STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE HUMAN KB CELL FOLATE RECEPTOR GENE P4 PROMOTER - COOPERATION OF 3 CLUSTERED SP1-BINDING SITES WITH INITIATOR REGION FOR BASAL PROMOTER ACTIVITY

The human folate receptors (hFRs) are important in the cellular accumu lation of folates and antifolates. We described the structure of the h uman KB cell FR (hFR-KB) gene and identified two discrete promoter reg ions (P1 and P4) upstream from exons 1 and 4, respectively (Elwood et al., 1993). To further understand the molecular basis of hFR expressio n, we have now analyzed the basal transcription of the P4 promoter loc alized upstream of a major transcription start site. The sequence upst ream from exon 4 contains several potential transcriptional factor-bin ding sites and a consensus initiator region sequence at the transcript ion start site but does not contain canonical TATA or CAAT boxes. Whil e deletion of a 5' flanking sequence from nt - 1023 to nt - 605 of P4 promoter region decreases the luciferase reporter gene expression in K B cells to 54 - 70% of control construct, the removal of the sequence between nt - 292 and nt - 46 markedly decreases the activity to 3%. DN ase I footprints and competitive mobility shift and supershift mobilit y assays indicate that Spl or Spl-related nuclear protein(s) bind to t hree clustered GC-rich regions within the sequence between nt - 292 an d nt - 46 of the hFR-KB P4 promoter. Both in vitro and in vivo analyse s of the expression of promoter constructs containing site-specific mu tation(s) of these three Spl-binding sites and initiator sequence demo nstrate that each of three Spl sites and the initiator sequence are re quired for optimum promoter activity and that they interact cooperativ ely in this P4 promoter of the hFR-KB gene.