title: The human reduced folate carrier gene is regulated by the AP2 and sp1 transcription factor families and a functional 61-base pair polymorphism.
abstract: Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g. CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49-61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates.
authors.affiliation: Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Johnathan R
authors.initials: JR
authors.lastname: Whetstine
authors.forename: Teah L
authors.initials: TL
authors.lastname: Witt
authors.forename: Larry H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D005492:Folic Acid / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D058978:Reduced Folate Carrier Protein; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D016329:Sp1 Transcription Factor; C493674:TfAP-2 protein, Drosophila; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D005492:Folic Acid