利用CRISPR/Cas9系统检测果蝇细胞中组蛋白甲基化修饰对20E信号传导的调控

Regulation of histone methylation modification in 20E signaling transduction detected by CRISPR/Cas9 system in Drosophila cells

【目的】利用CRISPR/Cas9基因敲除系统在黑腹果蝇Drosophila melanogaster细胞中筛选并检测组蛋白甲基化修饰对蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone,20E)信号传导的调控。【方法】通过Flybase网站分析并总结黑腹果蝇组蛋白甲基转移酶及其修饰位点;将5个组蛋白甲基转移酶基因[trr,Mes-4,ash1,Su(var)3-9和egg]的sgRNA插入到敲除载体pAc-sgRNA-Cas9骨架中并转染黑腹果蝇Kc细胞,利用TA克隆和测序鉴定突变位点。以Trr正调控20E初级应答基因Br-C的转录表达为阳性对照,利用qRT-PCR和Western blot检测该系统敲除靶基因的有效性;通过检测20E应答元件(20E response element,EcRE)双荧光素酶活性确定trr,Mes-4,ash1,Su(var)3-9和egg是否参与20E信号传导。【结果】果蝇组蛋白甲基化修饰主要发生在组蛋白H3的赖氨酸残基上,H3的精氨酸残基和组蛋白H4的甲基化修饰较少。trr敲除载体pAc-trr-sgRNA-Cas9转染Kc细胞后成功突变trr,降低H3K4三甲基化水平并阻断20E对其初级应答基因Br-C的转录诱导,证明该敲除系统的有效性。除trr之外,Mes-4和egg的敲除降低EcRE的双荧光素酶活性。【结论】插入靶标基因sgRNA序列的pAc-sgRNA-Cas9敲除载体在果蝇Kc细胞中可以有效快捷地突变靶基因。利用该敲除系统发现,除Trr之外,Mes-4和egg影响EcRE的活性而参与20E信号传导。本研究为昆虫细胞CRISPR/Cas9介导的基因敲除和组蛋白甲基化修饰调控20E信号传导的深入研究提供了理论依据与工作基础。

【Aim】To screen and detect the regulation of histone methylation modification in 20-hydroxyecdysone(20E)signaling transduction by CRISPR/Cas9 knockout system in Drosophila melanogaster cells.【Methods】Histone methyltransferases of D.melanogaster and their modification sites were analyzed and summarized from Flybase website.After transfection of the constructed knockout vector pAc-sgRNA-Cas9 inserted with the sgRNA of five histone methyltransferase genes[trr,Mes-4,ash1,Su(var)3-9 and egg]into Kc cells of D.melanogaster,the gene mutation was detected by TA cloning and sequencing.Taking Trr inducing 20E primary response gene Br-C as a positive control,qRT-PCR and Western blotting were used to test the validity of pAc-sgRNA-Cas9 knockout system.The dual luciferase assay of 20E response element(EcRE)was used to determine whether trr,Mes-4,ash1,Su(var)3-9 and egg participate in 20E signaling transduction.【Results】Drosophila histone methylation modification occurs mainly on histone H3 lysine residues and less on H3 arginine residues.Besides,there was less methylation modification on histone H4.After the transfection of its knockout vector pAc-trr-sgRNA-Cas9 in Kc cells,trr was mutated successfully,the tri-methylation level of H3K4 was reduced,and the 20E-induced transcriptional level of its primary response gene Br-C was inhibited,indicating the validity of this knockout system.Besides trr,Mes-4 and egg knockouts also reduced the dual luciferase activity of EcRE.【Conclusion】The pAc-sgRNA-Cas9 knockout system inserted with sgRNA of target gene is effective and fast for gene mutation in Drosophila Kc cells.Using this knockout system,in addition to Trr,Mes-4 and egg were found to participate in the regulation of 20E signaling transduction via manipulating the activity of EcRE.This study lays the theoretical basis and work foundation for CRISPR/Cas9-mediated gene knockout in insect cells and further studying histone methylation modification involved in regulating 20E signaling transduction.