title: Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: a role for NF-Y in regulating basal transcription.
abstract: Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5'-regulatory region of the SLC25A19 gene (1,080 bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5'- and 3'-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between -131 and +20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.
authors.affiliation: Department of Medicine, University of California, Irvine, CA 92697, USA.
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Nabokina
authors.forename: Judith E
authors.initials: JE
authors.lastname: Valle
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D001483:Base Sequence; D001667:Binding, Competitive; D023081:CCAAT-Binding Factor / Q000737:chemistry / Q000502:physiology*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D056945:Hep G2 Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics*; D033681:Mitochondrial Membrane Transport Proteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D023081:CCAAT-Binding Factor; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; C434255:SLC25A19 protein, human; D049410:Luciferases, Renilla
keywords: 2-Amino-2-hydroxymethyl-propane-1,3-diol; A; Adenosine; Base pair(s); Bp; C; Chip; Chromatin immunoprecipitation; Cytidine; DMEM; DNA complementary to RNA; Dulbecco's Modified Eagle Medium; EDTA; EMSA; Electromobility shift assay; Ethylenediaminetetraacetic acid; G; Guanosine; IgG; Immunoglobulin G; Kb; Kilobase; MCF; MTPPT; Mitochondria; Mitochondrial carrier family; Mitochondrial thiamine pyrophosphate transporter; NF-Y; Nuclear factor Y; Nucleotide(s); PCR; Polymerase chain reaction; Promoter; RACE; RT-PCR; Rapid amplification of the cDNA ends; Reverse transcriptase polymerase chain reaction; SDS; SE; SLC25A19; Sodium dodecyl sulfate; Solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier), member 19; Standard error of mean; T; THTR-1; THTR-2; TPP; Thiamine pyrophosphate; Thiamine transporter-1; Thiamine transporter-2; Thymidine; Transcription regulation; Tris; cDNA; nt