Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression.
OBJECTIVE
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.
Verhave
Menno
M
Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, The Floating Hospital for Children, New England Medical Center Hospitals, Boston, Massachusetts, USA.
Krasinski
Stephen D
SD
Christian
Sara I
SI
Van Schaik
Sandrijn
S
Van Den Brink
Gijs R
GR
Doting
Edwina M H
EM
Maas
Saskia M
SM
Wolthers
Katja C
KC
Grand
Richard J
RJ
Montgomery
Robert K
RK
D000818:Animals; D018938:Caco-2 Cells; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D010694:Lactase-Phlorizin Hydrolase / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D010694:Lactase-Phlorizin Hydrolase