ETS-1 transcription factor binds cooperatively to the palindromic head to head ETS-binding sites of the stromelysin-1 promoter by counteracting autoinhibition

Stromelysin-1 (matrix metalloproteinase-3) is a member of the matrix metalloproteinase family. Regulation of its gene expression is critical for tissue homeostasis. Patterns of increased co-expression of stromelysin-1 and ETS-1 genes have been observed in pathological processes. Stromelysin-1 promoter is transactivated by ETS proteins through two palindromic head to head ETS-binding sites, an unusual configuration among metalloproteinase promoters. By using surface plasmon resonance, electrophoretic mobility shift assay, and photo-cross-linking, we showed that full-length human ETS-1 (p51) binds cooperatively to the ETS-binding site palindrome of the human stromelysin-1 promoter, with facilitated binding of the second ETS-1 molecule to form an ETS-1.DNA.ETS-1 ternary complex. The study of N-terminal deletion mutants allowed us to conclude that cooperative binding implied autoinhibition counteraction, requiring the 245-330-residue region of the protein that is encoded by exon VII of the gene. This region was deleted in the natural p42 isoform of ETS-1, which was unable to bind cooperatively to the palindrome. Transient transfection experiments showed a good correlation between DNA binding and promoter transactivation for p51. In contrast, p42 showed a poorer transactivation, reinforcing the significance of cooperative binding for full transactivation. It is the first time that ETS-1 was shown to be able to counteract its own autoinhibition.     

小鼠缺氧诱导丝裂原因子基因启动子的结构与功能分析

缺氧诱导丝裂原因子（hypoxia-induced mitogenic factor, HIMF）是一种肺组织特异性生长因子,可刺激肺细胞增生和再生,但HIMF基因表达调控的机制尚未明确．为探索HIMF基因转录调控机制,首先以小鼠基因组DNA为模板,通过PCR扩增方法获得－348～+61 bp、－302～+61 bp、－131～+61 bp、－68～+61 bp的HIMF启动子片段,再将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子ETS-1结合位点的突变或缺失体．在阳离子脂质体的介导下,报告基因载体分别瞬时转染正常氧浓度条件下培养的小鼠肺上皮MLE-12和MLE-15细胞、结肠癌CT26细胞．结果发现,各HIMF启动子片段在MLE-12、MLE-15细胞中均有活性,但在CT26细胞中活性缺失;－302～－131 bp区存在HIMF启动子的核心调控元件．针对该区域ETS-1转录因子结合位点进行突变或缺失,能导致HIMF启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合ETS-1．结果提示,转录因子ETS-1参与正常氧浓度下HIMF启动子活性的调控,为研究HIMF基因的转录调控机制奠定了实验基础．