The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice
The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice
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DOI：
10.1128/mcb.01003-12
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发表时间：
2013-02-01
影响因子：
5.3
通讯作者：
Tanimoto, Keiji
中科院分区：
文献类型：
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作者：
Okamura, Eiichi;Matsuzaki, Hitomi;Tanimoto, Keiji
在小鼠Igf 2/H19印迹基因座中，印迹控制区（H19 ICR）的差异甲基化在精子发生过程中建立，并在整个发育过程中保持在后代中。然而，以前，我们观察到，父系H19 ICR，酵母人工染色体转基因小鼠（YAC-TgM）进行分析时，优先甲基化后受精。为了鉴定赋予甲基化印记的DNA序列，我们将H19 ICR分成两个片段（1.7和1.2 kb），将它们连接到插入了CTCF结合位点的λ DNA片段的两端，并在YAC-TgM中分析。在没有H19 ICR序列的情况下，母体遗传的λ序列在植入后通常是甲基化的，变得低甲基化，证明了对ICR内甲基化的保护活性。同时，父系遗传的λ序列在植入前仅在连接1.7 kb片段时被高甲基化。一致的是，当H19 ICR的两个亚片段分别研究它们在YAC-TgM中的活性时，只有1.7-kb片段能够引入父本等位基因特异性DNA甲基化。这些结果表明，受精后甲基化印记是由父亲等位基因特异性甲基化活性存在于一个1.7 kb的DNA片段的H19 ICR，而母亲等位基因特异性活动保护从从头DNA甲基化的等位基因。
In the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a lambda DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited lambda sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited lambda sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.