CTCF binding sites promote transcription initiation and prevent DNA methylation on the maternal allele at the imprinted H19/Igf2 locus
CTCF binding sites promote transcription initiation and prevent DNA methylation on the maternal allele at the imprinted H19/Igf2 locus
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DOI：
10.1093/hmg/ddl237
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发表时间：
2006-10-01
影响因子：
3.5
通讯作者：
Bartolomei, Marisa S.
中科院分区：
文献类型：
--
作者：
Engel, Nora;Thorvaldsen, Joanne L.;Bartolomei, Marisa S.
H19/Igf2位点的印迹依赖于差异甲基化结构域（DMD）作为母体特异性的甲基化敏感绝缘子和父亲特异性的高甲基化位点。DMD中的四个重复序列结合母体等位基因上的CTCF，并被认为是在父系等位基因上招募甲基化。我们删除了这四个重复序列，并分析了内源位点突变的影响。H19（DMD-Delta R）等位基因可以在精子发生过程中成功获得甲基化并沉默父本H19，这表明这些父本特异性功能独立于CTCF结合位点。母亲遗传突变导致双等位基因Igf2表达，与功能绝缘子的缺失一致。此外，我们还发现了CTCF结合位点的两个先前未描述的作用。在突变等位基因上，H19 RNA在6.5 d.p.c.胚胎和9.5 d.p.c.胎盘中几乎检测不到，这是第一次确定重复序列是负责启动H19转录的元件。此外，在胚胎着床后，突变的母体等位基因突然获得甲基化，此时胚胎正在经历全基因组的从头甲基化。总之，这些实验表明，CTCF重复序列除了对功能性绝缘子至关重要外，还促进了早期胚胎中H19表达的启动，并需要维持整个DMD的低甲基化状态。
Imprinting at the H19/Igf2 locus depends on a differentially methylated domain (DMD) acting as a maternal-specific, methylation-sensitive insulator and a paternal-specific locus of hypermethylation. Four repeats in the DMD bind CTCF on the maternal allele and have been proposed to recruit methylation on the paternal allele. We deleted the four repeats and assayed the effects of the mutation at the endogenous locus. The H19(DMD-Delta R) allele can successfully acquire methylation during spermatogenesis and silence paternal H19, indicating that these paternal-specific functions are independent of the CTCF binding sites. Maternal inheritance of the mutations leads to biallelic Igf2 expression, consistent with the loss of a functional insulator. Additionally, we uncovered two previously undescribed roles for the CTCF binding sites. On the mutant allele, H19 RNA is barely detectable in 6.5 d.p.c. embryos and 9.5 d.p.c. placenta, for the first time identifying the repeats as the elements responsible for initiating H19 transcription. Furthermore, methylation is abruptly acquired on the mutant maternal allele after implantation, a time when the embryo is undergoing genome-wide de novo methylation. Together, these experiments show that in addition to being essential for a functional insulator, the CTCF repeats facilitate initiation of H19 expression in the early embryo and are required to maintain the hypomethylated state of the entire DMD.