Lacy J, Roth G, Shieh B
Department of Medicine, Yale University, New Haven, CT 06520.
J Immunol. 1994 Dec 15;153(12):5537-48.
EBV infection of human B lymphocytes induces expression of the low affinity IgE receptor, Fc epsilon RII/CD23. CD23 is constitutively expressed in EBV-immortalized B cells and may play an essential role in immortalization. We previously explored the regulation of CD23 by EBV, showing that induction results from transcriptional activation that is mediated, in part, by an EBV-responsive transcriptional regulatory element in the 5' region of CD23 (-229 to +305 relative to the type a promoter). We now report the localization of the regulatory element and characterization of its cognate DNA-binding proteins. Reporter gene assays in EBV-positive and -negative lines localized a functional EBV-responsive enhancer to a 37-bp fragment (+248 to +284) that contains a GC-rich sequence (GC box) within intron I of type a CD23. This fragment was shown by mobility shift assays to specifically bind nuclear protein(s) from EBV-positive lines, but not EBV-negative lines. Mutation of the GC box resulted in a loss of protein-binding activity, implicating involvement of a GC box-binding protein in the DNA/protein interaction. Supershift assays suggested that the ubiquitous GC box-binding transcription factor, Sp1, is not a part of the complex, and UV-crosslinking studies demonstrated that the DNA/protein complex contains at least two proteins that differ in size from other known GC box-binding proteins. Binding of these proteins to the enhancer element requires phosphorylation, because phosphatase treatment of nuclear extracts abolished formation of the DNA/protein complex. These studies reveal the presence of an EBV-responsive enhancer element in intron I of type a CD23 and implicate a GC box-binding transcription factor in the activation of CD23 by EBV.
人类B淋巴细胞的EB病毒感染可诱导低亲和力IgE受体FcεRII/CD23的表达。CD23在EB病毒永生化的B细胞中组成性表达，可能在永生化过程中起关键作用。我们之前探讨了EB病毒对CD23的调控，发现诱导作用源于转录激活，部分是由CD23 5'区域（相对于a型启动子为-229至+305）中的一个EB病毒反应性转录调控元件介导的。我们现在报告该调控元件的定位及其同源DNA结合蛋白的特性。在EB病毒阳性和阴性细胞系中的报告基因分析将一个功能性的EB病毒反应性增强子定位到一个37bp的片段（+248至+284），该片段在a型CD23的内含子I中包含一个富含GC的序列（GC框）。通过迁移率变动分析表明，该片段可特异性结合EB病毒阳性细胞系的核蛋白，而不结合EB病毒阴性细胞系的核蛋白。GC框的突变导致蛋白结合活性丧失，这表明一种GC框结合蛋白参与了DNA/蛋白相互作用。超迁移分析表明，普遍存在的GC框结合转录因子Sp1不是该复合物的一部分，紫外线交联研究表明，DNA/蛋白复合物包含至少两种大小与其他已知GC框结合蛋白不同的蛋白。这些蛋白与增强子元件的结合需要磷酸化，因为对核提取物进行磷酸酶处理会消除DNA/蛋白复合物的形成。这些研究揭示了在a型CD23内含子I中存在一个EB病毒反应性增强子元件，并表明一种GC框结合转录因子参与了EB病毒对CD23的激活。
