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MUCOSAL BIOLOGY

NF1 transcriptional factor(s) is required for basal promoter activation of the human intestinal NaPi-IIb cotransporter gene

Departments of Pediatrics, Physiology, and Nutritional Sciences, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona

Submitted 8 August 2004 ; accepted in final form 27 September 2004

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).

type IIb sodium-phosphate cotransporter; Slc34a2; Caco-2 cells; nuclear factor 1

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