Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 415 Curie Boulevard, Philadelphia, PA 19104, USA.
Dev Biol. 2011 Jul 15;355(2):349-57. doi: 10.1016/j.ydbio.2011.04.036. Epub 2011 May 10.
Expression of coregulated imprinted genes, H19 and Igf2, is monoallelic and parent-of-origin-dependent. Like most imprinted genes, H19 and Igf2 are regulated by a differentially methylated imprinting control region (ICR). CTCF binding sites and DNA methylation at the ICR have previously been identified as key cis-acting elements required for proper H19/Igf2 imprinting. Here, we use mouse models to elucidate further the mechanism of ICR-mediated gene regulation. We specifically address the question of whether sequences outside of CTCF sites at the ICR are required for paternal H19 repression. To this end, we generated two types of mutant ICRs in the mouse: (i) deletion of intervening sequence between CTCF sites (H19(ICR∆IVS)), which changes size and CpG content at the ICR; and (ii) CpG depletion outside of CTCF sites (H19(ICR-8nrCG)), which only changes CpG content at the ICR. Individually, both mutant alleles (H19(ICR∆IVS) and H19(ICR-8nrCG)) show loss of imprinted repression of paternal H19. Interestingly, this loss of repression does not coincide with a detectable change in methylation at the H19 ICR or promoter. Thus, neither intact CTCF sites nor hypermethylation at the ICR is sufficient for maintaining the fully repressed state of the paternal H19 allele. Our findings demonstrate, for the first time in vivo, that sequence outside of CTCF sites at the ICR is required in cis for ICR-mediated imprinted repression at the H19/Igf2 locus. In addition, these results strongly implicate a novel role of ICR size and CpG density in paternal H19 repression.
受调控的印迹基因 H19 和 Igf2 的表达是单等位基因且依赖于亲本来源。与大多数印迹基因一样，H19 和 Igf2 受差异甲基化印迹控制区（ICR）调控。先前已经鉴定出 ICR 中的 CTCF 结合位点和 DNA 甲基化为正确的 H19/Igf2 印迹所必需的关键顺式作用元件。在这里，我们使用小鼠模型进一步阐明 ICR 介导的基因调控机制。我们特别提出了一个问题，即 ICR 内 CTCF 位点之外的序列是否需要用于父系 H19 的抑制。为此，我们在小鼠中生成了两种类型的 ICR 突变体：（i）在 ICR 中的 CTCF 位点之间缺失间隔序列（H19(ICR∆IVS)），这会改变 ICR 的大小和 CpG 含量；（ii）在 CTCF 位点之外的 CpG 耗竭（H19(ICR-8nrCG)），这仅改变 ICR 的 CpG 含量。单独的两种突变等位基因（H19(ICR∆IVS)和 H19(ICR-8nrCG)）都显示出父系 H19 印迹抑制的丧失。有趣的是，这种抑制的丧失与 H19 ICR 或启动子处的甲基化没有明显变化并不一致。因此，完整的 CTCF 位点或 ICR 处的高甲基化对于维持父系 H19 等位基因的完全抑制状态都不是必需的。我们的研究结果首次在体内证明，ICR 内 CTCF 位点之外的序列在顺式中对于 ICR 介导的 H19/Igf2 基因座的印迹抑制是必需的。此外，这些结果强烈表明 ICR 大小和 CpG 密度在父系 H19 抑制中具有新的作用。
