Abstract
Our laboratory previously identified two functional promoters (designated A and B) for the human reduced folate carrier (hRFC) gene that result in hRFC transcripts with differing 5′-untranslated regions. By transiently transfecting HT1080 and HepG2 cells with a series of 5′ and 3′ deletions in the hRFC-B and -A promoters, the minimal promoters were localized within 46 and 47 base pairs, respectively Gel mobility shift assays with the hRFC-B basal promoter region revealed specific DNA-protein complexes involving a highly conserved GC-box and Sp1 or Sp3. In Drosophila SL2 cells, both Sp1 and the long Sp3 isoform potently transactivated the hRFC-B basal promoter; however, the short Sp3 isoforms were transcriptionally inert and resulted in a potent inhibition of Sp1 transactivation. For the hRFC-A basal promoter, a CRE/AP-1-like element was bound by the bZip superfamily of DNA-binding proteins. 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-box and CRE/AP-1-like elements were mutated, a 60-90% decrease in promoter activity was observed in both cell lines. These results identify the critical regulatory regions for the hRFC basal promoters and stress the functional importance of the Sp and bZip families of transcription factors in regulating hRFC expression.
Original language | English |
---|---|
Pages (from-to) | 6350-6358 |
Number of pages | 9 |
Journal | Journal of Biological Chemistry |
Volume | 276 |
Issue number | 9 |
DOIs | |
State | Published - Mar 2 2001 |
Keywords
- Animals
- Base Sequence
- Carrier Proteins/genetics
- Cyclic AMP/pharmacology
- DNA-Binding Proteins/physiology
- Drosophila
- Gene Expression Regulation
- Humans
- Membrane Proteins
- Membrane Transport Proteins
- Molecular Sequence Data
- Organ Specificity
- Promoter Regions, Genetic
- Reduced Folate Carrier Protein
- Response Elements
- Sp1 Transcription Factor/physiology
- Sp3 Transcription Factor
- Transcription Factors/metabolism
- Transcriptional Activation
- Transfection