Zhang W, Zhang J, Kornuc M, Kwan K, Frank R, Nimer S D
Laboratory of Molecular Aspects of Hematopoiesis, Sloan-Kettering Institute, New York, New York, USA.
Mol Cell Biol. 1995 Nov;15(11):6055-63. doi: 10.1128/MCB.15.11.6055.
To isolate transcription factors important in the regulation of the human interleukin-3 (IL-3) gene, we screened a lambda gt11 cDNA library, constructed from phytohemagglutinin-stimulated human T-cell RNA, with a probe containing regulatory sequences in the upstream region of the IL-3 gene (located from bp -165 to -128 and referred to as the DNase I footprint A region). We isolated a 0.96-kb cDNA that encoded a basic amino acid domain and a leucine zipper domain and used the "rapid amplification and cloning of 3' ends" technique to isolate the 3' half of the cDNA clone, generating a 1.9-kb full-length cDNA clone. Using in vitro-translated protein, which we call NF-IL3A, we defined the IL-3 promoter sequences bound by NF-IL3A in DNase I footprinting assays as TAATTACGTCTG and, using gel shift assays, defined ATTACG as the minimal sequence required for binding of NF-IL3A in vitro. Proteins that bind to the NF-IL3A binding site are found in both unstimulated and stimulated T-cell lines in similar amounts, although the level of NF-IL3A mRNA increases after T-cell activation in several mature T-cell lines. The NF-IL3A protein is nearly identical to a recently identified transcriptional repressor protein, E4BP4, and NF-IL3A binds specifically to regulatory sequences in both the adenovirus E4 promoter and the human gamma interferon promoter. Cotransfection experiments demonstrate that introduction of an expression vector containing the NF-IL3A cDNA into resting T cells transactivates IL-3 promoter-chloramphenicol acetyltransferase gene plasmids that contain the A region; this effect requires the presence of an intact NF-IL3A binding site. One or more copies of the A region also confer NF-IL3A responsiveness on a heterologous promoter in T cells. NF-IL3A appears to play an important role in the expression of IL-3 by T cells.
为了分离在人类白细胞介素3（IL-3）基因调控中起重要作用的转录因子，我们用一个含有IL-3基因上游区域调控序列（位于-165至-128碱基对，称为核酸酶I足迹A区域）的探针，筛选了一个由植物血凝素刺激的人类T细胞RNA构建的λgt11 cDNA文库。我们分离出一个编码碱性氨基酸结构域和亮氨酸拉链结构域的0.96 kb cDNA，并使用“3'末端快速扩增和克隆”技术分离出该cDNA克隆的3'半段，得到一个1.9 kb的全长cDNA克隆。我们使用体外翻译的蛋白质（我们称之为NF-IL3A），在核酸酶I足迹分析中确定了NF-IL3A结合的IL-3启动子序列为TAATTACGTCTG，并通过凝胶迁移分析确定ATTACG为NF-IL3A体外结合所需的最小序列。在未刺激和刺激的T细胞系中，以相似的量发现了与NF-IL3A结合位点结合的蛋白质，尽管在几个成熟T细胞系中，T细胞活化后NF-IL3A mRNA的水平会增加。NF-IL3A蛋白与最近鉴定的转录抑制蛋白E4BP4几乎相同，并且NF-IL3A特异性地结合腺病毒E4启动子和人类γ干扰素启动子中的调控序列。共转染实验表明，将含有NF-IL3A cDNA的表达载体导入静止T细胞，可使含有A区域的IL-3启动子-氯霉素乙酰转移酶基因质粒反式激活；这种效应需要完整的NF-IL3A结合位点的存在。A区域的一个或多个拷贝也赋予T细胞中异源启动子对NF-IL3A的反应性。NF-IL3A似乎在T细胞IL-3的表达中起重要作用。
