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.     

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.     

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.     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating Insulin-like Growth Factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p=0.03, and 4.9%, p=0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p=0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.     

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     

Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy

Folic acid (FA) supplementation before and during pregnancy has been associated with decreased risk of neural tube defects although recent reports suggest it may also increase the risk of other chronic diseases. We evaluated exposure to maternal FA supplementation before and during pregnancy in relation to aberrant DNA methylation at two differentially methylated regions (DMRs) regulating insulin-like growth factor 2 (IGF2) expression in infants. Aberrant methylation at these regions has been associated with IGF2 deregulation and increased susceptibility to several chronic diseases. Using a self-administered questionnaire, we assessed FA intake before and during pregnancy in 438 pregnant women. Pyrosequencing was used to measure methylation at two IGF2 DMRs in umbilical cord blood leukocytes. Mixed models were used to determine relationships between maternal FA supplementation before or during pregnancy and DNA methylation levels at birth. Average methylation at the H19 DMR was 61.2%. Compared to infants born to women reporting no FA intake before or during pregnancy, methylation levels at the H19 DMR decreased with increasing FA intake (2.8%, p = 0.03 and 4.9%, p = 0.04, for intake before and during pregnancy, respectively). This methylation decrease was most pronounced in male infants (p = 0.01). Methylation alterations at the H19 DMR are likely an important mechanism by which FA risks and/or benefits are conferred in utero. Because stable methylation marks at DMRs regulating imprinted genes are acquired before gastrulation, they may serve as archives of early exposures with the potential to improve our understanding of developmental origins of adult disease.Key words: folic acid, epigenetics, IGF2, periconception, prenatal, exposure     

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.     

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.     

Characterization of the methylation status of five imprinted genes in sheep gametes

Genomic imprinting is a mammalian developmental process that uses epigenetic mechanisms to induce monoallelic and parental-specific expression of particular autosomal genes. A crucial epigenetic event consists of DNA methylation of CpG-islands, which become differentially methylated regions (DMRs) on the maternal and paternal alleles during oogenesis or spermatogenesis (germline DMRs). By contrast, somatic DMRs are acquired after fertilization. While there are several studies referring to methylation acquisition within germline DMRs in the mouse and human, a comparable methylation analysis of orthologous sequences is still lacking in sheep. To identify germline DMRs, this study analysed the methylation status of the available CpG-islands of five ovine imprinted genes ( H19, IGF2R, DLK1, DIO3 and BEGAIN ) in mature spermatozoa and in female gametes at different stages of their follicle growth, including in vitro matured oocytes. The 5'-end CpG-island of H19 showed a full methylation in spermatozoa and an absent methylation in growing and fully grown oocytes. The intron 2 CpG-island of IGF2R was unmethylated in male gametes, while it showed a high level of methylation in early stages of oogenesis. The promoter CpG-islands of DLK1 and DIO3 were found to be unmethylated both in spermatozoa and oocytes. Finally, the exon 9 CpG-island of BEGAIN was hypermethylated in mature male gametes, while it showed an almost complete methylation only in late stages of oocyte development. Our findings suggest that DNA methylation establishment during early stages of sheep oogenesis and subsequent in vitro maturation is gene-specific and that, of the five genes investigated, only the CpG-islands of H19 and IGF2R might represent ovine germline DMRs.     

Intragenic DNA methylation status down-regulates bovine IGF2 gene expression in different developmental stages

DNA methylation is a key epigenetic modification in mammals and has an essential and important role in muscle development. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to evaluate the expression of IGF2 and the methylation pattern on the differentially methylated region (DMR) of the last exon of IGF2 in six tissues with two different developmental stages. The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The quantitative real-time PCR (qPCR) analysis indicated that IGF2 has a broad tissue distribution and the adult bovine group showed significant lower mRNA expression levels than that in the fetal bovine group (P < 0.05 or P < 0.01). Moreover, the DNA methylation level analysis showed that the adult bovine group exhibited a significantly higher DNA methylation levels than that in the fetal bovine group (P < 0.05 or P < 0.01). These results indicate that IGF2 expression levels were negatively associated with the methylation status of the IGF2 DMR during the two developmental stages. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a marker-assisted selection for muscle developmental in beef cattle breeding program and as a model for studies in other species.     

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.     

Lsh controls silencing of the imprinted Cdkn1c gene

Epigenetic regulation, such as DNA methylation plays an important role in the control of imprinting. Lsh, a member of the SNF2 family of chromatin remodeling proteins, controls DNA methylation in mice. To investigate whether Lsh affects imprinting, we examined CpG methylation and allelic expression of individual genes in Lsh-deficient embryos. We report here that loss of Lsh specifically alters expression of the Cdkn1c gene (also known as p57(Kip2)) but does not interfere with maintenance of imprints at the H19, Igf2, Igf2r, Zac1 and Meg9 genes. The reactivation of the silenced paternal Cdkn1c allele correlates closely with a loss of CpG methylation at the 5' DMR at the Cdkn1c promoter, whereas KvDMR1 and DMRs of other imprinted genes were not significantly changed. Chromatin immunoprecipitations demonstrate a direct association of Lsh with the 5' DMR at the Cdkn1c promoter, but not with Kv DMR1 or other imprinted loci. These data suggest that methylation of the 5' DMR plays an important role in the imprinting of the Cdkn1c gene. Furthermore, it suggests that Lsh is not required for maintenance of imprinting marks in general, but is only crucial for imprinting at distinct genomic sites.     

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.     

Correlation between ZBED6 Gene Upstream CpG Island methylation and mRNA expression in cattle

DNA methylation is essential for the regulation of gene expression and important roles in muscle development. To assess the extent of epigenetic modifications and gene expression on the differentially methylated region (DMR) in ZBED6, we simultaneously examined DNA methylation and expression in six tissues from two different developmental stages (fetal bovine and adult bovine). The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The result of quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution and is highly expressed in adult bovine (P?P?P?P?相似文献   

DNA methylation variability at growth-related imprints does not contribute to overweight in monozygotic twins discordant for BMI

Defective genomic imprinting is often associated with syndromes that include abnormal growth as a clinical phenotype. However, whether differential methylation at imprinted loci also contributes to nonsyndromic abnormal body weight regulation is yet unknown. In this study, we investigated a potential contribution of aberrant DNA methylation at nine differentially methylated regions (DMRs) to the development of nonsyndromic overweight. Sixteen monozygotic (MZ) twins discordant for BMI (BMI difference ranging from 2.9-9.5 kg/m(2)) were recruited from the East Flanders Prospective Twin Survey. DNA extracted from saliva samples was bisulfite-treated followed by PCR amplification of target regions in DMRs most representative for abnormal growth syndromes: KvDMR1, H19 CTCF4, H19 CTCF6, IGF2 DMR0, IGF2 DMR2, GRB10, MEST, SNRPN, GNAS XL-α-s and GNAS Exon1A. At the DMRs analyzed, methylation-dependent primer extension experiments revealed only small intrapair differences in methylation indexes (MI) between the heavy and lean co-twins. In addition, no significant correlations between intrapair BMI differences and intrapair differences in MI were observed. In conclusion, DNA methylation variability at the nine DMRs analyzed does not seem to contribute to the discordancy in BMI observed in these MZ twins.