mouse, displaying paternal allele specific expression in neonatal brain. Paternal expression is accompanied by paternal-specific DNA methylation at a differentially methylated domain (DMD) within the locus. The cis-acting elements necessary for Rasgrf1 imprinting are known. A series of tandem DNA repeats control methylation of the adjacent DMD, which is a methylation sensitive enhancer-blocking element. These two sequences constitute a binary switch that controls imprinting and represents the Imprinting Control Region (ICR). One paternally transmitted mutation, which helped define the ICR, induced paramutation, in trans, on the maternal allele. Like many imprinted genes, Rasgrf1 lies within an imprinted cluster. One of four noncoding transcripts in the cluster, AK015891, is known to be imprinted.Methodology/Principal FindingsHere we demonstrate that an additional noncoding RNA, AK029869, is imprinted and paternally expressed in brain throughout development. Intriguingly, any of several maternally inherited ICR mutations affected expression of the paternal AK029869 transcript in trans. Furthermore, we found that the ICR mutations exert different trans effects on AK029869 at different developmental times.Conclusions/SignificanceFew trans effects have been defined in mammals and, those that exist, do not show the great variation seen at the Rasgrf1 imprinted domain, either in terms of the large number of mutations that produce the effects or the range of phenotypes that emerge when they are seen. These results suggest that trans regulation of gene expression may be more common than originally appreciated and that where trans regulation occurs it can change dynamically during development.

Targeted tumor gene therapy based on loss of IGF2 imprinting

Loss of genomic imprinting of the insulin-like growth factor 2 gene (IGF2) maybe associated with the loss of activity of CTCF, an insulator of transcription, in many human neoplasms. This is because the inactive CTCF cannot bind to the differentially methylated domains (DMD). This results in an interaction between the IGF2 promoters and enhancers and IGF2 is produced. In this study we investigated the feasibility of using a combination of expression vectors AdDC3312-EGFP and AdDC312-DT-A driven by H19 enhancer DMD-H19 promoter complex. HCT-8, HT-29 and H-522 (LOI) infected with AdDC312-EGFP produced the EGFP protein. However, in the MCF-7 and GES-1 cells (MOI) no EGFP protein was produced. The AdDC312-DT-A significantly decreased cell viability and induced cell apoptosis only in LOI cells in vitro and therefore effectively suppressed tumour development in HCT-8 xenograft in nude mice. To conclude, the toxin gene therapy vector proved effective in inhibiting LOI cells growth in vitro and in vivo and provides an additional option for gene therapy based on loss of IGF2 imprinting.

Embryo vitrification affects the methylation of the H19/Igf2 differentially methylated domain and the expression of H19 and Igf2

To investigate the effects of embryo vitrification on the methylation status of the H19/Igf2 differentially methylated domain (DMD) and the expression of those genes. Comparative and controlled study. A university-affiliated hospital. ICR female and male mice. Day 14 fetuses that arose from copulation of male and female mice were taken as the control group. Embryos resulting from superovulation, IVF, in vitro culture, and embryo transfer were taken as the IVF group. Embryos that were vitrified were taken as the vitrified group. The methylation status of H19/Igf2 DMD was analyzed by cloning and sequencing following DNA bisulfite treatment. The expression of H19 and Igf2 was quantified by real-time reverse-transcription polymerase chain reaction. Loss of methylation was found in the H19/Igf2 DMD of the fetuses from the IVF group, but it was more severe in the vitrified group. H19 expression was significantly increased in the IVF and vitrified groups, as compared with the control group; however, the quantity in the vitrified group was less than that detected in the IVF group. Igf2 expression in the IVF and vitrified groups was found to have decreased significantly, whereas the Igf2 expression in the vitrified group was greater than in the IVF group. Embryo vitrification aggravated the loss of methylation in the H19/Igf2 DMD, and compensated for the perturbed expression of H19 by IVF procedures.

Identification of a region of the DNMT1 methyltransferase that regulates the maintenance of genomic imprints

Reprogramming of DNA methylation patterns during mammalian preimplantation development involves the concurrent maintenance of methylation on differentially methylated domains (DMDs) of imprinted genes and a marked reduction of global (non-DMD) genomic methylation. In the developing mammalian embryo, one allele of a DMD is unmethylated, and the opposite parental allele is methylated, having inherited this methylation from the parental gamete. The maintenance of DMDs is important for monoallelic imprinted gene expression and normal development of the embryo. Because the DNMT1 cytosine methyltransferase governs maintenance methylation in mammals, rearrangements of non-DMD, but not DMD methylation in preimplantation embryos suggest that the preimplantation DNMT1-dependent maintenance mechanism specifically targets DMD sequences. We explored this possibility using an engineered mouse ES cell line to screen for mutant DNMT1 proteins that protect against the loss of DMD and/or global (non-DMD) methylation in the absence of the wild-type endogenous DNMT1 methyltransferase. We identified DNMT1 mutants that were defective in maintenance of either DMD and/or non-DMD methylation. Among these, one mutant maintained non-DMD methylation but not imprinted DMD methylation and another mutant maintained just DMD methylation. The mutated amino acids of these mutants reside in a mammal-specific, disordered region near the amino terminus of DNMT1. These findings suggest that DNMT1 participates in epigenetic reprogramming through its ability to distinguish different categories of methylated sequences.

DNA methylation patterns at the IGF2‐H19 locus in sperm of Swiss Landrace and Swiss Large White boars

DNA methylation patterns at the IGF2-H19 locus were investigated in sperm DNA from Swiss Landrace (SL) and Swiss Large White (LW) boars. The putative IGF2 differentially methylated regions (DMR) 0, 1 and 2, a quantitative trait nucleotide (QTN) region in the intron 3 and a CpG island in the intron 4 of the IGF2 gene as well as three regions around porcine CTCF binding sites within the H19 differentially methylated domain (DMD) were selected for the DNA methylation analysis. In both breeds putative IGF2 DMR0, 1, 2 and H19 DMD were hypermethylated. Significant differences in DNA methylation content were found between the two breeds in the two DMD regions proximal to the H19 gene. The IGF2 QTN region and the CpG island in the IGF2 intron 4 were hypomethylated in sperm DNA of both breeds. The methylation analysis revealed significantly more methylated CpG sites in the intron 4 of sperm from the LW breed than in that from SL. No difference was found in global DNA methylation between the two breeds. These results indicate differences in DNA methylation patterns between breeds and it remains to be established whether variation in DNA methylation patterns impacts on phenotypic traits.

Transgenerational effects of the endocrine disruptor vinclozolin on the methylation pattern of imprinted genes in the mouse sperm

Endocrine-disrupting chemicals (EDCs), among which is the antiandrogen vinclozolin (VCZ), have been reported to affect the male reproductive system. In this study, VCZ was administered to pregnant mice at the time of embryo sex determination, and its possible effects on the differentially methylated domains (DMDs) of two paternally (H19 and Gtl2) and three maternally (Peg1, Snrpn, and Peg3) imprinted genes were tested in the male offspring. The CpGs methylation status within the five gene DMDs was analyzed in the sperm, tail, liver, and skeletal muscle DNAs by pyrosequencing. In the sperm of controls, the percentages of methylated CpGs were close to the theoretical values of 100 and 0% in paternally or maternally imprinted genes respectively. VCZ decreased the percentages of methylated CpGs of H19 and Gtl2 (respective values 83.1 and 91.5%) and increased those of Peg1, Snrpn, and Peg3 (respective values 11.3, 18.3, and 11.2%). The effects of VCZ were transgenerational, but they disappeared gradually from F1 to F3. The mean sperm concentration of the VCZ-administered female offspring was only 56% of that of the controls in the F1 offspring, and it was back to normal values in the F2 and F3 offspring. In the somatic cells of controls, the percentages of methylated CpGs were close to the theoretical value of 50% and, surprisingly, VCZ altered the methylation of Peg3. We propose that the deleterious effects of VCZ on the male reproductive system are mediated by imprinting defects in the sperm. The reported effects of EDCs on human male spermatogenesis might be mediated by analogous imprinting alterations.

Prenatal Exposure to Disrupted Methyl Metabolism Affects Expression & Allele‐Specific Methylation of H19/Igf2 in Brain

Epigenetic processes may play a role in the metabolic programming associated with prenatal nutrition. We studied the effect of prenatal exposure to disrupted maternal methyl metabolism on expression and allele‐specific methylation of H19/Igf2 in brain. H19/Igf2 expression is regulated by a differentially methylated domain (DMD). We studied F1 hybrid offspring from male Cast/EiJ mice and female C57BL6/J mice [with (Cbs+/‐) and without (Cbs+/+) heterozygous disruption of the cystathionine‐ß ‐synthase gene] fed a control or high methionine/low folate (HH) diet during pregnancy. The F1 hybrid offspring were then fed the control or HH diet from weaning for 6 weeks. Independent of prenatal diet, Cbs+/‐ x Cast mice fed the post‐weaning HH diet had higher (P<0.001) plasma homocysteine than Cbs+/+ x Cast mice fed the control diet. This was accompanied by higher (P<0.05) S‐adenosylhomocysteine (SAH), lower (P<0.01) S‐adenosylmethionine/SAH ratios, and lower (P<0.01) Igf2 mRNA in brain. Mice exposed to the prenatal and post‐weaning HH diet had higher (P=0.05) H19 mRNA, lower H19/Igf2 mRNA ratios, and higher (P<0.05) paternal and maternal allele DMD methylation in brain than mice not exposed to the prenatal HH diet. These findings show that prenatal exposure to disrupted maternal methyl metabolism is associated with differential methylation and expression of H19/Igf2 and suggests a role for methyl metabolism in metabolic programming.Grant Funding SourceCIHR/Sick Kids Foundation, Heart & Stroke Foundation

DNA methyltransferase 1o functions during preimplantation development to preclude a profound level of epigenetic variation

Most mouse embryos developing in the absence of the oocyte-derived DNA methyltransferase 1o (DNMT1o-deficient embryos) have significant delays in development and a wide range of anatomical abnormalities. To understand the timing and molecular basis of such variation, we studied pre- and post-implantation DNA methylation as a gauge of epigenetic variation among these embryos. DNMT1o-deficient embryos showed extensive differences in the levels of methylation in differentially methylated domains (DMDs) of imprinted genes at the 8-cell stage. Because of independent assortment of the methylated and unmethylated chromatids created by the loss of DNMT1o, the deficient embryos were found to be mosaics of cells with different, but stable epigenotypes (DNA methylation patterns). Our results suggest that loss of DNMT1o in just one cell cycle is responsible for the extensive variation in the epigenotypes in both embryos and their associated extraembryonic tissues. Thus, the maternal-effect DNMT1o protein is uniquely poised during development to normally ensure uniform parental methylation patterns at DMDs.

Antagonism between DNA and H3K27 Methylation at the Imprinted Rasgrf1 Locus

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.

Methylation changes in porcine primordial germ cells

Epigenetic re-programming is an important event in the development of primordial germ cells (PGC) into functional gametes, characterized by genome-wide erasure of DNA methylation and re-establishment of epigenetic marks, a process essential for restoration of the potential for totipotency. In this study changes in the methylation status of centromeric repeats and two IGF2-H19 differentially methylated domain (DMD) sequences were examined in porcine PGC between Days 24 and 31 of pregnancy. The methylation levels of centromeric repeats and IGF2-H19 DMD sequences decreased rapidly from Days 24 to 28 in both male and female PGC. At Days 30 and 31 of pregnancy centromeric repeats and IGF2-H19 DMD sequences acquired new methylation in male PGC, while in female PGC these sequences were completely demethylated by Day 30 and remained hypomethylated at Day 31. To characterize methylation changes that PGC undergo in culture, the methylation status of embryonic germ cells (EGCs) derived from PGC at Day 26 of pregnancy was examined. Centromeric repeats and IGF2-H19 DMD sequences were similarly methylated in both male and female EGC and hypermethylated in female EGC compared with female PGC at the same embryonic age. Our results show that, similar to murine PGC, porcine PGC undergo genome-wide DNA demethylation shortly after arrival in the genital ridges. When placed in culture porcine PGC terminate their demethylation program and may acquire new DNA methylation marks. To our knowledge, this is the first report regarding epigenetic re-programming of genital ridge PGC in the pig.

Prenatal Exposure to Maternal Hyperhomocysteinemia and Allele‐Specific Methylation and Expression of H19/Igf2

Epigenetic processes may play a role in the metabolic programming associated with prenatal exposure to maternal dietary factors. The goal of this study was to determine the effect of prenatal exposure to maternal hyperhomocysteinemia (HHcy) on allele‐specific methylation and expression of genomically imprinted H19 and Igf2. The H19 gene is located in close proximity to Igf2 with expression of H19 and Igf2 regulated by the methylation status of a differentially methylated domain (DMD). Female C57BL/6J mice with (Cbs +/−) and without (Cbs +/+) heterozygous targeted disruption of the gene for cystathionine‐β‐synthase were mated with male Cast/EiJ mice and fed a control or high methionine/low folate diet to induce HHcy (HH) during pregnancy. The F1 hybrid offspring mice were then challenged with the HH diet or control diet from weaning for 1 month. Mice exposed in utero to maternal HHcy and challenged with the HH diet post weaning had higher plasma total homocysteine than mice not exposed in utero to maternal HHcy but challenged with the HH diet post weaning (12.66±2.7 μM vs 6.99±0.9). This was accompanied by lower (p<0.05) methylation of liver H19 DMD on both the maternal and paternal alleles and higher H19 and lower Igf2 transcript levels in liver. These findings show that prenatal exposure to maternal HHcy results in tissue‐specific changes in H19/Igf2 methylation and expression.

The Transcriptional Status but Not the Imprinting Control Region Determines Allele-Specific Histone Modifications at the Imprinted H19 Locus

Genomic imprinting governs allele-specific gene expression in an epigenetically heritable manner. The characterization of histone modifications at imprinted gene loci is incomplete, and whether specific histone marks determine transcription or are dependent on it is not understood. Using chromatin immunoprecipitations, we examined in multiple cell types and in an allele-specific manner the active and repressive histone marks of several imprinted loci, including H19, KvDMR1, Snrpn promoter/exon 1, and IG-DMR imprinting control regions. Expressed alleles are enriched for specific actively modified histones, including H3 di- and trimethylated at Lys4 and acetylated histones H3 and H4, while their silent counterparts are associated with repressive marks such as H3 trimethylated at Lys9 alone or in combination with H3 trimethylated at Lys27 and H4/H2A symmetrically dimethylated at Arg3. At H19, allele-specific histone modifications occur throughout the entire locus, including nontranscribed regions such as the differentially methylated domain (DMD) as well as sequences in the H19 gene body that are not differentially methylated. Significantly, the presence of active marks at H19 depends on transcriptional activity and occurs even in the absence of the DMD. These findings suggest that histone modifications are dependent on the transcriptional status of imprinted alleles and illuminate epigenetic mechanisms of genomic imprinting.