TY - JOUR
T1 - Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression
AU - Verhave, Menno
AU - Krasinski, Stephen D.
AU - Christian, Sara I.
AU - van Schaik, Sandrijn
AU - van den Brink, Gijs R.
AU - Doting, Edwina M. H.
AU - Maas, Saskia M.
AU - Wolthers, Katja C.
AU - Grand, Richard J.
AU - Montgomery, Robert K.
PY - 2004
Y1 - 2004
N2 - OBJECTIVES: Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb of the 5'-flanking region of the rat LPH gene control the correct tissue, cell, and crypt-villus expression in transgenic animals. METHODS: To examine further the regulation conferred by this region, protein-DNA interactions were studied using DNase I footprint analyses in LPH-expressing and nonexpressing cell lines. Functional delineation of this 5'-flanking sequence was performed using deletion analysis in transient transfection assays. RESULTS: Studies revealed a generally positive activity between -74 and -37 bp, a cell-specific negative region between -210 and -95 bp, and additional elements further toward the 5'-terminus that conferred a highly cell-specific response in reporter activity. Computer analysis of distal regions encompassing identified footprints revealed potential binding sites for various intestinal transcription factors. Co-transfection and electromobility shift assay experiments indicated binding of HNF3beta at three sites relevant to LPH expression. CONCLUSIONS: The data demonstrate that the cell specificity of LPH gene expression depends upon both positive and negative interactions among elements in the first 2 kb of the LPH 5'-flanking region
AB - OBJECTIVES: Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb of the 5'-flanking region of the rat LPH gene control the correct tissue, cell, and crypt-villus expression in transgenic animals. METHODS: To examine further the regulation conferred by this region, protein-DNA interactions were studied using DNase I footprint analyses in LPH-expressing and nonexpressing cell lines. Functional delineation of this 5'-flanking sequence was performed using deletion analysis in transient transfection assays. RESULTS: Studies revealed a generally positive activity between -74 and -37 bp, a cell-specific negative region between -210 and -95 bp, and additional elements further toward the 5'-terminus that conferred a highly cell-specific response in reporter activity. Computer analysis of distal regions encompassing identified footprints revealed potential binding sites for various intestinal transcription factors. Co-transfection and electromobility shift assay experiments indicated binding of HNF3beta at three sites relevant to LPH expression. CONCLUSIONS: The data demonstrate that the cell specificity of LPH gene expression depends upon both positive and negative interactions among elements in the first 2 kb of the LPH 5'-flanking region
U2 - https://doi.org/10.1097/00005176-200409000-00010
DO - https://doi.org/10.1097/00005176-200409000-00010
M3 - Article
C2 - 15319629
SN - 0277-2116
VL - 39
SP - 275
EP - 285
JO - Journal of pediatric gastroenterology and nutrition
JF - Journal of pediatric gastroenterology and nutrition
IS - 3
ER -