一个新的HMT家族成员SET07的理化性质及其功能

近年来发现组蛋白的甲基化可以改变染色体的状态,进而调节基因的转录、细胞周期、个体发育以及肿瘤发生.对本实验室新近克隆的组蛋白甲基转移酶基因家族新成员SET07理化性质,是否具有甲基转移酶的功能进行初步研究.构建pGEX4T2P2RP2质粒,并对其进行表达和纯化;利用硝纤膜及原核表达产物免疫昆明鼠制备抗SET07多克隆抗体;采用Western印迹对其蛋白水平的表达进行观察;利用HMT(组蛋白甲基转移酶)实验观察表达纯化的SET07蛋白是否具有甲基转移酶的功能.经大肠杆菌表达及纯化获得了较纯的GSTSET07片段,制备获得的多克隆抗体效价高于1∶1000;Western印迹发现SET07蛋白存在于胞核及胞浆.其中在胞核中以49kD存在,在胞浆中SET07以4种形式存在;HMT实验证明,表达纯化的SET07蛋白具有组蛋白甲基转移酶的功能.上述结果提示,SET07可能在合成后经过加工修饰以复合体的形式在胞核内发挥甲基转移酶作用,为进一步研究SET07基因的作用机制、SET07与个体发育、肿瘤发生之间的关系奠定基础.

A Novel Member SET07 from Histone Methyltransferases(HMT) Family

Histone methyltransferases(HMT)are the recently characterized members of the N-terminal histone tail modifying enzymes. The catalytic motif was mapped to conserve evolutionarily SET (suppressor of variegation,enhancer of zeste and trithorax) domain.Subsequently,other SET domain containing proteins were shown to harbor HMT activity towards specific histone substrates to negatively or positively regulate gene expression.We previously cloned a novel histone methyltransferase,SET07.The characterization and function of this gene was carried out.GST-tagged vector pGEX-4T-2-P2RP2 was constructed and recombinant SET07 protein was purified by affinity chromatography.The multiclonal antibody was obtained by immuning Kunming mouse with the SET07 protein and testifying the effective titer 1∶1 000.Western blot analysis found that compared with only one form of 49 KD in nuclei,SET07 had four isoforms in cytoplasm.HMT assay indicated that SET07 might function as histone methyltransferase in nuclei after modification,which contributed to the understanding of the molecular role of SET07.