转录因子E2F1对急性单核细胞白血病新抗原基因MLAA-34的转录调控作用

Effects of transcription factor E2F1 on transcriptive regulation of acute monocytic leukemia -related gene MLAA-34

目的:探讨转录因子E2F1对急性单核细胞白血病新抗原基因MLAA-34的转录调控作用。方法:利用双荧光素酶报告基因检测系统及定点突变技术分析E2F1对MLAA-34基因启动子转录活性的影响。通过凝胶迁移实验(EMSA)和染色质免疫共沉淀(ChIP)实验,验证E2F1是否与MLAA-34启动子核心区直接特异性结合。构建E2F1真核表达载体和干涉载体,转染U937细胞,RT-PCR和Western Blot检测MLAA-34基因的转录和表达变化。结果:转录因子E2F1对MLAA-34基因表达具有调控作用,E2F1结合序列点突变后,相对荧光素酶活性升高(P<0.01),绿色荧光蛋白的表达增高。EMSA和ChIP实验,从细胞内、外水平分别证明E2F1可与MLAA-34启动子直接结合而发挥调控作用。在过表达试验中,E2F1的增加可下调MLAA-34的表达(P<0.05);在干涉试验中,E2F1的降低可上调MLAA-34的表达(P<0.05)。结论:转录因子E2F1可与MLAA-34基因启动子上的转录调控区结合,并抑制急性单核细胞白血病细胞中MLAA-34基因的转录。

Objective:To investigate the transcriptional regulation of transcription factor E2 F1 on acute monocytic leukemia-related gene MLAA-34.Methods:The effect of E2 F1 on the transcriptional activity of MLAA-34 gene promoter was analyzed by luciferase reporter gene detection system and site-directed mutation technique.EMSA and ChIP assay were used to verify whether E2 F1 directly and specifically binds to the core region of MLAA-34 promoter.The over-expression vector and interference vector of E2 F1 were constructed to transfect U937 cells,and RT-PCR and Western Blot were used to detect the transcription and expression changes of MLAA-34 gene.Results:The transcription factor E2 F1 had a regulatory effect on MLAA-34 gene expression,and the relative luciferase activity was increased after E2 F1 binding point mutation(P<0.01).EMSA and ChIP experiments demonstrated that E2 F1 can directly bind to MLAA-34 promoter and play a regulatory role.In the over-expression test,the increase of E2 F1 can down-regulate the expression of MLAA-34(P<0.05).In the interference test,the decrease of E2 F1 can up-regulate the expression of MLAA-34(P<0.05).Conclusion:Transcription factor E2 F1 can bind to the transcriptional regulatory region on the promoter of MLAA-34 gene and inhibit the transcription of MLAA gene in acute monocytic leukemia.