MOLECULAR-CLONING AND FUNCTIONAL-ANALYSIS OF THE PROMOTER OF THE HUMAN SQUALENE SYNTHASE GENE

We have cloned and characterized the 5'-flanking region of the gene en coding human squalene synthase. We report here the promoter activity o f successively 5'-truncated sections of a 1 kilobase of this region by fusing it to the coding region of a luciferase reporter gene. DNA seg ments of 200 base pairs (bp) 5' to the transcription start site, as de termined by primer extension analysis, show a strong promoter effect o n the expression of the luciferase chimeric gene and a high response t o the presence of sterols when transiently transfected into the human hepatoma cell line HepG2 or to the hamster-derived CHO-K1 cells. An ap proximately 50-fold induction of luciferase activity, in the absence o f sterols, was observed in transiently transfected HepG2 cells for fus ion constructs containing sections of 200, 459, and 934 bp of the puta tive human squalene synthase promoter. Loss of promoter activity and r esponse to sterols was localized to a 69-bp section located 131 nucleo tides 5' to the transcription start site. Sequence analysis of this re gion showed that it contained a sterol regulatory element 1 (SRE-1) pr eviously identified in other sterol regulated genes (Smith, J. R., Osb orne, T. F., Brown, M. S., Goldstein, J. L., and Gil, G. (1988). J. Bi ol. Chem. 263, 18480-18487) and two potential NF-1 binding sites. Addi tional CCAAT box, SRE-1 element, and two Sp1 sites were identified 3' to this section. Sequences within this 69-bp DNA, including the SRE-1 cis-acting element, show strong binding to the purified nuclear transc ription factor ADD1 (Tonzonoz, P., Kim, J. B., Graves, R. A., and Spie gelman B. M. (1993) Mol. Cell Biol. 13, 4753-4759) by mobility shift a ssay and footprinting analyses.