Transcriptional regulation of the human reduced folate carrier A1/A2 promoter: Identification of critical roles for the USF and GATA families of transcription factors.
The human reduced folate carrier (hRFC) gene has a complex regulation involving 6 alternatively spliced non-coding exons and promoters (A1/A2, A, B, C, D, and E). The hRFC-A1/A2 promoter is unique in that it transcribes a novel transcript with an in-frame AUG in non-coding exon A1/A2 that encodes a modified hRFC protein with altered transport function. In this report, we characterize the hRFC-A1/A2 promoter in HepG2 human hepatoma cells. By transfecting HepG2 cells with 5' and 3' deletion constructs, a transcriptionally important 270 bp region was identified. Gel shift assays identified transcription factor binding to three E-box elements and one GATA site within this region. These elements were verified by transfections of mutant constructs into HepG2 cells. Cotransfections in Drosophila Mel-2 cells confirmed promoter activation by USF1 and GATA1. A physical association between USF1 and GATA1 was demonstrated by their co-immunoprecipitation. By real time PCR analysis of transfected HepG2 cells, USF1 and GATA1 increased endogenous hRFC-A1/A2 transcripts. Altogether, our results demonstrate a transcriptionally important region in the hRFC-A1/A2 promoter including E-box and GATA elements, and a transactivation by USF1 and GATA1 proteins. Our results further establish the complexity of hRFC regulation, as a means of ensuring adequate folate cofactor transport for cell proliferation.
Payton
Scott G
SG
Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, and Wayne State University School of Medicine, Detroit, MI 48201, USA.
Liu
Mingjun
M
Ge
Yubin
Y
Matherly
Larry H
LH
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila / Q000201:enzymology; D024721:E-Box Elements / Q000235:genetics / Q000502:physiology; D005091:Exons; D050980:GATA Transcription Factors / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D050980:GATA Transcription Factors; D026901:Membrane Transport Proteins; C120043:SLC19A2 protein, human; D051720:Upstream Stimulatory Factors