A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock
A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock
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DOI：
10.1101/gad.1552207
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发表时间：
2007-07-01
影响因子：
10.5
通讯作者：
Ueda, Hiroki R.
中科院分区：
文献类型：
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作者：
Matsumoto, Akira;Ukai-Tadenuma, Maki;Ueda, Hiroki R.
果蝇的生物钟由集成的自动调节反馈环组成，如果不全面识别体内的网络组件，就很难阐明这一时钟。以前的研究使用高密度寡核苷酸阵列在全基因组范围内筛选时钟控制的基因，这些阵列识别了数百个时钟控制的基因。为了确定这些候选基因中的核心时钟基因，我们在体内应用了使用RNA干扰(RNAi)系统的全基因组功能筛选。在这里，我们报告了新的时钟候选基因的鉴定，包括时钟橙(Clockwork橙，CWO)，一个属于基本螺旋-环-螺旋橙家族的转录抑制因子。CWO通过规范的E-box序列有节律性地表达，并由CLK-CyC直接调控。利用果蝇基因组平铺阵列对其靶基因进行全基因组搜索发现，CWO形成了自己的负反馈环，并通过E-box序列直接抑制其他时钟基因的表达。此外，这种负转录反馈环有助于维持体内高幅度的昼夜节律振荡。基于这些结果，我们认为，周期性CLK-CyC活性与CWO施加的可调阈值之间的竞争使E-box介导的转录保持在其活性的可控范围内，从而使果蝇的生物钟能够产生高幅度振荡。
The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.