Lennartsson Andreas, Garwicz Daniel, Lindmark Anders, Gullberg Urban
Division of Hematology and Transfusion Medicine, Lund University, BMC, C14, S-221 84 Lund, Sweden.
Gene. 2005 Aug 15;356:193-202. doi: 10.1016/j.gene.2005.05.004.
Cathepsin G is a hematopoietic serine protease stored in the azurophil granules of neutrophil granulocytes. The mRNA of cathepsin G is transiently expressed during the promyelocyte stage of neutrophil maturation. The protease plays several roles in inflammatory actions of neutrophils, such as bactericidal effects. A human cathepsin G gene fragment of 6 kb directs a promyelocyte-specific expression in transgenic mice, indicating the presence of necessary cis-acting elements. However, neither the precise architecture of the promoter, nor the trans-acting factors responsible for its activation, have been characterized. In the present work, 2.6 kb upstream of the translation start site of the human cathepsin G gene was cloned. When transfected to monoblast-like U937 or to acute promyelocytic leukemia NB4 cells, both expressing endogenous cathepsin G, the initial 360 bp upstream of the translation start were sufficient to direct a strong expression of a luciferase reporter gene. No expression was observed in erythroid K562 control cells. Further deletions revealed three major regulatory regions containing the consensus binding-sites for the transcription factors C/EBP, c-myb and PU.1. Moreover, a GC-rich region, similar to a cis-element in the proteinase 3 promoter, was identified. Direct binding of the trans-factors C/EBPalpha, C/EBPepsilon, c-myb and PU.1 to the promoter was shown by chromatin immunoprecipitation. The functional significance of the cis-elements was verified by site-directed mutagenesis. Mutations of the putative PU.1 site moderately decreased the activity of the promoter in monoblastic U937 cells, but not in promyelocytic NB4 cells. Separate mutations of the putative C/EBP binding site, c-myb-binding site or the GC-rich element resulted in a dramatically reduced transcriptional activity in both cell lines, suggesting cooperation between corresponding trans-factors.
组织蛋白酶G是一种造血丝氨酸蛋白酶，储存于中性粒细胞的嗜天青颗粒中。组织蛋白酶G的mRNA在中性粒细胞成熟的早幼粒细胞阶段短暂表达。该蛋白酶在中性粒细胞的炎症反应中发挥多种作用，如杀菌作用。一个6kb的人组织蛋白酶G基因片段在转基因小鼠中指导早幼粒细胞特异性表达，表明存在必要的顺式作用元件。然而，启动子的确切结构以及负责其激活的反式作用因子均未得到表征。在本研究中，克隆了人组织蛋白酶G基因翻译起始位点上游2.6kb的片段。当转染到均表达内源性组织蛋白酶G的单核细胞样U937细胞或急性早幼粒细胞白血病NB4细胞时，翻译起始位点上游最初的360bp足以指导荧光素酶报告基因的强表达。在红系K562对照细胞中未观察到表达。进一步缺失揭示了三个主要调控区域，包含转录因子C/EBP、c-myb和PU.1的共有结合位点。此外，还鉴定出一个富含GC的区域，类似于蛋白酶3启动子中的顺式元件。染色质免疫沉淀显示反式因子C/EBPα、C/EBPε、c-myb和PU.1与启动子直接结合。通过定点诱变验证了顺式元件的功能意义。假定的PU.1位点的突变适度降低了启动子在单核细胞样U937细胞中的活性，但在早幼粒细胞NB4细胞中未降低。假定的C/EBP结合位点、c-myb结合位点或富含GC元件的单独突变导致两种细胞系中的转录活性显著降低，表明相应反式因子之间存在协同作用。
