摘要
远端增强子对关键靶基因的表达调控通常可以决定细胞的命运和功能,激活的增强子可以双向转录产生长非编码(long noncoding)增强子RNA(enhancer RNA,eRNA)调控靶基因表达,课题组前期研究发现增强子eRNA能够通过形成R环(R-loop)来促进增强子与靶基因的染色质远距离互作,引起局部三维基因组TAD(topologically associated domain)的改变。为了进一步探究eRNA在基因转录过程中的生物学功能,本研究选取原钙粘蛋白(protocadherin,Pcdh)基因簇的增强子eRNA PEARL(Pcdh eRNA associated with R-loop formation)作为研究对象,通过CRISPR(clustered regularly interspaced short palindromic repeats)DNA片段编辑技术、逆转录PCR、荧光定量PCR等遗传学和分子生物学实验,揭示了增强子eRNA PEARL对Pcdhα基因簇表达的促进作用。首先,本研究通过分析不同组织中HS5-1增强子eRNA发现其表达具有组织特异性;其次,通过CRISPR诱导HS5-1增强子内CTCF结合位点的反转或缺失会造成增强子eRNA PEARL转录水平降低至2%~10%,同时Pcdhα基因簇的转录水平也会降低至原来的13%~68%;最后,利用CRISPR DNA片段编辑技术删除HS5-1 eRNA双向转录起始位点或反转eRNA PEARL的转录起始位点后,Pcdhα基因簇的转录水平下降了约60%或40%,表明HS5-1增强子eRNA在调控基因表达中发挥重要作用。以上研究结果进一步证实了HS5-1增强子的转录产物可以调控Pcdhα基因簇的表达,为后续研究原钙粘蛋白基因簇在大脑中的表达调控机制提供了新的思路或方向。
The regulation of target genes by distal enhancers usually determines the fate and function of cells.Active enhancers in specific regions of chromatin may transcribe bidirectionally to produce long non-coding enhancer RNA(eRNA)to regulate gene expression.We recently found that an antisense enhancer eRNA PEARL(Pcdh eRNA associated with R-loop formation)regulates gene expression of members of the Pcdhαcluster via R-loop formation.To further explore the biological function of eRNA,we performed additional genetic and molecular experiments such as CRISPR(clustered regularly interspaced short palindromic repeats)DNA-fragment editing,RT-PCR,and qPCR.First,we performed expression analyses of the HS5-1 eRNA PEARL and found that it was expressed in a tissue-specific manner.In addition,upon CRISPR DNA-fragment deletion or inversion of the CTCF sites in the HS5-1 enhancer region,the expression of eRNA PEARL was reduced to 2%-10%and the expression of Pcdhαgene cluster was also reduced to 13%-68%of the original levels.Finally,deletion of the bidirectional transcription start site(TSS)of HS5-1 eRNA or inversion of TSS of the eRNA PEARL resulted in approximately 60%or 40%decrease of levels of Pcdhαgene expression.In summary,these data suggested a functional role of the HS5-1 eRNA in gene regulation of the Pcdhαcluster,providing a new direction for future researches on the regulatory mechanisms of clustered Pcdh gene expression in the brain.
作者
徐思远
寿佳
吴强
Siyuan Xu;Jia Shou;Qiang Wu(Center for Comparative Biomedicine,Key laboratory of Systems Biomedicine(Ministry of Education),Institute of Systems Biomedicine,Shanghai Jiao Tong University,Shanghai 200240,China)
出处
《遗传》
CAS
CSCD
北大核心
2022年第8期695-707,共13页
Hereditas（Beijing)
基金
国家自然科学基金项目(编号:31630039,91940303)
上海市科学技术委员会项目(编号:19JC1412500,21DZ2210200)资助。
