Lewis H, Kaszubska W, DeLamarter J F, Whelan J
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Mol Cell Biol. 1994 Sep;14(9):5701-9. doi: 10.1128/mcb.14.9.5701-5709.1994.
Cytokine-induced expression of the E-selectin gene requires the promoter binding and interaction of the transcription factors NF-kappa B and ATF. Here we have further analyzed the E-selectin promoter and revealed an additional region (nucleotides -140 to -105 [-140/-105]) which is essential in controlling promoter activation by cytokines. We identified high-mobility-group protein I(Y) [HMG-I(Y)] interacting specifically at two sites within this region. We noted that one of the HMG-I(Y)-binding sites overlaps a sequence element (-127/-118) diverging at only one position from the NF-kappa B consensus binding sequence. This led us to ask whether the -127/-118 element represents a second functional NF-kappa B-binding site within the E-selectin promoter. Using specific antisera, we show that p50, p65, and, interestingly, RelB are components of the complex interacting at this site. Mutational analysis of the -127/-118 NF-kappa B site indicates that both NF-kappa B and HMG-I(Y) binding at this site are essential for interleukin-1 induction of the promoter. We demonstrate that the binding affinity of the p50 subunit of NF-kappa B to both NF-kappa B sites within the E-selectin promoter is significantly enhanced by HMG-I(Y). In addition, an essential role for cooperative interaction between the two NF-kappa B complexes is shown by the requirement for both NF-kappa B sites to mediate E-selectin promoter activation by interleukin-1 and p50/p65 expression. We conclude that HMG-I(Y) mediates binding of a distinct NF-kappa B complex at two sites within the E-selectin promoter. Furthermore, a unique cooperativity between these NF-kappa B complexes is essential for induced E-selectin expression. These results suggest mechanisms by which NF-kappa B complexes are involved in specific gene activation.
细胞因子诱导的E-选择素基因表达需要转录因子NF-κB和ATF与启动子结合并相互作用。在此，我们进一步分析了E-选择素启动子，并发现了一个额外区域（核苷酸-140至-105 [-140 / -105]），该区域对于控制细胞因子诱导的启动子激活至关重要。我们鉴定出高迁移率族蛋白I(Y) [HMG-I(Y)]在该区域内的两个位点特异性相互作用。我们注意到，HMG-I(Y)结合位点之一与一个序列元件（-127 / -118）重叠，该序列元件与NF-κB共有结合序列仅在一个位置上不同。这使我们不禁要问，-127 / -118元件是否代表E-选择素启动子内的第二个功能性NF-κB结合位点。使用特异性抗血清，我们表明p50、p65，有趣的是，RelB是在此位点相互作用的复合物的组成成分。对-127 / -118 NF-κB位点的突变分析表明，该位点的NF-κB和HMG-I(Y)结合对于启动子的白细胞介素-1诱导都是必不可少的。我们证明，HMG-I(Y)可显著增强NF-κB的p50亚基与E-选择素启动子内两个NF-κB位点的结合亲和力。此外，通过白细胞介素-1和p50 / p65表达介导E-选择素启动子激活需要两个NF-κB位点，这表明了两个NF-κB复合物之间协同相互作用的重要作用。我们得出结论，HMG-I(Y)介导了E-选择素启动子内两个位点上不同NF-κB复合物的结合。此外，这些NF-κB复合物之间独特的协同作用对于诱导的E-选择素表达至关重要。这些结果提示了NF-κB复合物参与特定基因激活的机制。
