Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidian Ciona intestinalis
Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidian Ciona intestinalis
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DOI：
10.1242/dev.038141
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发表时间：
2009-11-01
期刊：
影响因子：
4.6
通讯作者：
Di Gregorio, Anna
中科院分区：
文献类型：
--
作者：
Passamaneck, Yale J.;Katikala, Lavanya;Di Gregorio, Anna
脊索是脊索体计划的一个重要特征。在海鞘、青蛙和小鼠胚胎上的实验表明，短毛和FoxA类转录因子的共同表达是脊索发育所必需的。然而，由于对脊索基因和顺式调控模块(CRM)的有限了解，介导这些转录因子协同作用的顺式调控序列的研究变得复杂。我们已经确定了一种易于测试的模型，用于这种研究在海鞘动物的155个碱基的脊索特异性CRM中。该CRM包含与Ciona brachyury(Ci-Bra)和FoxA(Ci-FoxA-a)的功能结合位点。通过结合点突变分析和错误表达实验，我们证明了这两个转录因子与该CRM结合是激活转录的必要条件和充分条件。为了深入了解控制其活性的顺式调控标准，我们调查了155-bp CRM内转录因子结合位点的组织。155个碱基的序列包含两个具有相同核心序列但取向相反的Ci-Bra结合位点，其中只有一个是增强子活性所必需的。这些位点的取向和间距的变化对CRM的活性有很大影响，因为在Ciona基因组中发现的具有不同排列的相同位点簇无法激活脊索细胞中的转录。这项工作首次证明了Brachyury和FoxA在激活单个脊索CRM中存在协同作用，并强调了转录因子结合位点安排对其功能的重要性。
The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.