Transcriptional Regulation Of The SLC52A3 Gene: Molecular Characterization Of The Minimal Promoter In Human Intestinal Cells

Abstract

The human the SLC52A3 gene encodes the riboflavin (RF) transporter‐3 (RFVT‐3), a membrane protein that plays an important role in intestinal RF absorption. There is little known about the regulation at the transcriptional and post‐transcriptional levels. Here we address the former regulation and for that we identified the minimal SLC52A3 promoter region, the regulatory element(s) involved in its activation, and confirmed promoter activity and establish physiological relevance in vivo in transgenic mice. Using transiently transfected human intestinal epithelial HuTu 80 cells and 5′‐deletion analysis, the minimal SLC52A3 promoter was found to be between ‐199 and +8 bp. Bioinformatics analysis predicted several cis‐regulatory elements in this region but only the stimulating protein‐1 (Sp1) (at position ‐74/‐71 bp) was found to play key role in promoter function. Electro‐mobility shift, super‐shift assays and by ChIP analysis confirmed the binding of the nuclear factor Sp1 to the minimal promoter. The importance of the Sp1 nuclear factor was further confirmed in studies with Drosophila SL2 cells (which lack endogenous Sps expression) where significant induction in SLC52A3 promoter activity was observed upon transfection with exogenous Sp1. Finally, activity of the cloned SLC52A3 promoter fused to the luciferase reporter gene was confirmed in vivo in transgenic mice. We report on the identification and molecular characterization of the minimal SLC52A3 promoter, and on the role of Sp1 in regulating its activity in gut epithelial cells. [Supported by grants from the DVA and NIDDK]