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).
Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA.
Hua
H
Xu
Jennifer K
JK
Uno
Michael
M
Inouye
James F
JF
Collins
Fayez K
FK
Ghishan
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