NF1 transcriptional factor(s) is required for basal promoter activation of the human intestinal NaPi-IIb cotransporter gene.
The human intestinal type IIb Na+-P(i) cotransporter (hNaPi-IIb) gene promoter lacks a TATA box and has a high GC content in the 5'-flanking region. To understand the mechanism of hNaPi-IIb gene transcription, the current study was performed to characterize the minimal promoter region and transcriptional factor(s) necessary to activate gene expression in human intestinal cells (Caco-2). With the use of progressively shorter promoter constructs, a minimal promoter extending from bp -58 to +15 was identified and shown to direct high levels of hNaPi-IIb cotransporter expression in Caco-2 cells. Gel mobility shift assays (GMSAs) indicated that two regions could be bound by nuclear proteins from Caco-2 cells: region A at bp -26/-23 and region B at bp -44/-35. The introduction of mutations in region A abolished promoter activity, whereas mutations in region B had no effect. Deletion mutants of the same regions showed identical results. Furthermore, DNase I footprinting experiments confirmed the observation made by GMSAs. Additional studies, which used a specific nuclear factor 1 (NF1) antiserum, demonstrated that NF1 protein(s) binds to the minimal promoter at region A. These results indicated that the NF1 protein(s) is required to activate the basal transcription of hNaPi-IIb gene under normal growth conditions. This study has thus identified a new target gene in the small intestinal epithelium that is directly regulated by NF1 transcriptional factor(s).
Xu
Hua
H
Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA.
Uno
Jennifer K
JK
Inouye
Michael
M
Collins
James F
JF
Ghishan
Fayez K
FK
D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000502:physiology*; D018938:Caco-2 Cells; D005786:Gene Expression Regulation; D006801:Humans; D051839:NFI Transcription Factors; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic / Q000502:physiology*; D050608:Sodium-Phosphate Cotransporter Proteins; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*
D022762:CCAAT-Enhancer-Binding Proteins; D051839:NFI Transcription Factors; C497592:NFIA protein, human; D009838:Oligodeoxyribonucleotides; C493557:SLC34A2 protein, human; D050608:Sodium-Phosphate Cotransporter Proteins; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters; D014157:Transcription Factors