Abstract
The mouse Igf2/H19 locus is regulated by genomic imprinting, in which the paternally methylated H19 imprinting control region (ICR) plays a critical role in mono-allelic expression of the genes in the locus. Although the maternal allele-specific insulator activity of the H19 ICR in regulating imprinted Igf2 expression has been well established, the detailed mechanism by which the H19 ICR controls mono-allelic H19 gene expression has not been fully elucidated. In this study, we evaluated the effect of H19 ICR orientation on imprinting regulation in mutant mice in which the H19 ICR sequence was inverted at the endogenous locus. When the inverted-ICR allele was paternally inherited, the methylation level of the H19 promoter was decreased and the H19 gene was derepressed, suggesting that methylation of the H19 promoter is essential for complete repression of H19 gene expression. Unexpectedly, when the inverted allele was maternally inherited, the expression level of the H19 gene was lower than that of the WT allele, even though the H19 promoter remained fully hypomethylated. These observations suggested that the polarity of the H19 ICR is involved in controlling imprinted H19 gene expression on each parental allele, dependent or independent on DNA methylation of the H19 promoter.
Highlights
The mouse Igf2/H19 locus is regulated by genomic imprinting, in which the paternally methylated H19 imprinting control region (ICR) plays a critical role in mono-allelic expression of the genes in the locus
To investigate the effect of H19 ICR orientation on the imprinted expression and DNA methylation status of the H19 locus, inversion of the H19 ICR sequence was induced at the mouse endogenous locus by genome editing
We included single-stranded oligodeoxynucleotides with sequences that correspond to the junctional sequences after correct inversion, expecting that this would facilitate the inverted ligation of the ICR fragment (Supplementary Fig. 1b)
Summary
The mouse Igf2/H19 locus is regulated by genomic imprinting, in which the paternally methylated H19 imprinting control region (ICR) plays a critical role in mono-allelic expression of the genes in the locus. The maternal allele-specific insulator activity of the H19 ICR in regulating imprinted Igf[2] expression has been well established, the detailed mechanism by which the H19 ICR controls mono-allelic H19 gene expression has not been fully elucidated. When the inverted allele was maternally inherited, the expression level of the H19 gene was lower than that of the WT allele, even though the H19 promoter remained fully hypomethylated These observations suggested that the polarity of the H19 ICR is involved in controlling imprinted H19 gene expression on each parental allele, dependent or independent on DNA methylation of the H19 promoter. Mono-allelic expression of the Igf2/H19 gene locus is controlled by paternal allele-specific DNA methylation of the H19 ICR, located 2–4 kb upstream of the H19 gene, and its insulator activity is involved in the imprinted expression of the distallylocated Igf[2] gene (Supplementary Fig. 1a). DNA methylation of the H19 promoter seems mandatory for the repression of H19 expression
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