Functional elements in the minimal promoter of the human proton-coupled folate transporter.
The proton-coupled folate transporter (PCFT) is the dominant intestinal folate transporter, however, its promoter has yet to be revealed. Hence, we here cloned a 3.1kb fragment upstream to the first ATG of the human PCFT gene and generated sequential deletion constructs evaluated in luciferase reporter assay. This analysis mapped the minimal promoter to 157bp upstream to the first ATG. Crucial GC-box sites were identified within the minimal promoter and in its close vicinity which substantially contribute to promoter activity, as their disruption resulted in 94% loss of luciferase activity. We also identified upstream enhancer elements including YY1 and AP1 which, although distantly located, prominently transactivated the minimal promoter, as their inactivation resulted in 50% decrease in reporter activity. This is the first functional identification of the minimal PCFT promoter harboring crucial GC-box elements that markedly contribute to its transcriptional activation via putative interaction with distal YY1 and AP1 enhancer elements.
Stark
Michal
M
Dept. of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Gonen
Nitzan
N
Assaraf
Yehuda G
YG
D001483:Base Sequence; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D011401:Promoter Regions, Genetic; D058979:Proton-Coupled Folate Transporter; D020218:Response Elements; D017421:Sequence Analysis; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D050986:YY1 Transcription Factor / Q000378:metabolism*
D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; C505058:SLC46A1 protein, human; D018808:Transcription Factor AP-1; D050986:YY1 Transcription Factor; C494698:YY1 protein, human