Skerka C, Decker E L, Zipfel P F
Department of Molecular Biology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Immunobiology. 1997 Dec;198(1-3):179-91. doi: 10.1016/S0171-2985(97)80039-3.
The Early Growth Response Genes (EGR-1 to AT133/EGR-4) encode a family of proteins that are composed of three homologous consecutive zinc fingers of the Cys2-His2 type and different flanking sequence. Upon growth stimulation of resting cells the four EGR-genes are simultaneously transcribed. We have analyzed the expression of the four EGR-proteins in Jurkat T cells and show by Western blot analysis that the four EGR-proteins are coordinately induced upon treatment with a combination of PHA and PMA. As the individual proteins are reported to bind to identical target sequences, we have analyzed the DNA-binding of the native proteins. Using nuclear extract in which we have demonstrated expression of all four EGR-proteins, only EGR-1, but no other member of this protein family is found to bind to the EGR-consensus site (GCG GGG GCG). In addition, DNA-binding of both native EGR-1 and of recombinant EGR-1 and AT133/EGR-4 proteins expressed in insect cells was analyzed. This comparison revealed distinct binding properties of recombinant EGR-1 and AT133/EGR-4 to oligonucleotides that include the EGR-consensus sites. The distinct binding affinities suggest that in vivo EGR-proteins bind to different target sequences and that each EGR-protein regulates distinct target genes. This is underlined by demonstrating that EGR-1 but not AT133/EGR-4 binds to a related G-rich promoter element with the sequence GGG GTG GGG. This G-rich sequence serves as an overlapping binding site for the two zinc finger proteins EGR-1 and Sp1. As similar overlapping binding sites for EGR-1 and Sp1 have been identified in several human and mouse gene promoters, we raise the question whether the Sp1 binding sites described in a large number of eukaryotic gene promoters also represent binding sites for EGR-1.
早期生长反应基因（EGR - 1至AT133/EGR - 4）编码一类蛋白质，这类蛋白质由三个连续的Cys2 - His2型同源锌指结构和不同的侧翼序列组成。在静止细胞受到生长刺激时，这四个EGR基因会同时转录。我们分析了Jurkat T细胞中这四种EGR蛋白的表达情况，并通过蛋白质免疫印迹分析表明，在用PHA和PMA联合处理后，这四种EGR蛋白会协同诱导表达。由于据报道单个蛋白可结合相同的靶序列，我们分析了天然蛋白的DNA结合情况。使用已证明表达所有四种EGR蛋白的核提取物，发现只有EGR - 1，而该蛋白家族的其他成员均未发现与EGR共有位点（GCG GGG GCG）结合。此外，还分析了天然EGR - 1以及在昆虫细胞中表达的重组EGR - 1和AT133/EGR - 4蛋白的DNA结合情况。这种比较揭示了重组EGR - 1和AT133/EGR - 4与包含EGR共有位点的寡核苷酸具有不同的结合特性。不同的结合亲和力表明，在体内EGR蛋白结合不同的靶序列，并且每个EGR蛋白调节不同的靶基因。通过证明EGR - 1而非AT133/EGR - 4与具有序列GGG GTG GGG的相关富含G的启动子元件结合，进一步强调了这一点。这个富含G的序列作为两种锌指蛋白EGR - 1和Sp1的重叠结合位点。由于在几个人类和小鼠基因启动子中已鉴定出EGR - 1和Sp1的类似重叠结合位点，我们提出一个问题，即在大量真核基因启动子中描述的Sp1结合位点是否也代表EGR - 1的结合位点。
