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

Abstract

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.

Sperm DNA Methylation Analysis in Swine Reveals Conserved and Species-Specific Methylation Patterns and Highlights an Altered Methylation at the GNAS Locus in Infertile Boars1

“…This result is not related to poor identification and localization in swine of IGF2 and H19 DMRs, which are highly homologous to the corresponding human gene cluster and well described in the literature [50,51]. Moreover, our results are in accordance with a study on bovine sperm that did not find any significant difference in the methylation level of IGF2/H19 DMRs of high-or low-fertility bulls [52].…”

Dietary betaine supplementation to gestational sows enhances hippocampal IGF2 expression in newborn piglets with modified DNA methylation of the differentially methylated regions

“…In this study, we detected significant hypermethylation on DMR1 and DMR2 of the IGF2 locus in the hippocampus of prenatally betaine-supplemented piglets. It is well known that IGF2 gene is imprinted to express exclusively from the paternal allele in most fatal tissues in various mammalian species including pigs [48]. However, biallelic expression or relaxation of IGF2 imprinting appears to be a common feature in adult porcine tissues [49].…”

Effects of parental folate deficiency on the folate content, global DNA methylation, and expressions of FRα, IGF-2 and IGF-1R in the postnatal rat liver

“…Folic acid deficiency may prevent normal methylation of epigenetically regulated IGF-2 gene. Since its expression is regulated by DNA methylation, it may be vulnerable to abnormal methylation during development [19-20]. A number of studies have also demonstrated that maternal supplementation with folic acid increased the methylation of the IGF-2 gene in the rat liver [21] and human blood offspring [22].…”