IDENTIFICATION OF A FUNCTIONAL IMPERFECT ESTROGEN-RESPONSIVE ELEMENT IN THE 5'-PROMOTER REGION OF THE HUMAN CATHEPSIN-D GENE

17 beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 hu man breast cancer cells, Previous studies have identified an Sp1-imper fect estrogen-responsive element (ERE) half-site [GGGCGG-(N)(23)ACGGG] (-199 to -165) in the promoter region which forms an Sp1-estrogen rec eptor (ER) complex and confers E2 responsiveness on the corresponding Sp1-ERE-chloramphenicol acetyl transferase (CAT) construct. Further an alysis of downstream regions of the promoter identified a CGCCC-(N)(3) TGACC sequence (-119 to -107) which is homologous to the adenovirus ma jor late promoter element (MLPE) and binds the ER to form a retarded b and in a gel electrophoretic mobility shift assay, The corresponding p romoter-CAT construct is also E2-inducible. The MLPE resembles an impe rfect palindromic ERE containing imperfect (5') and perfect (3') ERE h alf-sites; analysis of oligonucleotides with mutations in these half-s ites 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 enhanc ed binding at the MLPE site. Identification of this second functional enhancer sequence in the 5'-promoter region of cathepsin D is consiste nt with the increasingly complex cell-specific regulation of hormone-r esponsive genes.