BCL11A在神经细胞中稳定低表达对智能障碍风险基因的转录调控机制  

Transcriptional Regulation Mechanisms of Stable BCL11A Knockdown in Neuron on Risk Genes of Intellectual Disability

作　　者：唐小涵 陈金蓉 苏炳银[1] 李淑蓉[1,2] 解学敏 Tang Xiaohan;Chen Jinrong;Su Bingyin;Li Shurong;Xie Xuemin(Department of Histology and Embryology,Key Laboratory of Development and Regeneration of Sichuan Province,Chengdu Medical College,Chengdu 610500,China;Department of Pathology and Pathophysiology,Chengdu Medical College,Chengdu 610500,China)

机构地区：[1]成都医学院发育与再生四川省重点实验室组织胚胎学教研室,成都610500 [2]成都医学院病理学与病理生理学教研室,成都610500

出　　处：《成都医学院学报》2023年第3期278-286,共9页Journal of Chengdu Medical College

基　　金：四川省科技厅应用基础研究重点项目(No:2021YJ0018,No:2019YJ0366,No:2021YJ0169);成都医学院发育与再生四川省重点实验室基金重点项目(No:SYS18-01)。

摘　　要：目的 探究稳定敲降BCL11A对神经发育关键因子的表达影响。方法 设计并筛选靶向人源BCL11A的shRNAs,通过慢病毒构建能稳定敲降BCL11A的人神经母细胞瘤细胞株(SH-SY5Y细胞)。首先,利用逆转录荧光定量PCR(RT-qPCR)、蛋白质印迹技术实验体外验证BCL11A的敲降效果,RT-qPCR实验探索BCL11A敲降后皮质发育关键转录因子的表达变化,进而利用转录组测序分析BCL11A敲降后差异基因的富集情况,最终通过染色质免疫共沉淀(ChIP)实验验证差异基因启动子的转录活性标志pPolⅡ以及染色质活性修饰H3K9ac的变化。结果 BCL11A敲降后神经元特异转录因子GRM7、BCL11B、CTCF、ADCYAP1R1、NeuroD1/2、PAX6、SATB1/2的表达降低;皮质神经元关键转录因子GRIN1、TCF12、MEF2D的表达升高。转录活性标志pPolⅡ以及染色质活性修饰H3K9ac在GRM7启动子的富集度降低,在GRIN1启动子的富集呈现升高趋势。结论 转录因子BCL11A表达缺失可影响智能障碍相关转录因子的转录活性与表达变化。Objective To explore the effect of stable knockdown of BCL11A on the expression of key factors in neurodevelopment.Methods shRNAs targeting human BCL11A were designed and screened.Human neuroblastoma cell line(SH-SY5Y cells)that could stably knock down BCL11A was constructed by lentivirus.The knock-down effect of BCL11A was verified in vitro by reverse-transcription quantitative polymerase chain reaction(RT-qPCR)and Western blotting(WB).The expression changes of key transcription factors in cortical development after BCL11A knockdown were detected by RT-qPCR.The enrichment of differential genes after BCL11A knockdown was analyzed by transcriptome sequencing.The changes of the transcriptional activity marker pPolII and the chromatin modification factor H3K9ac of the differential gene promoters were verified by chromatin immunoprecipitation(ChIP).Results After BCL11A knockdown,the expressions of neural-specific transcription factors GRM7,BCL11B,CTCF,ADCYAP1R1,NeuroD1/2,PAX6 and SATB1/2 were down-regulated,while the expressions of key transcription factors GRIN1,TCF12 and MEF2D in cortical neurons were up-regulated.The enrichment of the transcriptional activity marker pPolII and the chromatin modification factor H3K9ac were down-regulated in GRM7 promoter while up-regulated in GRIN1 promoter.Conclusion Stably down-regulated BCL11A leads to changes in transcriptional activity and expression of transcription factors related to intellectual disability.

关 键 词：慢病毒 BCL11A CRISPR/Cas9 神经系统 

分 类 号：Q291[生物学—细胞生物学]