Identification and characterization of the minimal 5'-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells.
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.
Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California.
Abhisek
A
Ghosal
Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California.
Subrata
S
Sabui
Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California hmsaid@uci.edu.
Hamid M
HM
Said
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D043562:Receptors, G-Protein-Coupled; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism
D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D043562:Receptors, G-Protein-Coupled; C540350:SLC52A2 protein, human; C548553:SLC52A3 protein, human; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D008156:Luciferases
intestinal cells; minimal promoter region; promoter activity; riboflavin; riboflavin transporter-3; stimulating protein-1