Molecular Characterization of the SLC19A2 Promoter

说明(申请人提供)：膳食硫胺素是人体必需的营养素。硫胺素在肠道中的摄取已经得到了很好的研究，但摄取过程的分子机制和调控还不是很清楚。最近，从几种人体组织中克隆了一种硫胺转运蛋白(SLC19A2)。我们盲目地证实了这种转运蛋白在小肠和大肠中的存在。为了在基因表达水平上了解转运蛋白的调控，我们克隆了5‘调控区，并使用荧光素酶报告系统建立了活性所需的最小区域。我们能够显示克隆的假定启动子在肠道Caco-2细胞中具有高水平的活性，而在肝脏细胞中的活性大约是HepG2细胞的两倍。使用基于网络的计算机程序在最小区域中检测到几个顺式调控元件。我们计划在体外通过凝胶移位和超移位分析、突变分析和功能分析来进一步表征推定的启动子区域，以准确地确定哪些转录调控蛋白与推定的顺式元件结合。我们还希望在体内表征推定的启动子。为了彻底检测启动子，我们建立了携带启动子荧光素酶构建的转基因小鼠系。我们将首先检查启动子活性在小鼠体内的组织分布，重点是肠道。以前的研究已经确定，包括维生素在内的营养物质的运输过程经历了明确的发育调节。对发育过程中硫胺素摄取过程的可能调控以及这是否包括启动子调控，我们一无所知。这些问题将在这项提案中得到解决。

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.