BACKGROUND Paternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes. The molecular mechanism by which differential methylation of the H19 ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kb H19 ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at the H19 ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of the H19 ICR are essential for maintaining its maternal hypomethylation status and for acquisition of paternal methylation, respectively, during the post-fertilization period. RESULTS Using a series of 5'-truncated H19 ICR transgenes to dissect the 478-bp sequence, we identified a 118-bp region required for post-fertilization methylation activity. Deletion of the sequence from the paternal endogenous H19 ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenous H19 ICR from genome-wide reprogramming. We then reconstructed a synthetic DNA fragment in which the CTCF binding sites, Sox-Oct motifs, as well as the 118-bp sequence, were inserted into lambda DNA, and used it to replace the endogenous H19 ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the both Igf2 and H19 genes was recapitulated. CONCLUSIONS Our results identified a 118-bp region within the H19 ICR that is required for de novo DNA methylation of the paternally inherited H19 ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identified cis elements, could fully replace the function of the H19 ICR in the mouse genome.

中文翻译：

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配子DNA甲基化的概括以及在小鼠Igf2 / H19基因座上用重组的DNA元素进行的受精后维持。

背景技术H19印迹控制区（ICR）的父本等位基因特异性DNA甲基化调节Igf2 / H19基因的印迹表达。然而，尚未完全了解在配子发生期间建立并在受精后维持H19 ICR差异甲基化的分子机制。我们以前表明转基因小鼠中的一个2.9-kb H19 ICR片段仅在受精后才被甲基化，表明在H19 ICR发生了两个可分离的事件，配子和受精后甲基化。然后，我们确定CT19 / Sox-Oct基序和H19 ICR的478-bp序列对于在受精期后分别维持其母体低甲基化状态和获得母体甲基化至关重要。结果使用一系列的5' 截短的H19 ICR转基因以解析478-bp序列，我们确定了受精后甲基化活性所需的118-bp区域。从父本内源性H19 ICR删除序列会导致受精后甲基化的丧失，这表明该序列的甲基化活性是保护内源性H19 ICR免受基因组范围重编程所必需的。然后，我们重建了一个合成的DNA片段，其中将CTCF结合位点，Sox-Oct基序以及118 bp序列插入了λDNA中，并用其取代了内源性H19 ICR。该片段在精子发生过程中被甲基化。此外，在受精后，其等位基因特异的甲基化状态得以忠实地保持，并且Igf2和H19基因的印迹表达得以概括。结论我们的研究结果确定了H19 ICR内118 bp的区域，该区域是在植入前阶段父本遗传的H19 ICR的从头DNA甲基化所必需的。除先前确定的顺式元件外，包含118 bp序列的基于λDNA的人工片段可以完全替代小鼠基因组中H19 ICR的功能。