Molecular interaction and synergistic activation of a promoter by Six, Eya, and Dach proteins mediated through CREB binding protein
Molecular interaction and synergistic activation of a promoter by Six, Eya, and Dach proteins mediated through CREB binding protein
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DOI：
10.1128/mcb.22.19.6759-6766.2002
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发表时间：
2002-10-01
影响因子：
5.3
通讯作者：
Kawakami, K
中科院分区：
文献类型：
--
作者：
Ikeda, K;Watanabe, Y;Kawakami, K
无眼果蝇、无眼果蝇和腊肠犬是复眼形成所必需的，并形成了一个具有直接蛋白质相互作用和遗传调控的基因网络。这些基因的脊椎动物同源物Six、Eya和Dach在肌肉形成过程中也形成了类似的遗传网络。为了阐明Six，Eya和Dach之间网络的分子机制，我们研究了编码蛋白质之间的分子相互作用。Eya直接与Six互动，但从未与Dach互动。Dach通过共产生GAL 4-Eya融合蛋白来反式激活多聚化的GAL 4报告基因。Eya和Dach的反式激活被VP 16或E1 A的过表达所抑制，但不被CREB结合蛋白（CBP）结合缺陷的E1 A突变所抑制。CBP向固定化染色质DNA模板的募集依赖于FLAG-Dach和GAL 4-Eya 3。这些结果表明CBP是Eya和Dach之间相互作用的介导者。与我们的预期相反，Dach本身与染色质DNA结合，而不是被GAL 4-Eya 3束缚。Dach也以较低的亲和力结合裸DNA。保守的DD 1结构域负责与DNA结合。还通过GAL 4-Six、Eya和Dach的共产生观察到反式激活，表明当Eya通过Six蛋白质与DNA连接时，Eya和Dach协同作用是相关的。我们的研究结果表明，协同作用是通过直接相互作用的Six-Eya和通过相互作用的Eya-Dach与CBP介导的，并解释了Six，Eya和Dach之间的遗传相互作用的分子基础。这项工作提供了有关该基因盒在组织分化和器官发生中的作用和作用机制的基本信息。
Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, we examined the molecular interactions among the encoded proteins. Eya interacted directly with Six but never with Dach. Dach transactivated a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach was repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template was dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to our expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. Our results demonstrated that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis.