Transcriptional regulation of the human reduced folate carrier promoter C: Synergistic transactivation by Sp1 and C/EBP β and identification of a downstream repressor

Scott G. Payton, Johnathan R. Whetstine, Yubin Ge, Larry H. Matherly

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


The human reduced folate carrier (hRFC) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to six alternatively spliced non-coding regions (designated A1/A2, A, B, C, D, and E) and by at least four promoters. By transient transfections of HepG2 human hepatoma cells with 5′ and 3′ deletion constructs spanning 2883 bp of upstream sequence, a transcriptionally important region was localized to within 177 bp flanking the transcriptional start sites for exon C. By gel shift and chromatin immunoprecipitation assays, Sp1 and C/EBP β transcription factors were found to bind consensus elements (GC-box, CCAAT-box) within this region. The functional importance of these elements was confirmed by transient tranfections of HepG2 cells with hRFC-C reporter constructs in which these elements were mutated, and by co-transfections of Drosophila SL-2 cells with wild-type hRFC-C promoter and expression constructs for Sp1 and C/EBP β. Whereas both Sp1 and C/EBP β transactivated hRFC-C promoter activity, C/EBP α and γ were transcriptionally inert. Sp1 combined with C/EBP β resulted in a synergistic transactivation. In HepG2 cells, transfections with Sp1 and C/EBP β both increased endogenous levels of hRFC-C transcripts. By 3′ deletion analysis, a repressor sequence was localized to within 71 bp flanking the minimal promoter. On gel shifts, a novel transcriptional repressor was localized to within 30 bp. Collectively, these results identify transcriptionally important regions in the hRFC-C minimal promoter that include a GC-box and CCAAT-box, and suggest that cooperative interactions between Sp1 and C/EBP β are essential for hRFC-C transactivation. Another possible factor in the tissue-specific regulation of the hRFC-C region involves the downstream repressor flanking the minimal promoter.

Original languageEnglish
Pages (from-to)45-57
Number of pages13
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Issue number1
StatePublished - Jan 21 2005


  • Animals
  • Base Sequence
  • CCAAT-Binding Factor
  • CCAAT-Enhancer-Binding Proteins/physiology
  • Cells, Cultured
  • Chromatin Immunoprecipitation
  • Drosophila
  • Gene Expression Regulation
  • Humans
  • Membrane Transport Proteins/genetics
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • Reduced Folate Carrier Protein
  • Sp1 Transcription Factor/physiology
  • Transcription Factor CHOP
  • Transcription Factors/physiology
  • Transcription, Genetic
  • Transcriptional Activation
  • Transfection
  • Tumor Cells, Cultured


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