RFX5调控原钙粘蛋白α基因簇的表达  

RFX5 regulates gene expression of the Pcdhαcluster

作　　者：王娜 甲芝莲 吴强 Na Wang;Zhilian Jia;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)

机构地区：[1]上海交通大学系统生物医学研究院比较生物医学研究中心,系统生物医学教育部重点实验室,上海200240

出　　处：《遗传》2020年第8期760-774,共15页Hereditas（Beijing)

基　　金：国家自然科学基金项目(编号:91640118,31630039)资助。

摘　　要：基因的表达调控与基因组在细胞核内的三维空间架构相辅相成,原钙粘蛋白(protocadherin,Pcdh)基因簇在大脑发育中起到关键作用,可以作为研究基因表达调控机制的模式基因。转录因子RFX5(regulatory factor x 5)是翼螺旋家族(winged HLH family)的成员,其蛋白由寡聚化结构域、DNA结合域、螺旋结构域和激活域组成,在调控免疫系统的主要组织相容性复合物Ⅱ类(major histocompatibility complex classⅡ,MHCⅡ)的表达中起着至关重要的作用。本研究发现RFX5与CTCF在全基因组上结合的位点有部分重叠,利用CRISPR/Cas9 DNA大片段编辑技术,构建了RFX5基因缺失的HEC-1-B细胞系。通过RNA-seq实验,发现RFX5敲除能够显著升高Pcdhα6、Pcdhα12、Pcdhαc2的表达水平。通过ChIP-nexus实验,发现敲除RFX5导致染色质架构蛋白CTCF和cohesin在原钙粘蛋白α基因簇处的结合增加。最后,染色质构象捕获QHR-4C实验发现Pcdhα6、Pcdhα12启动子与远端增强子HS5-1的染色质远距离相互作用增强。上述研究表明RFX5蛋白可能通过调控染色质高级结构影响原钙粘蛋白α基因簇的表达,为未来进一步探索RFX5的功能提供了参考。Gene expression and three-dimensional(3D)genome organization are thought to be closely related.The protocadherin(Pcdh)clusters are central for neuronal self-avoidance in brain development and have been used as model genes to explore the role of 3D genome structures in gene regulation.Transcription factor RFX5(regulatory factor x 5)is a member of the winged-helix subfamily of the helix-turn-helix superfamily proteins.The RFX5 protein contains four domains:oligomerization,DNA-binding,helical,and transactivation domains.RFX5 plays an important role in regulating expression of the major histocompatibility complex class Ⅱ(MHC Ⅱ)gene complex.Here we report a role of RFX5 in the regulation of clustered Pcdh genes.We first knocked out the RFX5 gene by using CRISPR/Cas9 DNA-fragment editing in HEC-1-B cell line.By RNA-seq experiments,we found that RFX5 deletion results in a significant increase of expression levels of the Pcdhα6,Pcdhα12 and Pcdhαc2 genes.By ChIP-nexus experiments,we found that RFX5 deletion leads to the increased binding of CTCF and RAD21 in the Pcdhαcluster.Finally,through quantitative high-resolution chromosome conformation capture copy(QHR-4C)experiments,we found that RFX5 deletion results in a significant increase of long-distance chromatin interactions between the HS5-1 enhancer and its target promoters in the Pcdhαcluster.In conclusion,RFX5 regulates gene expression of the Pcdhαcluster through higher-order chromatin structure.

关 键 词：RFX5 Pcdh CTCF COHESIN CRISPR/Cas9 

分 类 号：Q786[生物学—分子生物学]