Methylation status of differentially methylated regions at Igf2/H19 locus in porcine gametes and preimplantation embryos

The aim of this study was to demonstrate how differential methylation imprints are established during porcine preimplantation embryo development. For the methylation analysis, the primers for the three Igf2/H19 DMRs were designed and based upon previously published sequences. The methylation marks of Igf2/H19 DMRs were analysed in sperm and MII oocytes with our results showing that these regions are fully methylated in sperm but remain unmethylated in MII oocytes. In order to identify the methylation pattern at the pronuclear stage, we indirectly compared the methylation profile of Igf2/H19 DMR3 in each zygote derived by in vitro fertilization, parthenogenesis, and androgenesis. Interestingly, this region was found to be differently methylated according to parental origins; DMR3 was hemimethylated in in vitro fertilized zygotes, fully methylated in parthenogenetic zygotes, and demethylated in androgenetic zygotes. These results indicate that the methylation mark of the paternal allele is erased by active demethylation, and that of the maternal one is de novo methylated. We further examined the methylation imprints of Igf2/H19 DMR3 during early embryonic development. The hemimethylated pattern as seen in zygotes fertilized in vitro was observed up to the 4-cell embryo stage. However, this mark was exclusively demethylated at the 8-cell stage and then restored at the morula stage. These results suggest that methylation imprints are established via dynamic changes during early embryonic development in porcine embryos.     

Hypomethylated and hypermethylated profiles of H19DMR are associated with the aberrant imprinting of IGF2 and H19 in human hepatocellular carcinoma

In this study, 39 human hepatocellular carcinoma (HCC) tissues and 7 normal adult liver tissues were screened for heterozygous polymorphisms in IGF2, H19, and the differentially methylated region of H19 (H19DMR) using PCR-RFLP and PCR sequencing. The imprinting of IGF2 and H19 was examined by RT-PCR-RFLP, while the methylation profile of H19DMR was detected by bisulfite sequencing from every informative sample. Of the informative HCC samples 47.06% (8 of 17) demonstrated a gain of imprinting of IGF2, and 21.74% (5 of 23) of the informative HCC samples demonstrated a loss of imprinting of H19. Interestingly, we found three methylation profiles for H19DMR in the informative HCC samples: hyper-, medium-, and hypomethylated profiles. Furthermore, the hypomethylated and hypermethylated profiles were immediately associated with aberrant imprinting of IGF2 and H19.     

Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae

Imprinted genes are known to be crucial for placental development and fetal growth in mammals, but no primary epigenetic abnormality in placenta has been documented to compromise human fetal growth. Imprinted genes demonstrate parent-of-origin-specific allelic expression that is epigenetically regulated i.e. extrinsic to the primary DNA sequence. To undertake an epigenetic analysis of poor fetal growth in placentae and cord blood tissues, we first established the tissue-specific patterns of methylation and imprinted gene expression for two imprinting clusters (KvDMR and H19 DMR) on chromosome 11p15 in placentae and neonatal blood for 20 control cases and 24 Small for Gestational Age (SGA) cases. We confirmed that, in normal human placenta, the H19 promoter is unmethylated. In contrast, most other human tissues show paternal methylation. In addition, we showed that the IGF2 DMR2, also paternally methylated in most human tissues, exhibits hypomethylation in placentae. However, in neonatal blood DNA, these two regions maintain the differential methylation status seen in most other tissues. Significantly, we have been able to demonstrate that placenta does maintain differential methylation at the imprinting control regions H19 DMR and KvDMR. Of note, in one SGA placenta, we found a methylation alteration at the H19 DMR and concomitant biallelic expression of the H19 gene, suggesting that loss of imprinting at H19 is one cause of poor fetal growth in humans. Of particular interest, we demonstrated also a decrease in IGF2 mRNA levels in all SGA placentae and showed that the decrease is, in most cases, independent of H19 regulation.     

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.     

Genomic imprinting controls matrix attachment regions in the Igf2 gene

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.     

Biallelic expression of<Emphasis Type="Italic"> HRAS</Emphasis> and<Emphasis Type="Italic"> MUCDHL</Emphasis> in human and mouse

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.     

Timing of establishment of paternal methylation imprints in the mouse

Imprinted genes are characterized by predominant expression from one parental allele and differential DNA methylation. Few imprinted genes have been found to acquire a methylation mark in the male germ line, however, and only one of these, H19, has been studied in detail. We examined methylation of the Rasgrf1 and Gtl2 differentially methylated regions (DMR) to determine whether methylation is erased in male germ cells at e12.5 and when the paternal allele acquires methylation. We also compared their methylation dynamics with those of H19 and the maternally methylated gene Snrpn. Our results show that methylation is erased on Rasgrf1, H19, and Snrpn at e12.5, but that Gtl2 retains substantial methylation at this stage. Erasure of methylation marks on Gtl2 appears to occur later in female germ cells to give the unmethylated profile seen in mature MII oocytes. In the male germ line, de novo methylation of Rasgrf1, Gtl2, and H19 occurs in parallel between e12.5 and e17.5, but the DMR are not completely methylated until the mature sperm stage, suggesting a methylation dynamic different from that of IAP, L1, and minor satellite sequences, which have been shown to become fully methylated by e17.5 in male germ cells. This study also indicates important differences between different imprinted DMR in timing and extent of methylation in the germ cells.     

Functional characterization of a testis-specific DNA binding activity at the H19/Igf2 imprinting control region

The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.     

A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting.

The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.     

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.     

MIRA-SNuPE,a quantitative,multiplex method for measuring allele-specific DNA methylation

5-methyl-C (5mC) and 5-hydroxymethyl-C (5hmC) are epigenetic marks with well-known and putative roles in gene regulation, respectively. These two DNA covalent modifications cannot be distinguished by bisulfite sequencing or restriction digestion, the standard methods of 5mC detection. The methylated CpG island recovery assay (MIRA), however, specifically detects 5mC but not 5hmC. We further developed MIRA for the analysis of allele-specific CpG methylation at differentially methylated regions (DMRs) of imprinted genes. MIRA specifically distinguished between the parental alleles by capturing the paternally methylated H19/Igf2 DMR and maternally methylated KvDMR1 in mouse embryo fibroblasts (MEFs) carrying paternal and maternal duplication of mouse distal Chr7, respectively. MIRA in combination with multiplex single nucleotide primer extension (SNuPE) assays specifically captured the methylated parental allele from normal cells at a set of maternally and paternally methylated DMRs. The assay correctly recognized aberrant biallelic methylation in a case of loss of imprinting. The MIRA-SNuPE assays revealed that placenta exhibited less DNA methylation bias at DMRs compared to yolk sac, amnion, brain, heart, kidney, liver and muscle. This method should be useful for the analysis of allele-specific methylation events related to genomic imprinting, X chromosome inactivation and for verifying and screening haplotype-associated methylation differences in the human population.Key words: epigenetics, imprinting, DMR, MIRA, MBD, DNA methylation, SNuPE