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.
Highlights
Porcine insulin-like growth factor 2 (IGF2) and the H19 genes are imprinted
The maternal allele H19 differentially methylated domain (DMD) interacts with Igf2 DMR1 allowing maternal H19 to be expressed while the paternal H19 DMD interacts with Igf2 DMR2, allowing Igf2 to be expressed and leaves the H19 gene silent
We hypothesized that an alteration in IGF2 imprinting status might be reflected in DNA methylation variations at differentially methylated regions as suggested by the boundary and chromatin loop models. To test this hypothesis we studied the association between the IGF2 imprinting status in three different tissue samples of six boars and their DNA methylation of H19 DMD, IGF2 DMR1 and DMR2 (Figure 1)
Summary
Porcine 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. An extensive number of studies have been conducted to elucidate the epigenetic mechanisms of IGF2 and H19 which are thought to be co-ordinately regulated, both in terms of their expression patterns and their reciprocal imprinting (for review see [3]) It was shown by deletions in mice that a region of paternal-specific DNA methylation (differentially methylated domain, DMD) upstream of H19 is an epigenetic mark required for imprinting of IGF2 and H19 [10,11]. This model was named the chromatin loop model [14]
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