MOLECULAR REGULATION OF THE HUMAN IL-3 GENE - INDUCIBLE T-CELL-RESTRICTED EXPRESSION REQUIRES INTACT AP-1 AND ELF-1 NUCLEAR-PROTEIN BINDING-SITES
MOLECULAR REGULATION OF THE HUMAN IL-3 GENE - INDUCIBLE T-CELL-RESTRICTED EXPRESSION REQUIRES INTACT AP-1 AND ELF-1 NUCLEAR-PROTEIN BINDING-SITES
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DOI：
10.1084/jem.178.5.1681
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发表时间：
1993-11-01
影响因子：
15.3
通讯作者：
EMERSON, SG
中科院分区：
文献类型：
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作者：
GOTTSCHALK, LR;GIANNOLA, DM;EMERSON, SG
白细胞介素3（IL-3）是一种造血干细胞生长和分化因子，仅在活化的T细胞和NK细胞中表达。迄今为止的研究已经鉴定了调控IL-3转录的IL-3编码序列的5'端元件，但是赋予T细胞特异性表达的序列仍有待明确定义。我们现在已经确定了T细胞限制性IL-3基因转录所需的DNA序列。在T细胞和非T细胞中用人IL-3-氯霉素乙酰转移酶（CAT）报告质粒进行的一系列瞬时转染显示，含有319 bp的5'侧翼序列的质粒仅在T细胞中有活性。缺失分析显示，T细胞特异性由49-bp片段（bp-319至-270）赋予，该片段包括在Ets家族转录因子Elf-1的结合位点上游6 bp处的AP-1转录因子的潜在结合位点。用MLA-144 T细胞核提取物进行的DNaseI足迹和电泳迁移率变动分析表明，该49 bp区域含有包括共有AP-1和Elf-1结合位点的核蛋白结合区。此外，从纯化的人T细胞制备的提取物含有与合成的寡核苷酸结合的蛋白质，所述寡核苷酸对应于AP-1和Elf-1结合位点。在体外转录和翻译的Elf-1蛋白结合特异性的Elf-1网站，和Elf-1抗血清竞争和超移动核蛋白复合物中存在的MLA-144核提取物。此外，在电泳迁移率变动测定反应中加入抗Jun家族抗血清完全阻断了AP-1相关复合物的形成。在MLA-144 T细胞中的瞬时转染研究揭示，在AP-1位点中含有突变的构建体几乎完全消除了CAT活性，而Elf-1位点或NF-IL-3位点（IL-3启动子中先前描述的核蛋白结合位点（bp -155至-148））的突变降低了CAT活性，
Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloremphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonudeotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp -155 to -148) in the IL-3 promoter, reduced CAT activity to