PU.1 is essential for p47(phox) promoter activity in myeloid cells
PU.1 is essential for p47(phox) promoter activity in myeloid cells
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DOI：
10.1074/jbc.272.28.17802
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发表时间：
1997-07-11
影响因子：
4.8
通讯作者：
Clark, RA
中科院分区：
文献类型：
--
作者：
Li, SL;Valente, AJ;Clark, RA
吞噬细胞胞质蛋白p47（phox），NADPH氧化酶的一个组成部分，主要局限于骨髓细胞的表达。为了研究p47（phox）基因表达的顺式元件和反式作用因子，我们克隆并鉴定了p47（phox）启动子，在翻译起始密码子上游21个核苷酸处发现了一个主要的转录起始位点。用一系列5 ′-缺失p47（phox）-荧光素酶报告基因构建体进行HL-60人骨髓细胞的瞬时转染，所述构建体延伸至--224和-86构建体具有最强的p47（phax）启动子活性，而-46构建体显示出活性的主要降低，而-36构建体活性完全丧失。DNA酶I足迹分析鉴定了从-57至-53的受保护区域。该区域含有一个共识PU.1位点特异性结合两个PU，I存在于骨髓细胞的核提取物和PU.1在体外合成。该位点的突变消除了PU.1结合并消除了p47（phox）启动子指导报告基因表达的能力。p47（phox）启动子在所有测试的髓样细胞系（HL-60、THP-1、U937、PLB-985）中有活性，但在非髓样细胞（HeLa、HEK 293）中无活性。最后，PU.1在HeLa细胞中反式激活p47（phox）-荧光素酶构建体。我们得出结论，与某些其他骨髓特异性基因类似，骨髓细胞中的p47（phox）启动子活性需要PU.1。
Expression of the phagocyte cytosolic protein p47(phox), a component of NADPH oxidase, is restricted mainly to myeloid cells. To study the cis elements and trans acting factors responsible for its gene expression, we have cloned and characterized the p47(phox) promoter, A predominant transcriptional start site was identified 21 nucleotides upstream of the translation initiation codon, To identify the gene promoter sequences, transient transfections of HL-60 human myeloid cells were performed with a series of 5'-deletion p47(phox)-luciferase reporter constructs that extended as far upstream as -3050 bp relative to the transcriptional start site, The -224 and -86 constructs had the strongest p47(phax) promoter activity, whereas the -46 construct showed a major reduction in activity and the -36 construct a complete loss of activity. DNase I footprint analysis identified a protected region from -57 to -53. This region containing a consensus PU.1 site bound specifically both PU,I present in nuclear extracts from myeloid cells and PU.1 synthesized in vitro. Mutations of this site eliminated PU.1 binding and abolished the ability of the p47(phox) promoter to direct expression of the reporter gene, The p47(phox) promoter was active in all myeloid cell Lines tested (HL-60, THP-1, U937, PLB-985), but not in non-myeloid cells (HeLa, HEK293). Finally, PU.1 transactivated the p47(phox)-luciferase constructs in HeLa cells, We conclude that, similar to certain other myeloid specific genes, p47(phox) promoter activity in myeloid cells requires PU.1.