Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance
Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance
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DOI：
10.1128/mcb.24.8.3497-3504.2004
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发表时间：
2004-04-01
影响因子：
5.3
通讯作者：
Ohlsson, R
中科院分区：
文献类型：
--
作者：
Pant, V;Kurukuti, S;Ohlsson, R
H19基因上游的差异甲基化印迹控制区(ICR)通过甲基化敏感的染色质绝缘体功能调节等位基因Igf2的表达。我们先前已经证明母系遗传的11-锌指蛋白CTCF的四个靶点中的三个突变会导致Igf2印记的丢失。在这里，我们发现，只有CTCF4位点的突变也导致母体Igf2等位基因的强烈激活，尽管与其他ICR位点1和3相比，4位点DNA与CTCF的体外相互作用明显弱。此外，在具有突变位4的肝脏和心脏细胞亚群中，母系遗传的I到3位点以复杂的模式去重新甲基化，这表明母体H19 ICR等位基因的甲基化特权状态需要所有四个CTCF位点之间的相互依赖。为了支持这一结论，我们证明了CTCF分子在体内和体外都相互结合，我们证明了两个在体外预先组装的CTCF-DNA复合体之间有很强的相互作用。我们认为CTCF位点可能相互协作，共同维持母体H19 ICR等位基因的无甲基化状态和绝缘体特性。考虑到许多其他的CTCF靶点，我们认为不同DNA结合的CTCF分子之间的位点特异性相互作用可能在参与基因调控的环染色质结构域的组织中提供一般的焦点。
The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites I to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.