PU.1 regulates the expression of the Vav proto-oncogene
PU.1 regulates the expression of the Vav proto-oncogene
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DOI：
10.1002/jcb.10089
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发表时间：
2002-01-01
影响因子：
4
通讯作者：
Kawahara, RS
中科院分区：
文献类型：
--
作者：
Denkinger, DJ;Lambrecht, TQ;Kawahara, RS
Vav是rho/rac gtpase的鸟嘌呤核苷酸交换因子，在胚胎从原始造血到最终造血的转变过程中上调。它是与胚胎内决定性造血干细胞分化相关的几个遗传标记之一。随后，在成人中，vav主要在造血系统的细胞中表达。研究发现，一种与23-bp片段结合的耐热蛋白复合物存在于髓细胞中，而t细胞中不存在。这种复合物在通常不表达vav的非造血细胞中不存在。利用饱和诱变方法Mutex，获得了与PU.1一致的蛋白结合位点（AGAGGAAGT）的“足迹”。一种针对PU.1的特异性抗体超移了该复合体并鉴定出PU.1在复合体内的存在。人类PU.1的GST融合蛋白与来自髓系的耐热复合体具有相同的一致序列。通过EGFP报告基因的表达，GGAA PU.1核心结合位点的特异性突变使vav启动子活性沉默，显性的PU.1阴性抑制了PU.1在vav启动子上的反激活。此外，使用PU.1特异性抗体对免疫沉淀染色质进行PCR分析，检测到含有vav启动子的DNA的共免疫沉淀。这些结果表明，PU.1对髓细胞中vav启动子的转录活性至关重要。(C) 2002 Wiley-Liss, Inc。
Vav is a guanine nucleotide exchange factor for the rho/rac GTPases that is upregulated in the embryo during the transition from primitive to definitive hematopoiesis. It is one of several genetic markers that correlates with the differentiation of the intraembryonic definitive hematopoietic stem cell. Subsequently, in the adult, vav is expressed predominantly in cells of the hematopoietic system. A heat-resistant protein complex that bound to a 23-bp segment, which is essential for vav promoter activity, was found to be present in myeloid cells but not T-cells. The complex was absent in non-hematopoietic cells which normally do not express vav. Using a saturation mutagenesis method, Mutex, a "footprint" of the protein binding site (AGAGGAAGT) was obtained that was consistent with the consensus binding site for PU.1. A specific antibody to PU.1 supershifted the complex and identified the presence of PU.1 within the complex. A GST fusion protein of the human PU.1 bound to the same consensus sequence as the heat-resistant complex from myeloid lineages. Specific mutation of the GGAA PU.1 core binding site silenced vav promoter activity and a dominant negative PU.1 inhibited the transactivation of PU.1 at the vav promoter as measured by the expression of the EGFP reporter gene. In addition, PCR analysis of immunoprecipitated chromatin using specific antibodies for PU.1 detected the co-immunoprecipitation of DNA containing the vav promoter. These results suggest that PU.1 is essential for transcriptional activity of the vav promoter in myeloid cells. (C) 2002 Wiley-Liss, Inc.