Transcriptional regulation of the lactase-phlorizin hydrolase promoter by PDX-1.
Lactase-phlorizin hydrolase gene expression is spatially restricted along the anterior-posterior gut axis. Lactase gene transcription is maximal in the distal duodenum and jejunum in adult mammals and is barely detectable in the proximal duodenum. By contrast, pancreatic duodenal homeobox-1 (PDX-1) protein is expressed maximally in the proximal duodenum. This study aimed to determine the role of PDX-1 in regulating lactase gene promoter activity in intestinal epithelial cells. Caco-2 cells were cotransfected with lactase promoter-reporter constructs in the presence of a PDX-1 expression vector and assayed for luciferase activity. PDX-1 cotransfection results in repression of lactase promoter activity. Sequence analysis of the lactase promoter revealed a putative PDX-1 DNA binding site in the proximal 100-bp lactase gene promoter. EMSAs demonstrated that PDX-1 can interact with the lactase promoter binding site but not with a site in which the core PDX-1 binding sequence TAAT is mutated. Site-directed mutagenesis of the PDX-1 core binding site in the lactase promoter-reporter construct suggests that PDX-1 can function independently of DNA binding to its consensus binding site. Stable overexpression of PDX-1 results in repression of the endogenous human lactase gene in differentiated Caco-2 cells. Given the contrasting spatial expression pattern, PDX-1 may function to specify the anterior boundary of lactase expression in the small intestine and is thus a candidate regulator of anterior spatial restriction in the gut.
Wang
Zhi
Z
Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94304, USA.
Fang
Rixun
R
Olds
Lynne C
LC
Sibley
Eric
E
D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D004848:Epithelium / Q000201:enzymology / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D018398:Homeodomain Proteins; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism; D010694:Lactase-Phlorizin Hydrolase / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics*
D017931:DNA Primers; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D004247:DNA; D008156:Luciferases; D010694:Lactase-Phlorizin Hydrolase