Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal trancriptional regulatory region in an antifolate-resistant HeLa cell line

Abstract

This laboratory recently identified a novel proton-coupled folate transporter (PCFT) that mediates intestinal folate absorption and transport of folates into the central nervous system. The present study focuses on the definition of the minimum transcriptional regulatory region of this gene in HeLa cells and the mechanism(s) underlying the loss of PCFT expression in the methotrexate-resistant HeLa R1-11 cell line. The PCFT transcriptional regulatory controls were localized between -42 and +96 bases from the transcriptional start site using a luciferase-reporter gene system. The promoter is a G + C rich region of 139 nucleotides contained in a CpG island. HeLa R1-11 cells have no mutations in the PCFT open reading frame and its promoter; the transcription/translation machinery is intact because transient transfections in HeLa R1-11 and wild-type HeLa cells produced similar luciferase activities. Hypermethylation at CpG sites within the minimal transcriptional regulatory region was shown in HeLa R1-11 cells as compared with the parental PCFT-competent HeLa cells, using bisulfite conversion and sequence analysis. Treatment with 5-aza-2'-deoxycytidine resulted in a substantial restoration of transport and PCFT mRNA expression and small but significant decreases in methylation in the promoter region. In vitro methylation of the transfected reporter plasmid inhibited luciferase gene expression. Cytogenetics/fluorescence in situ hybridization indicated a loss of half the PCFT gene copies in HeLa R1-11 as compared with PCFT-competent HeLa cells. Taken together, promoter silencing through methylation and gene copy loss accounted for the loss of PCFT activity in antifolate-resistant HeLa R1-11 cells.