CLOCK/BMAL1调控脱腺苷酶Nocturnin转录的机制研究及钟控microRNA的鉴定

【作者】 李然；

【导师】 曹济民； 彭小忠；

【作者基本信息】 中国协和医科大学 ， 生理学， 2008， 博士

【摘要】 Nocturnin是具有生物节律转录特点的基因之一,在RNA水平具有明确的日节律变化,夜间呈现高表达状态,故得名Nocturnin(黑夜因子)。Nocturnin蛋白质所具有的结构域从酵母到人都具有高度的保守性。Nocturnin功能研究发现它具有脱腺苷酶(deadenylase)的作用,参与mRNA的降解以终止翻译过程。Nocturnin基因敲除小鼠的脂肪积累能力和野生型对照相比明显下降,当饲喂高脂饮食时,基因敲除小鼠对肥胖和脂肪变性的抵抗能力明显强于野生型对照小鼠,并且基因敲除小鼠的脂肪、肌肉和肝脏的胰岛素敏感性明显增强,而胰腺的葡萄糖反应性下降。可见在正常情况下,Nocturnin的作用在于调节代谢的昼夜节律性变化。肥胖和高血脂在世界范围内都是公认的影响人类健康的疾病并且受到广泛的关注。Nocturnin表达调控的研究对于正确认识和防治肥胖和高血脂、以及与Nocturnin有关的一些未知生理功能有重要意义。尽管Nocturnin作为钟控基因是分子生物钟调控细胞代谢功能的一个重要因子,但分子钟调控Nocturnin的机制尚不十分清楚。在爪蟾的Nocturnin转录调控研究中发现,该基因的日节律表达是通过Nocturnin元件(Nocturnin element,NE)(GTGACGTG)由磷酸化的CREB调控的结果。然而,Oishi等人的小鼠CLOCK敲除模型的芯片检测结果显示,Nocturnin与period1等钟基因一样,节律表达受到了影响。这说明高等动物的Nocturnin的节律表达可能不同于爪蟾,而可能是由CLOCK/BMAL1异二聚体通过结合于E-box而激活转录。但他们并没有证实这种可能性。我们的此项研究工作的主要目的是阐明Nocturnin的转录激活是否是由CLOCK/BMAL1异二聚体通过结合于E-box所致。本工作分析了Nocturnin启动子区的序列。结果显示,Nocturnin的转录调控可能由E-box参与,即CLOCK/BMAL1直接通过E-box元件调控Nocturnin的转录。然后,我们进一步采用染色体免疫共沉淀技术发现Nocturnin的转录可能直接受到了CLOCK/BMAL1的调控。本部分研究利用Huh7细胞节律模型进行了Nocturnin转录水平的检测。在72小时的连续检测中,发现人Nocturnin和钟基因Period1的转录水平呈现十分相近的变化趋势,即在CT16-20,CT40和CT68(CT,circadian time)三个时间点达到峰值,在CT24-32两个时间点达到谷值。这说明人Nocturnin的转录受到了内源性钟基因的调控,并且调控方式和人Periodl可能相同。这一现象与Nocturnin生物信息学预测结果一致。为了进一步对Nocturnin启动子区E-box的功能进行检测,我们首先利用荧光素酶报告基因检测技术验证了CLOCK/BMAL1具有对人Period1和Nocturnin的E-box的转录激活能力,然后对两类E-box(E-box1和E-box2)进行突变操作,再检测E-box对Noctunin启动子区的转录激活能力,发现Nocturnin启动子区的E-box2的转录激活能力要比E-box1更为明显。最后通过DNA亲和层析,验证了不同E-box与CLOCK/BMAL1的结合能力,结果显示E-box2的亲和能力要比E-box1更为明显,这些结果说明E-box是CLOCK/BMAL1调控Nocturnin转录的重要顺式作用元件;人Nocturnin的转录与爪蟾Nocturnin的转录机制不同的是CLOCK/BMAL1可以通过E-box元件调控其转录;Nocturnin启动子区的E-box2的作用要比E-box1更为明显。此外,依据节律基因调控机制中钟控基因很大程度上依赖于转录因子CLOCK直接通过E-box元件激活转录。而Pri-microRNA的转录调控和基因的调控机制是相同的,所以我们认为应该存在具有钟控基因一样的转录调控机制的钟控microRNA。我们根据John Kim等人在人、小鼠和大鼠脑组织中特异表达microRNA的鉴定结果选择了86个microRNA,进行转录调控区域E-box元件分析,然后根据E-box元件在人、小鼠和大鼠中的同源性挑选出候选的钟控microRNA。再进一步进行染色体免疫共沉淀验证,并在小鼠转录模型中验证这些microRNA的表达变化。从而我们发现了三个受到CLOCK调控的钟控microRNA(miR140、miR329和miR337)进一步研究参与到分子生物钟的microRNA的调控机制和功能提供了基础。本研究中,我们筛选到钟控基因Nocturnin,并证明了Nocturnin接受CLOCK/BMAL1的转录调控;并且依据CLOCK调控的分子钟的转录机制鉴定了三个钟控microRNA(miR140、miR329和miR337)。更多还原

【Abstract】 Nocturnin is one of the circadian genes which show day-night oscillation in transcription.The name ’nocturnin’ came from the phenomenon of its high level expression at night and it is conservative from yeast to human.The certified function of nocturnin is still needed to be explored,although recent studies indicate that it may degrade mRNA as a deadenylase.Nocturnin knockout mice showed reductions in liver lipid accumulation(steatosis) compared with wild-type mice and,after high-fat feeding;they are resistant to obesity and steatosis.Nocturnin,a RNA deadenylase,may underlie the coregulation of circadian rhythms and metabolism.Obese and hyperlipemia influence human health around the world,and receive the widespread attention.The research of Nocturnin is important for people to understand and solve to the influence of obese and hyperlipemia.Nocturnin is involved in lipogenesis,lipid catabolism,bile acid synthesis,and gluconeogenesis,a series of studies should be undertaken to investigate whether Nocturnin is a clock-controlled gene.In Xenopus retina,high expression of nocturnin at night is regulated by phosphorylation of cAMP response element-binding protein through binding to the NE of the nocturnin promoter.Nocturnin transcript levels are greatly reduced in mice homozygous for a hypomorphic allele Clock.The night-time peak transcription of nocturnin in a mouse as well as the disruption of rhythmic expression of nocturnin in mouse homozygous for a hypomorphic allele Clock suggest nocturnin is controlled by CLOCK directly or indirectly.Based on our sequence analyses of E-box elements in the nocturnin promoter regions of human and the result from Oishi’s laboratory,we suggest that the E-box could be involved in the regulation of nocturnin transcription in higher species.In Huh7 cells,we verified that CLOCK/BMAL1 bound to the canonical E-boxes in the promoter of human nocturnin in vivo.We demonstrated in this study that the transcription of human nocturnin displayed circadian oscillations in Huh7 cells(a human hepatoma cell line).The results indicate that hNoc and hPerl have similar oscillation in Huh7 cells.During the 72 hour observation period,hPerl and hNoc transcript levels showed three peaks at CT16-20,CT40 and CT68, respectively,and two toughs at CT24-32 and CT48-56.The results indicate that hNoc is regulated by molecular clock in Huh7 cells.To validate the activity hNoc E-boxes,a luciferase reporter system was used.The results suggested that the E-box2 of nocturnin might play a major role in circadian transcription driven by CLOCK/BMAL1 heterodimer.To further identify the relative importance of E-box 1 and E-box2 in the regulation of nocturnin transcription,the binding affinities of CLOCK/BMAL1 to the E-boxes 1 and 2 of nocturnin were examined by a series of DNA pull-down assays.Our results showed that human nocturnin transcription was regulated by CLOCK/BMAL1 via E-box.E-box2 was more efficient in binding CLOCK/BMAL1 than E-box1,suggesting that E-box2 may take a dominant role in mediating the regulation of CLOCK/BMAL1 on nocturnin transcription.In addition,base on these studies,we choosed 86 microRNAs which express in mammalian brain and searched the E-box elements in the regions around the microRNAs in genome sequences which come from the Ensembl database (http://www.ebi.ac.uk/ensembl).The results showed that E-box elements exist in regions around the genomic sequences of the 18 microRNAs.By blasting these genomic sequences in rat and human,we found those 12 E-boxes and their flanking sequences showed high identity.We then identified the binding between the predictive elements and transfactors(CLOCK and BMAL1) by useing chromatin immunoprecipition assay(CHIP) and transcription oscillation of these microRNAs by realtime PCR assay in running-wheel mice model.We found three clock-controlled microRNAs.In this study,we screened the clock-controlled genes and identified the transcription of nocturnin controlled by CLOCK/BMAL1.Base on the trascrptional mechnism of molecular clock,we identified the three microRNAs(miR140,miR329 and miR337).更多还原