【作者】 刘娟；

【导师】 钟英斌；

【作者基本信息】 苏州大学 ， 遗传学， 2023， 硕士

【摘要】 生物钟是以大约24小时为周期的生物节律,控制生物体的生理和行为节律。这种节律是通过细胞自主振荡器建立且由转录-翻译的负反馈环路所控制。目前研究证明翻译后修饰机制是时钟蛋白必不可少的调节器,调控其转录活性、亚细胞定位和蛋白质稳定性。CBP(CREB binding protein,CREBBP)是目前研究广泛的组蛋白乙酰转移酶,CBP的突变会导致多种疾病。重要的是,CBP在调节生物钟节律方面发挥着重要作用,但是其作用机制目前尚未可知。另外,小鼠Cbp纯合突变体在出生后1周左右死亡,果蝇CBP同源基因nejire3(nej3)的缺失导致早期胚胎致死。这些都限制了 CBP在生物体内对生物钟作用的研究。因此,由于缺乏Cbp纯合突变体,其在体内参与生物钟的具体作用机制还不清楚。在斑马鱼中cbp有两个横向同源基因,分别是cbpa和cbpb。我们通过CRISPR-Cas9技术构建了两个cbp突变体,并获得能稳定遗传的品系:cbpa-/-和cbpb-/-。通过行为学分析,我们发现在正常的光暗条件下,cbpa-/-与cbpb-/-突变体的平均运动距离缩短,且这种差距主要发生在白天有光的情况;在持续的黑暗条件下,cbpa-/-与cbpb-/-突变体的行为节律均出现显著的相位延迟、周期缩短及振幅减弱。通过实时荧光定量PCR(qRT-PCR)实验发现在正常光暗条件和持续黑暗条件下斑马鱼幼鱼cbpa-/-与cbpb-/-突变体内钟基因per2、per1a和per1b的表达均下调且节律减弱。在成鱼脑、眼、肝脏、肌肉、肠5个组织中,钟基因表达有显著组织特异性。同时,在cbpa和cbpb基因敲除后,肝脏组织中三个钟基因表达的节律性、相位及振幅发生了显著改变。表明Cbp在外周生物钟的调控中也起重要作用。我们在Cbp调控生物钟的机制中研究发现,Cbpa和Cbpb既可以通过CREB(cAMP/Ca2+response element binding protein)结合 CRE 元件激活per2、per1a和per1b的表达,也可以与经典的生物钟核心蛋白Bmalb-Clock1a结合通过E-box刺激下游基因表达。进一步研究发现,Cbp通过CRE元件激活生物钟基因的表达必须有E-box的存在,而Cbp通过E-box激活生物钟基因的表达时CRE元件是非必需的。这些结果说明E-box元件的存在是Cbp调控生物钟的前提。CoIP实验也证实Cbpa和Cbpb能与Clock1a-Bmal1b以及Creb1a结合,从而调控生物钟。我们进一步研究发现,Cbpa能同时与Clock1a-Bmal1b以及Creb1a形成复合体。说明同时存在E-box和CRE元件时,Cbpa与Clock1a-Bmal1b以及Creb1a形成复合体,通过E-box和CRE元件协同调控钟基因表达,进而调控生物钟。CBP在哺乳动物的代谢调控中起重要作用,而我们的研究证明斑马鱼Cbp对生物钟也起重要的调控作用。那么,Cbp是否调控代谢节律呢?我们研究发现,在cbpa-/-斑马鱼突变体的肝脏组织中,糖代谢相关的糖原合成酶gys1(Glycogen synthase 1)、丙酮酸脱氢酶pdhα1a(Pyruvate dehydrogenase alpha 1)、磷酸果糖激酶pfkma(Phosphofructokinase)和乳酸脱氢酶ldha(Lactate dehydrogenase)表达的节律性与相位发生显著改变。另外,脂代谢通路中的关键基因,乙酰辅酶A 羧化酶 acaca(Acetyl-CoA carboxylase)、肉碱棕榈酰基转移酶cpt2(Carnitine Palmitoyltransferase Ⅱ)、脂肪酸结合蛋白fabp1(Fatty acid binding protein 1)、脂肪酸合成酶fasn(Fatty acid synthase)、甘油三酯酶pnpla2(Adipose triglyceride lipase 2)和硬脂酰辅酶A去饱和酶scd1(Stearoyl-CoA desaturase 1)表达的节律性和相位在cbpa-/-突变体肝脏组织中也发生显著变化。这些结果说明Cbp对肝脏糖脂代谢节律起重要的调控作用。综上,我们在斑马鱼中获得了cbpa-/-和cbpb-/-稳定遗传的品系,发现cbp的突变影响了生物钟基因的表达、运动行为节律以及外周生物钟。研究揭示了Cbp对生物钟的作用机制:E-box是Cbp参与生物钟调控必要元件,Cbp与Clock-Bmal结合调控E-box基因的表达,当同时存在CRE元件时,Cbp与Clock-Bmal以及Creb结合形成复合体,协同调控含E-box和CRE元件基因的表达。最后我们发现,Cbp的缺失影响了糖代谢与脂代谢的节律。更多还原

【Abstract】 The circadian clock is a biological rhythm with a period of approximately 24 hours that controls the physiological and behavioral rhythms of an organism.This rhythm is established by a cell-autonomous oscillator and controlled by a negative transcription-translation feedback loop.Current studies have shown that posttranslational modification is an essential regulator of clock proteins,which regulates their transcriptional activity,subcellular localization and protein stability.CREB-binding protein(CREBBP)is a histone acetyltransferase that has been widely studied.Mutation of CBP can lead to a variety of diseases.Importantly,CBP plays an important role in the regulation of circadian rhythm,but its mechanism of action is not well understood.In addition,mouse Cbp homozygous mutants die around 1 week after birth,and deletion of the Drosophila CBP homolog nejire3(nej3)causes early embryonic lethality.These have limited the study of the role of CBP on the circadian clock in living organisms.Therefore,due to the lack of a homozygous mutant of Cbp,the specific mechanism of action involved in the circadian clock in vivo remains unclear.There are two copies of the cbp gene in zebrafish,cbpa and cbpb.Two hereditable cbp zebrafish mutants,cbpa-/-and cbpb-/-,were generated by CRISPR-Cas9 technology.Through behavioral analysis,we found that under normal light and dark conditions(LD),the average movement distance of cbpa-/-and cbpb-/mutants was shortened,and this difference mainly occurred when light was present during daytime.Under constant darkness(DD),both cbpa-/-and cbpb-/-mutants showed significant phase delay,period shortening and amplitude reduction.Quantitative real-time PCR(qRT-PCR)showed that the expression of clock genes per2,per1a and per1b was down-regulated and the rhythm was weakened under both LD and DD conditions in cbpa-/-and cbpb-/-zebrafish larvae mutants.The expression of clock genes in adult fish brain,eye,liver,muscle and intestines showed significant tissue specificity.At the same time,after the knockout of cbpa and cbpb genes,the rhythm,phase and amplitude of the three clock genes were significantly changed in liver tissue.These results indicate that Cbp also plays an important role in the regulation of peripheral circadian clock.In our study,we found that Cbpa and Cbpb could not only activate the expression of per2,per1a and per1b through cAMP/Ca2+response element binding protein(CREB)bind to CRE element,but also bind to the classical clock core protein Bmalb-Clockla to stimulate downstream gene expression through E-box.Further studies showed that Cbp activation of clock gene expression through CRE element requires the presence of E-box,while the CRE element is dispensable for Cbp activation of clock gene expression through E-box.These results suggest that the presence of E-box elements is a prerequisite for Cbp to regulate the circadian clock.Co1P assay also confirmed that Cbpa and Cbpb interacted with Clockla-Bmallb and Creb1a to regulate the circadian clock.We further found that Cbpa could form a complex with both Clock1a-Bmal1b and Creb1a.These results suggested that Cbpa formed a complex with Clockla-Bmallb and Crebla in the presence of E-box and CRE elements to regulate clock gene expression through the co-ordination of E-box and CRE elements.Since CBP plays an important role in metabolic regulation in mammals,our results demonstrated that zebrafish Cbp also plays an important role in regulating the circadian clock.So,does Cbp regulate metabolic rhythms?We found that in the liver tissue of cbpa-/-mutants,glycogen synthase 1(gys1),pyruvate dehydrogenase alpha(pdh1a),phosphofructokinase(pfkma)and lactate dehydrogenase(ldha),involved in glucose metabolism,were down-regulated and the rhythm and phase were significantly changed.In addition,key genes in the lipid metabolism pathway,acetyl-CoA carboxylase(acaca),Carnitine Palmitoyltransferase Ⅱ(cpt2),fatty acid binding protein(fabpla),fatty acid synthase(fasn),adipose triglyceride lipase(pnpla2)and stearoyl-CoA desaturase 1(scd1)were also down-regulated in cbpa-/liver tissue,the rhythm and phase were also significantly changed.These results suggest that Cbp plays an important role in regulating the rhythm of glucose and lipid metabolism in the liver.In summary,we have generated stable cbpa-/-and cbpb-/-zebrafish lines and found that mutations of cbp affected clock gene expression,motor behavior rhythm and peripheral circadian clock.We also revealed the mechanism of Cbp action on the circadian clock:E-box is an essential element of Cbp involved in the regulation of circadian clock.Cbp binds with CLOCK-BMAL to regulate the expression of E-box containing genes.When CRE element is present,Cbp binds with CLOCK-BMAL and Creb to form a complex to co-regulate the expression of genes both containing E-box and CRE element.Finally,we found that Cbp deficiency affected the rhythms of glucose and lipid metabolism.更多还原