小鼠生物钟的分子机理研究

发布时间：2018-06-11 11:06

本文选题：生物节律 + 锌指蛋白ZBTB20　；参考：《南京大学》2016年博士论文

【摘要】：几乎所有的生物,从蓝藻到脊椎动物,都产生一套由内源性生物钟控制的生物节律。生物钟控制机体的生理和行为的节律,以适应周围环境的变化,从而赋予了生存的优势,比如天敌动物的回避,代谢过程的平衡,细胞周期调控,这表明更好地理解昼夜节律的具有重要的意义。分子生物钟是由偶联的正、负的转录／翻译反馈回路构成。正向因子CLOCK和BMAL1蛋白形成异源二聚体,结合在E-box顶式元件上,并激活Pers (Per1, Per2和Per3)和Crys (Cry1, Cry2)基因的转录。然后,PER和CRY蛋白通过与BMAL1-CLOCK复合物相互作用而抑制自己的转录。此外,核受体REV-ERBs和RORs通过竞争性结合在Bma11基因启动子上的RRE-box顺式元件上,控制Bma11的转录。哺乳动物生物钟的系统有三个组成部分：输入途径,中央起搏器视交叉上核(SCN),输出途径。我的博士毕业论文的第一部分,包括第2,3和4章,主要阐述锌指蛋白ZBTB20在生物节律中的作用。我们的研究首次发现锌指蛋白ZBTB20在调控小鼠双峰行为节律起着至关重要的作用。首先,通过基因敲除技术,得到在大脑敲除Zbtb20的小鼠。表型分析发现Zbtb20基因敲除小鼠缺失晚上的活动,而早上的活动有所增强。将小鼠放在不同光照黑暗周期里,Zbtb20基因敲除小鼠的牵引受损,尤其是表现在前半夜。此外,Zbtb2O基因缺失损害小鼠活动依赖的下游节奏,例如代谢和核心体温。其次,进一步研究发现Zbtb20缺陷小鼠表现出类似于Prok2, Prokr2基因敲除小鼠的表型。通过分子生物学和生物化学方法分析,发现：与同窝野生型小鼠相比,在Zbtb20基因敲除小鼠里,Prokr2基因的RNA水平和蛋白水平都是显着性下降。而且ZBTB20蛋白可以直接结合在Prokr2基因的启动子上,促进P300蛋白介导的Prokr2的表达。在视交叉上核过表达PROKR2能部分恢复Zbtb20基因缺失小鼠的表型,证明ZBTB20介导PROKR2信号通路参与维持晚上行为节奏,并发挥重要作用。最后,结合RNA测序和蛋白质谱鉴定的方法,我们在全基因组水平发现ZBTB20多种靶向途径,为探究神经退行性疾病和生物钟输出信号改变之间的关系提供线索。我的博士毕业论文的第二部分,第5章,主要在动物水平阐述CIPC蛋白在生物钟的作用。先前在细胞水平的研究认为CIPC蛋白作为新的负反馈调节元件,参与调控分子生物钟。然而,其在有机体内的功能未曾报道。在这个课题中,我们首次构建CIPC基因敲除小鼠,并分析其表型。我们的工作发现CIPC的缺失对分子生物钟有微弱的影响。CIPC基因敲除小鼠维持正常的生物节律。CIPC基因敲除小鼠保持正常的对光的反应。CIPC基因敲除小鼠的外周生物钟也没有变化。该工作说明CIPC对于生物钟不是必须的。
[Abstract]:Almost all organisms, from cyanobacteria to vertebrates, produce a set of biological rhythms controlled by endogenous biologic clocks. The circadian clock controls the circadian rhythm of the body's physiology and behavior to adapt to the changes in the surrounding environment, thus giving the survival advantage, such as the avoidance of the natural enemies, the balance of metabolic processes, and the regulation of cell cycles. A good understanding of the circadian rhythm is important. The molecular clock is composed of a coupled positive, negative transcription / translation feedback loop. The forward factor CLOCK and BMAL1 protein form a heterogenous two polymer, combined on the E-box top element, and activates the transcription of Pers (Per1, Per2 and Per3) and Crys (Cry1, Cry2) genes. Then, PER and CRY protein In addition, the nuclear receptor REV-ERBs and RORs control the transcription of Bma11 by competitive binding on the RRE-box cis element on the promoter of the Bma11 gene. The system of the mammalian clock has three components: the input pathway, the central pacemaker, the suprachiasmatic nucleus (SCN), and the transmission of the central pacemaker. The first part of my doctoral thesis, including chapters 2,3 and 4, mainly describes the role of zinc finger protein ZBTB20 in biological rhythms. Our study was the first to find that the zinc finger protein ZBTB20 plays a vital role in regulating the Shuangfeng behavioral rhythm in mice. First, by gene knockout technology, the brain knocks down Zbtb20. The mouse. Phenotypic analysis found that Zbtb20 gene knockout mice were absent in the evening activity and the activity in the morning increased. In the dark cycle of different light, the traction of Zbtb20 gene knockout mice was damaged, especially in the first half of the night. In addition, the Zbtb2O gene deletion damage the downstream rhythm of the activity dependent mice, such as metabolism and core body. Secondly, further study found that Zbtb20 deficient mice showed a phenotype similar to Prok2, Prokr2 knockout mice. By molecular and biochemical analysis, it was found that the RNA level and protein level of the Prokr2 gene were significantly decreased in the Zbtb20 gene knockout mice compared with the same nest wild type mice. And ZBT B20 protein can be directly combined with the promoter of the Prokr2 gene to promote the expression of P300 protein mediated Prokr2. The overexpression of PROKR2 in the suprachiasmatic nucleus can partially restore the phenotype of Zbtb20 gene deletion mice, proving that the ZBTB20 mediated PROKR2 signaling pathway is involved in maintaining night behavior rhythm and plays an important role. Finally, RNA sequencing and eggs are combined. The method of white mass spectrometry identification, we found a variety of ZBTB20 targeting pathways at the whole genome level, providing clues to explore the relationship between neurodegenerative diseases and biological clock output signals. The second part of my doctoral thesis, fifth chapters, mainly describes the role of CIPC protein in the biological clock in animal levels. The study suggests that CIPC protein, as a new negative feedback regulator, participates in the regulation of molecular biological clocks. However, its function in the organic body has not been reported. In this subject, we first constructed CIPC knockout mice and analyzed their phenotypes. Our work found that the deletion of CIPC has a weak impact on the molecular clock of the.CIPC gene knockout. In addition to maintaining normal biological rhythms in mice,.CIPC knockout mice maintain normal response to light, and the peripheral biological clock of.CIPC knockout mice has not changed. This work indicates that CIPC is not necessary for the biological clock.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q75

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