Bell-Pedersen D, Lewis Z A, Loros J J, Dunlap J C
Department of Biology, Texas A & M University, College Station, TX 77843, USA.
Mol Microbiol. 2001 Aug;41(4):897-909. doi: 10.1046/j.1365-2958.2001.02558.x.
The circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. Input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. Very little is known about the regulation of outputs from the clock. In Neurospora crassa, the circadian clock transcriptionally regulates expression of the clock-controlled genes, including the well-characterized eas(ccg-2) gene. Dissection of the eas(ccg-2) gene promoter previously localized a 68 bp sequence containing an activating clock element (ACE) that is both necessary and sufficient for rhythmic activation of transcription by the circadian clock. Using electrophoretic mobility shift assays (EMSAs), we have identified light-regulated nuclear protein factors that bind specifically to the ACE in a time-of-day-dependent fashion, consistent with their role in circadian regulation of expression of eas(ccg-2). Nucleotides in the ACE that interact with the protein factors were determined using interference binding assays, and deletion of the core interacting sequences affected, but did not completely eliminate, rhythmic accumulation of eas(ccg-2) mRNA in vivo, whereas deletion of the entire ACE abolished the rhythm. These data indicate that redundant binding sites for the protein factors that promote eas(ccg-2) rhythms exist within the 68 bp ACE. The ACE binding complexes formed using protein extracts from cells with lesions in central components of the Neurospora circadian clock were identical to those formed with extracts from wild-type cells, indicating that other proteins directly control eas(ccg-2) rhythmic expression. These data suggest that the Neurospora crassa circadian clock regulates an unknown transcription factor, which in turn activates the expression of eas(ccg-2) at specific times of the day.
昼夜节律钟在生物体的环境与其行为之间建立了联系，在预期每日环境变化时，使基因的表达在时间上同步。输入途径感知环境信息并与生物钟相互作用，使其与外部周期同步，而输出途径从生物钟读取信息，以对下游靶点进行时间控制。目前对于生物钟输出的调控知之甚少。在粗糙脉孢菌中，昼夜节律钟通过转录调控生物钟控制基因的表达，包括特征明确的eas(ccg-2)基因。先前对eas(ccg-2)基因启动子的剖析定位了一个68bp的序列，该序列包含一个激活生物钟元件（ACE），它对于昼夜节律钟对转录的节律性激活既是必需的也是充分的。使用电泳迁移率变动分析（EMSA），我们鉴定了光调节的核蛋白因子，它们以一天中的时间依赖性方式特异性结合到ACE上，这与其在eas(ccg-2)表达的昼夜节律调控中的作用一致。使用干扰结合分析确定了ACE中与蛋白因子相互作用的核苷酸，并且核心相互作用序列的缺失影响但并未完全消除体内eas(ccg-2)mRNA的节律性积累，而整个ACE的缺失则消除了节律。这些数据表明，在68bp的ACE内存在促进eas(ccg-2)节律的蛋白因子的冗余结合位点。使用来自粗糙脉孢菌昼夜节律钟核心组件有损伤的细胞的蛋白提取物形成的ACE结合复合物与使用野生型细胞提取物形成的复合物相同，这表明其他蛋白质直接控制eas(ccg-2)的节律性表达。这些数据表明，粗糙脉孢菌的昼夜节律钟调控一种未知的转录因子，该转录因子进而在一天中的特定时间激活eas(ccg-2)的表达。
