Molecular Characterization of the SLC19A2 Promoter

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DESCRIPTION (provided by applicant): Dietary thiamine is an essential nutrient for humans. The uptake of thiamine in the intestine has been well studied but the molecular mechanism and regulation of the uptake process is not well understood. Recently a thiamine transporter has been cloned from several human tissues (SLC19A2). We deafly established the presence of this transporter in the small and large intestine. In an attempt to understand the regulation of the transporter at the level of gene expression we cloned the 5'regulatory region and using a luciferase reporter system established the minimal region required for activity. We were able to show high levels of activity of the cloned putative promoter in intestinal Caco-2 cells with approximately twice that activity in liver HepG2 cells. Several cis-regulatory elements were detected in the minimal region using web based computer programs. We plan to further characterize the putative promoter region in vitro using gel shift and super-shift assays, mutational analysis, and functional assays to establish exactly what transcriptional regulatory proteins bind the putative cis-elements. We wish to also characterize the putative promoter in vivo. To thoroughly examine the promoter we established transgenic mouse lines carrying the promoter luciferase constructs. We will first examine the tissue distribution of the activity for the promoter in the mouse with an emphasis on the intestine. Previous work has established that transport processes of nutrients, including vitamins, undergo clear developmental regulation. Nothing is known about possible regulation of the thiamine uptake process during development and whether this includes promoter regulation. These issues will be addressed in this proposal.