Rad21基因位点的遗传修饰抑制骨骼肌干细胞分化  

Genetic Manipulation of Rad21 Gene Locus Compromises Differentiation of Skeletal Muscle Stem Cells

作　　者：刘勤瑶 高庆 张潜英 汪瑞婷 张勇[1,2] 朱大海 LIU Qinyao;GAO Qing;ZHANG Qianying;WANG Ruiting;ZHANG Yong;ZHU Dahai(Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences,School of Basic Medicine,Peking Union Medical College,Beijing 100005,China;Bioland Laboratory(Guangzhou Regenerative Medicine and Health Guangdong Laboratory),Guangzhou 510005,China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院,北京100005 [2]生物岛实验室,广州再生医学与健康广东省实验室,广州510005

出　　处：《中国细胞生物学学报》2023年第7期1058-1065,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金重大研究计划(批准号:91540206);“173”基础加强计划重点基础研究项目(批准号:2020-JCJQ-ZD-264)资助的课题。

摘　　要：细胞谱系分化受到遗传、表观遗传、三维基因组结构的复杂调控。在真核细胞三维基因组结构中,黏连蛋白(cohesin)复合物介导染色质成环和拓扑相关结构域的形成。为了探讨Cohesin复合物介导的染色质高级结构是否调控成体干细胞分化及其相关基因表达,该研究选择了Cohesin复合物的一个亚基RAD21,制备了可诱导型RAD21蛋白降解的基因敲入小鼠模型(auxininducible degron,AID)。当auxin存在时,泛素连接酶Os TIR1降解含有AID标签的RAD21蛋白。小鼠制备策略是:在Rad21基因3′端引入了Loxp-Stop-Loxp-AID-EGFP-P2A-Os TIR1元件(Rad21^(Aid/+)),与骨骼肌成体干细胞特异的Cre工具鼠(Pax7-Cre)配繁,通过auxin诱导,在骨骼肌成体干细胞中特异降解RAD21蛋白,从而探究RAD21蛋白缺失对骨骼肌成体干细胞分化及相关基因表达的影响。该研究取得意外的结果:Rad21基因敲入的纯合子小鼠(Rad21Aid/Aid,Hom)比野生型小鼠(WT)出生率显著降低;出生的Hom小鼠较WT小鼠的体型小且体质量轻;出生的Hom小鼠骨骼肌重量降低,骨骼肌纤维直径减小;Western blot定量分析表明,在没有配繁Pax7-Cre,也没有auxin诱导的条件下,与WT相比,Hom小鼠的骨骼肌干细胞中RAD21蛋白水平显著降低。以上研究结果表明:Rad21基因位点的遗传修饰导致RAD21蛋白表达水平显著降低。进一步分离骨骼肌干细胞诱导分化,与WT相比,Hom小鼠的骨骼肌干细胞分化能力显著减弱,这表明Rad21蛋白表达异常显著抑制骨骼肌干细胞分化。总之,该研究发现Rad21基因位点修饰导致其表达异常,进而影响小鼠发育和骨骼肌干细胞分化。Eukaryotic genome is dynamically organized to three-dimensional structure through hierarchical folding,which is important for regulating cell lineage differentiation during development.The cohesin complex is an essential regulator for chromatin looping and topological associated domain formation and consists of 4 subunits,one of which is the kleisin protein RAD21.To investigate whether cohesin-mediated high order chromatin structure regulates adult stem cell differentiation,an inducible Rad21 degron knockin mouse line was generated using AID(auxin-inducible degron)(Rad21-Loxp-Stop-Loxp-AID-EGFP-P2A-OsTIR1,Rad21^(Aid/+)).By crossing the Rad21^(Aid/+)mice with skeletal muscle stem cell specific Cre mice(Pax7-Cre),the RAD21 protein would be specifically de­graded in skeletal muscle stem cells once treated with auxin.However,protein levels of RAD21 were significantly decreased in skeletal muscle stem cells from the both homozygotes(Hom)of Rad21Aid/Aid and Pax7-Cre;Rad21Aid/Aid even in absence of auxin,compared to wild-type littermates(WT),suggesting genetic manipulation of the Rad21 gene locus leads to dysregulation of its expression.Phenotypically,the homozygous mice had lower birth rate,less body weight and muscle mass,and smaller myofiber diameters,compared with WT littermates.Moreover,the sig­nificant reduction of RAD21 protein in skeletal muscle stem cells of the homozygous mice lead to compromised myogenic cell differentiation,which might account for their abnormal muscle growth and development.Together,the findings suggest RAD21 is critical regulator of development and stem cell differentiation in mice.

关 键 词：黏连蛋白 Rad21 可诱导型蛋白降解子 骨骼肌干细胞 细胞分化 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学]