Molecular, cellular and in vivo analyses of photoresponses and circadian rhythms in animals

(1) Gil的激活引起雏鸡松果体生物钟的相移，其方式类似于光引起的相移。在pinopsin基因的启动子区域，我们发现了一个光响应元件，负责其光依赖性转录激活。(2)视网膜棒g蛋白转导蛋白的法尼化不仅在细胞光信号传导中起重要作用，而且在细胞光适应中起重要作用。我们发现斑马鱼视网膜锥细胞中表达的GRK7-1的视蛋白磷酸化活性比杆状特异性GRK1A高几十倍。(3)雏鸡松果体和蛙视网膜MAPK具有昼夜节律性，在夜间活性最高。在小鼠SCN中，MAPK的时间和光激活在SCN的背内侧和中央区域都是不同的。我们确定了BMAL1、CRY1和CRY2中被MAPK磷酸化的氨基酸残基及其磷酸化依赖的功能变化。我们还发现SCN中大量表达的SCOP是K-Ras和下游MAPK的负调控因子。结果表明，p38 MAPK在白天调控分子钟，具有提前相位的作用。(4)当12L: 12D条件下的雏鸡暴露在长时间光照下直至深夜，松果体E4BP4蛋白保持在高水平，延迟了Per2的清晨诱导，延缓了松果体时钟的阶段。E4BP4被CKIε磷酸化，导致其蛋白酶体降解。(5)我们发现葡萄糖刺激大鼠-1细胞通过Tieg 1和Vdup 1参与的机制重置时钟。事实上，TIEG - 1蛋白在葡萄糖给药后被上调，它直接作用于Bmall的启动子抑制转录。(6)在松果体视蛋白驱血紫红蛋白基因的启动子区域，我们发现了一个顺式作用元件PIPE，它驱动斑马鱼松果体特异性基因的表达。

(1)Activation of Gil induced a phase-shift of the chick pineal circadian clock in a manner similar to that induced by light. In the promoter region of pinopsin gene, we identified a light-responsive element responsible for its light-dependent transcriptional activation.(2)Farnesylation of retinal rod G-protein transducin plays an important role in not only the cellular light-signaling but also the light-adaptation. We found that GRK7-1 expressed in the zebrafish retinal cones shows opsin-phosphorylating activity dozens-fold higher than that of rod-specific GRK1A.(3)Chick pineal and frog retinal MAPK was circadian-activated showing the peak activity at night. Within the mouse SCN, the profiles of the temporal and light activation of MAPK are both different between the dorsomedial and central regions of the SCN. We identified amino acid residues in BMAL1, CRY1 and CRY2 to be phosphorylated by MAPK and their phosphorylation-dependent functional changes. We also found that SCOP expressed abundantly in the SCN is a negative regulator of K-Ras and downstream MAPK. It was shown that p38 MAPK regulates the molecular clock in the daytime to have the phase-advancing effect.(4)When the chicks entrained to 12L : 12D conditions were exposed to prolonged light extending into the early night, pineal E4BP4 protein was kept at a high level, which delayed the morning induction of Per2 and delayed the phase of the pineal clock. E4BP4 was phosphorylated by CKIε, leading to its proteasomal degradation.(5)We found that glucose-stimulation of rat-1 cells resets the clock by a mechanism to which Tieg 1 and Vdup 1 contribute. TIEG 1 protein was in fact up-regulated upon glucose administration and it directly acts on the promoter of Bmall to inhibit the transcription.(6)Within the promoter region of pineal opsin exorhodopsin gene, we identified a cis-acting element, PIPE, that drives pineal-specific gene expression in the zebrafish.