Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California.
Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California
Am J Physiol Cell Physiol. 2015 Jan 15;308(2):C189-96. doi: 10.1152/ajpcell.00342.2014. Epub 2014 Nov 12.
The human riboflavin (RF) transporter-3 (product of the SLC52A3 gene) plays an important role in intestinal RF absorption. Our aims in this study were to identify the minimal 5'-regulatory region of the SLC52A3 gene and the regulatory element(s) involved in its activity in intestinal epithelial cells, as well as to confirm promoter activity and establish physiological relevance in vivo in transgenic mice. With the use of transiently transfected human intestinal epithelial HuTu 80 cells and 5'-deletion analysis, the minimal SLC52A3 promoter was found to be encoded between -199 and +8 bp (using the start of the transcription start site as position 1). Although several putative cis-regulatory elements were predicted in this region, only the stimulating protein-1 (Sp1) binding site (at position -74/-71 bp) was found to play a role in promoter activity, as indicated by mutational analysis. Binding of Sp1 to the minimal SLC52A3 promoter was demonstrated by means of EMSA and supershift assays and by chromatin immunoprecipitation analysis. Studies with Drosophila SL2 cells (which lack Sp activity) confirmed the importance of Sp1 in driving the activity of the SLC52A3 minimal promoter; they further showed that Sp3 can also do the activation. Finally, with the use of luciferase gene fusions, the activity of the cloned SLC52A3 promoter was confirmed in vivo in transgenic mice. These studies report, for the first time, on the identification and characterization of the SLC52A3 promoter and also demonstrate the importance of Sp1 in regulating its activity in intestinal epithelial cells.
人类核黄素（RF）转运蛋白-3（SLC52A3 基因产物）在肠道 RF 吸收中发挥重要作用。本研究旨在鉴定 SLC52A3 基因的最小 5'-调控区及其在肠上皮细胞中活性的调节元件，并确认启动子活性及其在转基因小鼠体内的生理相关性。通过瞬时转染的人肠上皮细胞 HuTu 80 和 5'-缺失分析，发现 SLC52A3 启动子的最小编码区位于-199 至+8 bp（转录起始位点为 1 位）。尽管在该区域预测了几个潜在的顺式调控元件，但只有刺激蛋白-1（Sp1）结合位点（位于-74/-71 bp 位）被发现对启动子活性起作用，这表明突变分析。通过 EMSA 和超迁移分析以及染色质免疫沉淀分析证实了 Sp1 与最小 SLC52A3 启动子的结合。用缺乏 Sp 活性的果蝇 SL2 细胞进行的研究进一步证实了 Sp1 在驱动 SLC52A3 最小启动子活性中的重要性；它们还表明 Sp3 也可以进行激活。最后，通过荧光素酶基因融合，在转基因小鼠体内证实了克隆的 SLC52A3 启动子的活性。这些研究首次报道了 SLC52A3 启动子的鉴定和特征，并证明了 Sp1 在调节肠上皮细胞中其活性的重要性。
