Chen D, Rothenberg E V
Division of Biology, California Institute of Technology, Pasadena 91125.
J Exp Med. 1994 Mar 1;179(3):931-42. doi: 10.1084/jem.179.3.931.
Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein-DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus.
环磷酸腺苷（cAMP）水平升高可导致白细胞介素2（IL-2）表达的基因特异性抑制。为了研究这种效应的机制，我们结合了电泳迁移率变动分析和体内基因组足迹分析，以评估福斯高林处理的细胞中假定的IL-2转录因子的可用性，以及这些因子在体内结合其位点的功能能力。福斯高林观察到的所有效应都依赖于蛋白激酶A，因为它们被引入蛋白激酶A的显性负突变亚基所阻断。在EL4.E1细胞系中，我们报道了cAMP水平升高对位于-200 bp处结合的NF-κB/Rel家族因子，以及对相对于IL-2转录起始位点在-225 bp处结合的一种新型、生化性质不同的“TGGGC”因子的特异性抑制作用。在凝胶迁移率变动分析中未检测到NF-AT或AP-1结合活性受到抑制。cAMP水平升高以延迟动力学抑制NF-κB活性，同时伴随IL-2 RNA积累的延迟抑制。在福斯高林存在下激活细胞可阻止体内稳定的蛋白质-DNA相互作用的维持，不仅在IL-2增强子的NF-κB和TGGGC位点，而且在NF-AT、AP-1和其他位点。这一结果以及环孢素A处理细胞中的类似结果表明，单个IL-2转录因子在体内不能稳定结合其靶序列，除非所有其他不同因子在相邻位点共同结合。有人提出，结合的非分层协同增强是IL-2基因座处组合转录因子作用的结构基础。
