The basal promoters for the human reduced folate carrier gene are regulated by a GC box and a cAMP-response element/AP-1-like element - Basis for tissue-specific gene expression

Our laboratory previously identified two functional promoters (designated A and B) for the human reduced folate carrier (hRFC) gene that result in hRF C transcripts with differing 5'-untranslated regions. By transiently transf ecting HT1080 and HepG2 cells with a series of 5' and 3' deletions in the h RFC-B and -A promoters, the minimal promoters were localized within 46 and 47 base pairs, respectively. Gel mobility shift assays with the hRFC-B basa l promoter region revealed specific DNA-protein complexes involving a highl y conserved GC-box and Sp1 or Sp3. In Drosophila SL2 cells, both Sp1 and th e long Sp3 isoform potently transactivated the hRFC-B basal promoter; howev er, the short Sp3 isoforms were transcriptionally inert and resulted in a p otent inhibition of Sp1 transactivation. For the hRFC-A basal promoter, a C RE/AP-1-like element was bound by the bZip superfamily of DNA-binding prote ins. Cell-specific DNA-protein complexes were identified for hRFC-A (CREB-1 and c-Jun in HT1080 cells; CREB-1 and ATF-1 in HepG2 cells). When the GC-b ox and CRE/AP-1-like elements were mutated, a 60-90% decrease in promoter a ctivity was observed in both cell lines. These results identify the critica l regulatory regions for the hRFC basal promoters and stress the functional importance of the Sp and bZip families of transcription factors in regulat ing hRFC expression.