Graduate school of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
Faculty of Life and Environmental Sciences, Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
Hum Mol Genet. 2021 Jan 21;29(22):3646-3661. doi: 10.1093/hmg/ddaa253.
Monoallelic gene expression at the Igf2/H19 locus is controlled by paternal allele-specific DNA methylation of the imprinting control region (H19 ICR) that is established during spermatogenesis. We demonstrated that the H19 ICR fragment in transgenic mice acquires allele-specific methylation only after fertilization, which is essential for maintaining its allelic methylation during early embryogenesis. We identified a DNA element required for establishing postfertilization methylation within a 118 bp (m118) region. A previously generated knock-in mouse whose endogenous H19 ICR was substituted with the human H19 ICR (hIC1; 4.8 kb) sequence revealed that the hIC1 sequence was partially methylated in sperm, although this methylation was lost by the blastocyst stage, which we assume is due to a lack of an m118-equivalent sequence in the hIC1 transgene. To identify a cis sequence involved in postfertilization methylation within the hIC1 region, we generated three transgenic mouse lines (TgM): one carrying an 8.8 kb hIC1 sequence joined to m118 (hIC1+m118), one with the 8.8 kb hIC1 and one with the 5.8 kb hIC1 sequence joined to m118 (hIC1-3'+m118). We found that the hIC1-3' region was resistant to de novo DNA methylation throughout development. In contrast, the 5' portion of the hIC1 (hIC1-5') in both hIC1+m118 and hIC1 TgM were preferentially methylated on the paternal allele only during preimplantation. As DNA methylation levels were higher in hIC1+m118, the m118 sequence could also induce imprinted methylation of the human sequence. Most importantly, the hIC1-5' sequence appears to possess an activity equivalent to that of m118.
IGF2/H19 基因座的单等位基因表达由印迹控制区（H19 ICR）的父系等位基因特异性 DNA 甲基化控制，该区域在精子发生过程中建立。我们证明，只有在受精后，转基因小鼠中的 H19 ICR 片段才会获得等位基因特异性甲基化，这对于在早期胚胎发生过程中维持其等位基因甲基化至关重要。我们确定了一个在 118bp（m118）区域内建立受精后甲基化所必需的 DNA 元件。之前生成的一种敲入小鼠，其内源 H19 ICR 被人 H19 ICR（hIC1；4.8kb）序列取代，显示 hIC1 序列在精子中部分甲基化，尽管这种甲基化在胚泡阶段丢失，我们认为这是由于 hIC1 转基因中缺乏 m118 等效序列。为了确定 hIC1 区域内受精后甲基化所涉及的顺式序列，我们生成了三个转基因小鼠品系（TgM）：一个携带与 m118 连接的 8.8kb hIC1 序列（hIC1+m118），一个携带 8.8kb hIC1 序列，一个携带 5.8kb hIC1 序列与 m118 连接（hIC1-3'+m118）。我们发现 hIC1-3'区域在整个发育过程中都抵抗从头 DNA 甲基化。相比之下，hIC1+m118 和 hIC1 TgM 中的 hIC1-5' 5'部分仅在植入前的父系等位基因上优先甲基化。由于 hIC1+m118 中的 DNA 甲基化水平更高，因此 m118 序列也可以诱导人序列的印迹甲基化。最重要的是，hIC1-5'序列似乎具有与 m118 等效的活性。
