bHLH-ORANGE family genes regulate the expression of E-box clock genes in Drosophila
bHLH-ORANGE family genes regulate the expression of E-box clock genes in Drosophila
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DOI：
10.1007/s13355-011-0052-z
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发表时间：
2011-08-01
影响因子：
1.3
通讯作者：
Matsumoto, Akira
中科院分区：
文献类型：
--
作者：
Itoh, Taichi Q.;Tanimura, Teiichi;Matsumoto, Akira
果蝇有13个基本螺旋-环-螺旋-橙色（bHLH-O）家族基因。其中一个成员，发条橙子（cwo），是哺乳动物时钟基因Dec 1和Dec 2的对应物，通过结合靶基因启动子中的E-box序列来调节生物钟基因的转录反馈环。本研究的目的是在分子和行为水平上确定果蝇bHLH-O蛋白在昼夜节律中的作用。在培养的果蝇S2细胞中进行启动子测定，以研究哪些已知的bHLH-O蛋白直接调节时钟基因的转录。除了CWO，其他三种bHLH-O蛋白，SIDE，M β和M γ，在体外抑制E-box时钟基因转录。利用RNA干扰（RNAi）技术在起搏神经元中产生bHLH-O基因敲低果蝇，然后测量其运动活动节律。CWO敲除果蝇表现出显著的表型。为了阐明CWO、SIDE、M β和M γ之间在昼夜节律调节中的功能互补，检测了在两个bHLH-O基因的组合存在下的启动子活性和双敲低果蝇中的运动节律。结果表明，CWO在体外和体内都是昼夜节律调节的关键因素。
Drosophila has 13 basic helix-loop-helix-ORANGE (bHLH-O) family genes. One of the members, clockwork orange (cwo), which is the counterpart of mammalian clock genes Dec1 and Dec2, regulates the transcriptional feedback loops of circadian clock genes through binding to E-box sequences in target gene promoters. The goal of the current study was to determine the role of Drosophila bHLH-O proteins in circadian rhythms at the molecular and behavioral level. Promoter assays in cultured Drosophila S2 cells were carried out to investigate which of the known bHLH-O proteins directly regulates the transcription of clock genes. In addition to CWO, three other bHLH-O proteins, SIDE, M beta and M gamma, suppressed E-box clock gene transcription in vitro. RNA interference (RNAi) was used to generate bHLH-O knockdown flies in pacemaker neurons, and then locomotor activity rhythm was measured. cwo knockdown flies exhibited a remarkable phenotype. To clarify the functional complementation in circadian regulation among CWO, SIDE, M beta and M gamma, promoter activity in the presence of combinations of two bHLH-O genes and locomotor rhythm in double knockdown flies were examined. The results suggest that CWO predominantly acts as a key factor of circadian regulation both in vitro and in vivo.