IDENTIFICATION OF A 6-BASE PAIR ELEMENT INVOLVED IN THE STEROL-MEDIATED TRANSCRIPTIONAL REGULATION OF FARNESYL DIPHOSPHATE SYNTHASE

Previous studies identified a 115-base pair (bp) region of the farnesy l diphosphate (FPP) synthase promoter which is involved in the transcr iptional regulation of this gene by sterols (Spear, D. H., Kutsunai, S . Y., Correll, C. C., and Edwards, P A. (1992) J. Biol. Chem. 267, 144 62-14469). In the current study we fused a 117-bp fragment, containing this region of interest, upstream of the heterologous minimal promote r of the herpes simplex virus thymidine kinase gene linked to the chlo ramphenicol acetyltransferase (CAT) reporter gene. Chinese hamster ova ry (CHO) cells were stably transfected with this fusion gene and incub ated in the absence or presence of sterols. Analysis of CAT mRNA by pr imer extension indicated that transcription of the fusion gene was und er sterol-mediated control, Thus, when cellular sterols were present, the CAT mRNA levels were reduced 2-4-fold. To further localize the FPP synthase sterol-responsive element(s), additional promoter-reporter g ene constructs containing either deletions or mutations were construct ed and transfected into CHO or CV-1 cells. These studies localized a 6 -bp region (ATTGGC) that is required for both transcriptional inductio n in the absence of sterols and transcriptional repression in the pres ence of sterols. Gel shift and footprinting analyses demonstrated that nuclear proteins isolated from CHO cells bound to six distinct region s of the promoter between nucleotides -293 to -47. Taken together, the se results further define both the cis-acting elements controlling nor mal transcription of the FPP synthase gene and identify a novel sequen ce involved in sterol regulation.