Orientation of mouse H19 ICR affects imprinted H19 gene expression through promoter methylation–dependent and –independent mechanisms

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.

小鼠Igf2/H19基因座受基因组印记（genomic imprinting）调控，其中父本甲基化的H19印记控制区（ICR）对该基因座内基因的单等位基因表达发挥关键调控作用。尽管H19 ICR的母本等位基因特异性绝缘子活性在调控印记型Igf2表达中的功能已得到充分证实，但H19 ICR控制H19基因单等位基因表达的具体分子机制尚未完全阐明。本研究针对内源基因座处H19 ICR序列发生倒位的突变小鼠，探究了H19 ICR的方向对印记调控的影响。当该反向ICR等位基因通过父本遗传时，H19启动子的甲基化水平降低，H19基因发生去抑制，这提示H19启动子的甲基化对于完全抑制H19基因表达至关重要。出乎意料的是，当该倒位等位基因通过母本遗传时，即便H19启动子仍保持完全低甲基化状态，H19基因的表达水平却低于野生型（WT）等位基因。上述观察结果表明，H19 ICR的极性参与调控两条亲本等位基因上的印记型H19基因表达，其调控机制可依赖或不依赖H19启动子的DNA甲基化。