Molecular Biology of the Biological Clock : From Gene to Behavior

在真核生物昼夜节律模型系统中，振荡基因产物易位进入细胞核是24小时生物钟周期产生的关键步骤。我们研究了哺乳动物周期基因家族时钟蛋白的核输入和血清休克在培养细胞中诱导同步时钟的作用。我们在COS7细胞中检测了mPER蛋白的核输入，发现mPER1和mPER2的核易位涉及与mPER3的物理相互作用。这表明在没有CRY蛋白的生理条件下，mPER1的核易位也可能发生。最近，我们证明了辅助转录环的存在有助于核心时钟反馈环。作为PAR亮氨酸拉链转录因子家族的一员，DBP的转录水平在SCN中表现出强大的节律。我们报道DBP能够通过直接结合mPer1启动子来激活一个假定的时钟振荡基因mPer1的启动子。DBP和CLOCK-BMAL1协同激活mPer1启动子。另一方面，dbp转录由CLOCK-BMAL1通过e -box激活，并被mPER和mCRY蛋白抑制，mPer1也是如此。反期节律调节的E4BP4是另一个亮氨酸拉链转录因子成员，无PAR结构域，对抗性抑制mPer1的转录。因此，时钟控制的dbp和e4bp4基因可能在中央时钟振荡中起重要作用。利用携带mPer1启动子与荧光素酶（mPer1-luc）基因融合的转基因小鼠，我们最近成功地监测了荧光素酶介导的昼夜变化在脑切片和活体动物的SCN中。我们可以记录数天的振荡光子发射，其周期性和相位准确地反映了天然mPer1 mRNA的表达。基因表达的实时光学成像将成为研究哺乳动物脑功能的有力工具。

In the eukaryotic circadian model systems, translocation of the oscillatory gene products into the nucleus is a key step for generation of a 24 hour cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. We examined the nuclear import of mPER proteins in COS7 cells and found that nuclear translocation of mPER1 and mPER2 involves physical interactions with mPER3. This indicates that nuclear translocation of mPER1 also can occur under physiological conditions in the absence of CRY proteins.Recently we demonstrated that an accessory transcription loop exists helping to the core clock feedback loop. Transcript levels of DBP, a member of the PAR leucine zipper transcription factor family, exhibit a robust rhythm in the SCN.We report that DBP is able to activate the promoter of a putative clock oscillating gene, mPer1, by directly binding to the mPer1 promoter. DBP and CLOCK-BMAL1 cooperatively activate the mPer1 promoter. On the other hand, dbp transcription is activated by CLOCK-BMAL1 through E-boxes and inhibited by the mPER and mCRY proteins, as is the case for mPer1. Antiphase circadian regulated E4BP4, another member of leucine zipper transcription factor without PAR domain, antagonistically suppresses mPer1 transcription. Thus, a clock-controlled dbp and e4bp4 genes may play an important role in the central clock oscillation.Using transgenic mice carrying the mPer1 promoter fused to the luciferase (mPer1-luc) gene, we recently succeeded to monitor luciferase-mediated bioluminescence with a day-night variation in the SCN in brain slices and in living animals. We can record for several days oscillating photon emission with a periodicity and phase that accurately mirrored native mPer1 mRNA expression. The real-time optical imaging of gene expression will be a new powerful tool to study mammalian brain function.