IRF4与MITF协同作用于络氨酸酶启动子的相关机制

Mechanism for synergistic effect of IRF4 and MITF on tyrosinase promoter

目的:探索干扰素调节因子4(interferon regulatory factor 4,IRF4)与小眼畸形相关转录因子(microphthalmia-associated transcription factor,MITF)协同作用激活络氨酸酶(tyrosinase,T YR)启动子的作用机制。方法:利用荧光素酶检测、细胞免疫荧光蛋白定位、GST融合蛋白沉降技术(GST-pull down)、免疫共沉淀等多种体外细胞实验学方法,观察IRF4与MITF的协同转录激活效应、亚细胞定位和蛋白-蛋白相互作用情况。结果:IRF4与MITF蛋白共同表达于细胞核;IRF4可以增强MITF在TYR启动子上的转录激活作用,产生协同效应,而在其他MITF目标启动子上不产生;IRF4不能单独激活TYR启动子,且与丧失功能的MITF突变蛋白在TYR启动子上不能产生协同作用;两个蛋白之间无直接相互作用。结论:IRF4与MITF在TYR启动子上产生协同作用,该效应主要依靠MITF进行调节;两个蛋白之间的相互作用可能需要DNA参与。

Objective： To investigate the mechanism for the synergistic eff ect of interferon regulatory factor 4 （IRF4） and microphthalmia-associated transcription factor （MITF） on tyrosinase （TYR） promoter. Methods： The synergistic transcriptional effect, subcellular localization, and protein-protein interaction for IRF4 and MITF were observed by luciferase assay, immunofluorescence, GST-pull down, and co-immunoprecipitation, espectively.Results： IRF4 and MITF proteins were co-expressed in the cell nucleus. IRF4 augmented the transcriptional function of MITF （but not the mutant MITF） to activate the expression of the TYR promoter, but with no effect on other MITF-specific target promoters. IRF4 alone did not affect TYR promoter significantly. No direct interaction between the two proteins was noted.Conclusion： IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF. This synergistic effect is mainly regulated by MITF; DNA might be involved in the interaction between the two proteins.