Molecular mechanism of circadian oscillation by real-time rhythm monitoring

哺乳动物生物钟基因的昼夜表达是基于转录反馈环。已知两种含有bHLH-PAS结构域的转录激活因子CLOCK和BMAL 1通过与称为E-box（CACGTG）的启动子元件相互作用来调节基因表达。非典型的E-box或E-box样序列也被报道是昼夜节律振荡所必需的。我们发现了一个新的顺式元件所需的细胞自主昼夜节律转录的公鸡基因。这个新元素由一个规范的E-box或一个非规范的E-box和一个与后者串联的E-box样序列组成，它们之间有一个短的间隔，6个碱基对。我们证明了E-box或E-box样序列都需要产生细胞自主振荡。我们还验证了这2个E-元件之间具有恒定长度的间隔核苷酸对于鲁棒振荡至关重要。因此，直接重复的E-box样元素是细胞自主的时钟和时钟控制的基因的转录振荡的产生所需的最低限度的元素。另一方面，我们开发了在体内监测的昼夜节律的时间在自由移动的小鼠通过记录多单位神经活动（MIJA）。视交叉上核（SCN）是哺乳动物的昼夜节律中枢，其神经活动与环境光输入紧密耦合，与室旁下区（SPZ）的MUA节律密切相关。在表现出减弱的昼夜运动节律性的Clock突变小鼠中，SCN和SPZ中的MUA节律性类似地钝化。这些结果表明，SPZ在将昼夜节律和光信号传递到参与产生行为活动的中心中起着功能性作用。

The circadian expression of the mammalian clock genes is based on transcriptional feedback loops. Two bHLH-PAS domain-containing transcriptional activators, CLOCK and BMAL1, are known to regulate gene expression by interacting with a promoter element termed the E-box (CACGTG). The non-canonical E-boxed or E-box-like sequences have also been reported to be necessary for circadian oscillation. We found a new cis-element required for cell-autonomous circadian transcription of cock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E-box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrated that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verified that the spacing nucleotides with constant length between these 2 E-elements are crucial for robust oscillation. Thus, the direct repeat of the E-box-like elements is the minimal required element for the generation of cell-autonomous transcriptional oscillation of clock and clock-controlled genes.On the other hand, we developed in vivo monitoring of circadian timing in freely moving mice by recording multi-unit neural activity (MIJA). Suprachiasmatic nucleus (SCN), the mammalian circadian center, neural activity is tightly coupled to environmental photic input and anticorrelated with MUA rhythm in the subparaventricular zone (SPZ). In Clock mutant mice exhibiting attenuated circadian locomotor rhythmicity, MUA rhythmicity in the SCN and SPZ is similarly blunted. These results suggest that the SPZ plays a functional role in relaying circadian and photic signals to centers involved in generating behavioral activity.