哺乳动物隐花色素蛋白调控生物钟节律机制的研究

发布时间：2018-04-26 05:07

本文选题：生物钟节律 + CRY基因　；参考：《中国农业大学》2016年博士论文

【摘要】：地球上几乎所有的生物都具有生物钟。在哺乳动物中,生物的主生物钟定位于大脑内被称之为视交叉上核(Suprachiasmatic nucleus, SCN)的核团中,SCN核团能够通过光信号同步化生物体外周生物钟。实际上,在单个细胞的水平就存在生物钟,生物钟可以是细胞水平的独立行为。最被广泛接受的生物钟分子机制是一个基于转录翻译的负反馈环路模型,包括两个转录激活因子(BMAL1蛋白和CLOCK蛋白)和两类转录抑制因子(PERIOD蛋白,或称作PER1/2/3蛋白；和CRYPTOCHROME蛋白,即CRY1/2蛋白)维持的近二十四小时的负反馈环路。基于这个负反馈模型,生物钟系统再通过一系列钟控基因(Clock-Controlled Gene, CCG基因)调控着生物的行为及生理节律。在哺乳动物细胞内,至少有10%的转录组显示出周期性的转录表达,而其中大部分的基因都是生物钟控制的基因。正向转录因子BMAL1基因或(CLOCK基因的周期性表达对于生物钟的产生不是必需的,但是周期性的负向调控因子(PER基因或CRY基因)的表达对于生物钟的形成至关重要。在生物钟的研究中PER蛋白与CRY蛋白被视为一个PER复合物来研究,PER蛋白的重要性被广泛接受。虽然很早就发现CRY1基因和CRY2基因可以导致不同的生物钟周期,但是关于CRY基因调控生物钟周期的机制却没有完善的研究。尽管到目前为止CRY蛋白的稳定性模型是解释CRY蛋白调控生物钟周期最为广泛接受的模型。在我们的实验中,我们首先发现了在内源CRY1基因启动子驱动下的CRY2基因单独表达也可以恢复CRY基因缺失细胞系的生物钟表型。在对CRY蛋白稳定性进行分析时,我们发现虽然CRY2蛋白的稳定性较强,但是相比稳定性较弱的CRY1蛋白却表现出短周期的生物钟节律,这与之前关于CRY蛋白稳定性调控生物钟周期的模型相矛盾。我们还发现CRY2蛋白的N端序列决定了CRY2蛋白比CRY1蛋白更加稳定,此外CRY2蛋白与CRYI蛋白中赖氨酸残基的数量可能也是蛋白稳定性的决定因素。由CRY2蛋白N端序列决定的蛋白稳定性不能直接影响生物钟的周期长度,反而是不能够明显影响CRY1蛋白与CRY2蛋白稳定性的C端序列决定了生物钟的周期长度。最后,我们还发现了CRY1蛋白与转录延伸因子P-TEFb(positive transcriptional elongation factor b)复合物以及7SK snRNP (small nuclear ribonucleoprotein complex)复合物之间的紧密联系,从而揭示了CRY蛋白通过7SK RNA行使生物钟转录抑制活性的可能机制。我们的研究证明了长久以来一直存在的通过CRY1蛋白与CRY2蛋白的比例调控生物钟周期的假设。更精确的说,我们的实验证明了不是通过单独的PER蛋白或者CRY蛋白,而是由这两个蛋白组成的PER复合物的入核速率决定了生物钟的周期长度。同时我们的研究显示哺乳动物CRY1蛋白可能是通过靶向失活P-TEFb复合物完成转录抑制功能。我们的研究解释了生物钟的周期调控机制和负反馈抑制机制,完整了生物钟负反馈环路的模型。
[Abstract]:Almost all living things on earth have biological clocks. In mammals, the main biological clock of organisms is located in the nucleus Suprachiasmatic nucleus (SCN) of the brain called Suprachiasmatic nucleus (SCNs). In fact, there is a biological clock at the level of a single cell, which can be an independent behavior at the cellular level. The most widely accepted molecular mechanism of the biological clock is a negative feedback loop model based on transcriptional translation, consisting of two transcriptional activators, BMAL1 and CLOCK, and two types of transcription suppressor, PERIOD, or PER1/2/3 protein; and CRYPTOCHROME protein. CRY1/2 protein) maintains a negative feedback loop for nearly 24 hours. Based on the negative feedback model, the biological clock system regulates the biological behavior and physiological rhythm through a series of clock-controlled genes (CCG gene). In mammalian cells, at least 10% of the transcriptome shows cyclic transcriptional expression, and most of the genes are controlled by the biological clock. The periodic expression of forward transcription factor (BMAL1) gene or CRY gene is not necessary for the production of biological clock, but the expression of periodic negative regulatory factor (per gene or CRY gene) is very important for the formation of biological clock. In the study of biological clock, the importance of PER protein and CRY protein being regarded as a PER complex to study per protein is widely accepted. Although it has long been discovered that CRY1 gene and CRY2 gene can lead to different circadian cycle, the mechanism of CRY gene regulating clock cycle is not well studied. Although by far the stability model of CRY protein is the most widely accepted model to explain the circadian clock cycle regulated by CRY protein. In our experiment, we first found that the expression of CRY2 gene driven by endogenous CRY1 gene promoter could also restore the clock phenotype of CRY gene deletion cell line. In the analysis of the stability of CRY protein, we found that although the stability of CRY2 protein is stronger than that of CRY1 protein with less stability, it shows a short cycle circadian rhythm. This contradicts previous models for the stability of CRY proteins to regulate the circadian clock cycle. We also found that the N-terminal sequence of CRY2 protein determines that CRY2 protein is more stable than CRY1 protein, and the number of lysine residues in CRY2 protein and CRYI protein may also be the determinants of protein stability. The protein stability determined by the N-terminal sequence of CRY2 protein does not directly affect the cycle length of the clock, but the C-terminal sequence, which can not affect the stability of the CRY1 protein and the CRY2 protein, determines the cycle length of the clock. Finally, we also found the close relationship between CRY1 protein and P-TEFb(positive transcriptional elongation factor b) complex and 7SK snRNP small nuclear ribonucleoprotein complex) complex, which revealed the possible mechanism of CRY protein exerting transcriptional inhibitory activity through 7SK RNA. Our research supports the long-established hypothesis that the circadian cycle is regulated by the ratio of CRY1 protein to CRY2 protein. To be more precise, our experiments show that the cycle length of the biological clock is determined not by a single PER protein or CRY protein, but by the nucleation rate of the PER complex composed of these two proteins. At the same time, our study suggests that mammalian CRY1 protein may be transcriptional inhibitory by targeting inactivated P-TEFb complexes. Our study explains the periodic regulation mechanism and negative feedback inhibition mechanism of biological clock, and completes the model of biological clock negative feedback loop.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q953

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