Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.
Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.
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DOI：
10.1093/nar/gkw478
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发表时间：
2016-08-19
影响因子：
14.9
通讯作者：
Sugiyama H
中科院分区：
文献类型：
--
作者：
Saha A;Kizaki S;De D;Endo M;Kim KK;Sugiyama H
调控遗传元件上的转录因子之间的功能协同性对于包括哺乳动物发育在内的各种细胞过程中的里程碑决策至关重要。然而，由于缺乏有效的工具来分析转录复合物中的蛋白质-DNA相互作用，它们在协同结合过程中的分子相互作用不能被精确地理解。在这里，我们表明，光诱导过量电子转移测定可用于分析协同性的蛋白质在转录复合物中使用的Pax 6到Sox 2的调节DNA元件（DC 5增强子）作为一个例子的合作结合。在该测定中，BrU标记的DC 5被引入用于有效检测从Sox 2和Pax 6转移到DNA的电子，并且互补链中的鸟嘌呤碱基被次黄嘌呤（I）取代以阻断Sox 2结合位点处的链内电子转移。通过检查DNA裂解发生的电子转移过程的结果，从色氨酸残基的Sox 2和Pax 6的DNA在280 nm照射后，我们不仅证实了它们的结合DNA，但也观察到它们的增加占用DC 5相对于单独的Sox 2和Pax 6作为其合作的相互作用的结果。
Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein–DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, BrU-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.
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影响因子：
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作者：
Cunha PM;Sandmann T;Gustafson EH;Ciglar L;Eichenlaub MP;Furlong EE
通讯作者：
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影响因子：
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