Wang F, Porter W, Xing W, Archer T K, Safe S
Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466, USA.
Biochemistry. 1997 Jun 24;36(25):7793-801. doi: 10.1021/bi963100j.
17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells. Previous studies have identified an Sp1-imperfect estrogen-responsive element (ERE) half-site [GGGCGG(N)23ACGGG] (-199 to -165) in the promoter region which forms an Sp1-estrogen receptor (ER) complex and confers E2 responsiveness on the corresponding Sp1-ERE-chloramphenicol acetyl transferase (CAT) construct. Further analysis of downstream regions of the promoter identified a CGCCC(N)3TGACC sequence (-119 to -107) which is homologous to the adenovirus major late promoter element (MLPE) and binds the ER to form a retarded band in a gel electrophoretic mobility shift assay. The corresponding promoter-CAT construct is also E2-inducible. The MLPE resembles an imperfect palindromic ERE containing imperfect (5') and perfect (3') ERE half-sites; analysis of oligonucleotides with mutations in these half-sites shows that only the perfect ERE half-site is required for binding the ER, whereas both sites are required for transactivation. In vivo exonuclease III footprinting showed that treatment with E2 also enhanced binding at the MLPE site. Identification of this second functional enhancer sequence in the 5'-promoter region of cathepsin D is consistent with the increasingly complex cell-specific regulation of hormone-responsive genes.
17β-雌二醇（E2）可诱导MCF-7人乳腺癌细胞中组织蛋白酶D基因的表达。先前的研究已在启动子区域鉴定出一个Sp1非完美雌激素反应元件（ERE）半位点[GGGCGG(N)23ACGGG]（-199至-165），该半位点形成Sp1-雌激素受体（ER）复合物，并赋予相应的Sp1-ERE-氯霉素乙酰转移酶（CAT）构建体E2反应性。对启动子下游区域的进一步分析鉴定出一个CGCCC(N)3TGACC序列（-119至-107），该序列与腺病毒主要晚期启动子元件（MLPE）同源，并在凝胶电泳迁移率变动分析中与ER结合形成滞后条带。相应的启动子-CAT构建体也可被E2诱导。MLPE类似于一个包含不完美（5'）和完美（3'）ERE半位点的非完美回文ERE；对这些半位点具有突变的寡核苷酸的分析表明，仅完美的ERE半位点是与ER结合所必需的，而两个位点对于反式激活都是必需的。体内核酸外切酶III足迹分析表明，用E2处理也增强了在MLPE位点的结合。在组织蛋白酶D的5'启动子区域鉴定出这第二个功能性增强子序列，这与激素反应性基因日益复杂的细胞特异性调节是一致的。
