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Characterization of the promoter region of the human PARG gene and its response to PU.1 during differentiation of HL-60 cells

机译:人类PARG基因启动子区的特征及其在HL-60细胞分化过程中对PU.1的反应

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摘要

The metabolism of poly(ADP-ribose) plays important roles in the nuclear function of mammalian cells. Previously, we analyzed expression of the poly(ADP-ribose) glycohydrolase (PARG) gene during HL-60 cell differentiation and found that expression was greatly reduced by 4 h after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and returned to the initial level within 20 h. In the present study, a 2.1-kb fragment of the 5'-flanking (promoter) region of the human PARG gene was isolated from the HL-60 genome by polymerase chain reaction and ligated into a luciferase-expression vector, pGL3, to generate the pPARG-Luc#2 reporter plasmid. Deletion analysis revealed that a 75-nt sequence is required for basal promoter activity and TPA responsiveness. Mutations in this 75-nt sequence reduced promoter activity and the TPA response of HL-60 cells. TFSEARCH analysis revealed that Ets family binding motifs are located in the 75-nt sequence. Chromatin immunoprecipitation assay, electrophoretic mobility shift assay and competition analysis indicated that PU.1 (Spi-1) binds to the 75-nt sequence. Moreover, co-transfection of HL-60 cells with a PU.1 expression plasmid and pPARG-Luc indicated that PU.1 down-regulate the PARG promoter. These results suggest that PARG gene expression is modulated by PU.1 during TPA-induced differentiation of HL-60 cells.
机译:聚(ADP-核糖)的代谢在哺乳动物细胞的核功能中起重要作用。以前,我们分析了HL-60细胞分化过程中聚(ADP-核糖)糖水解酶(PARG)基因的表达,发现12-O-十四烷酰-phorbol-13-乙酸盐(TPA)处理后4 h表达大大降低。并在20小时内恢复到初始水平。在本研究中,通过聚合酶链反应从HL-60基因组中分离了人类PARG基因5'侧翼(启动子)区域的2.1 kb片段,并将其连接到荧光素酶表达载体pGL3中以产生pPARG-Luc#2报告质粒。缺失分析表明,基础启动子活性和TPA响应性需要75-nt序列。此75 nt序列中的突变降低了HL-60细胞的启动子活性和TPA反应。 TFSEARCH分析显示,Ets家族结合基序位于75 nt序列中。染色质免疫沉淀测定,电泳迁移率变动测定和竞争分析表明PU.1(Spi-1)绑定到75 nt序列。此外,用PU.1表达质粒和pPARG-Luc共转染HL-60细胞表明PU.1下调了PARG启动子。这些结果表明在TPA诱导的HL-60细胞分化过程中,PU.1调节了PARG基因的表达。

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