Abstract
The lactase-phlorizin hydrolase (LPH) gene is expressed specifically in the enterocytes of the small intestine. LPH levels are high in newborn mammals, but decrease after weaning. We have previously suggested that the promoter element CE-LPH1, located at -40 to -54, plays an important role in this down-regulation, because the DNA binding activity of a nuclear factor that binds to this site is present specifically in small intestinal extracts and is down-regulated after weaning. In an effort to clone CE-LPH1-binding factors, a yeast one-hybrid genetic selection was used, resulting in the isolation of a partial cDNA encoding the human homeodomain protein HOXC11. The full-length HOXC11 sequence was obtained by rapid amplification of cDNA ends. It was shown in a yeast assay and by electrophoretic mobility shift assay that HOXC11 binds to the CE-LPH1 element with similar specificity to the endogenous intestinal factor. Two HOXC11 transcript sizes were identified by Northern blot analysis. The larger transcript (2.1 kilobase pairs) is likely to contain a translational start site in good context and is present in HeLa cells. The shorter 1.7-kilobase pair transcript, present in HeLa and Caco-2 cells, probably encodes a protein lacking 114 amino acids at the N-terminal end. Both forms of HOXC11 potentiate transcriptional activation of the LPH promoter by HNF1alpha. The expression of HOXC11 mRNA in human fetal intestine suggests a role in early intestinal development.
Highlights
Lactase-phlorizin hydrolase (LPH)1 is a membrane-bound small intestinal enzyme that hydrolyzes the lactose present in milk
Yeast containing the pRS-LPH reporter plasmid were transformed with a cDNA library prepared from differentiated Caco-2 cells, in which cDNA-encoded proteins are expressed as a fusion with the activation domain of the yeast transcriptional activator GAL4 (Fig. 2A)
HOXC11 and Cdx-2 Bind to Proximal Regulatory Elements in LPH and Sucrase-Isomaltase (SI)—We have previously demonstrated that 1 kb of the lactase gene promoter is sufficient to direct small intestine-specific expression and post-weaning decline in transgenic mice [3]
Summary
Lactase-phlorizin hydrolase (LPH) is a membrane-bound small intestinal enzyme that hydrolyzes the lactose present in milk. A factor present in nuclear extracts prepared from pig small intestine was shown to bind to the CELPH1 site at Ϫ40 to Ϫ54 in the promoter, adjacent to the putative TATA-box This DNA binding activity, termed NFLPH1, was found to be intestine-specific and was present at higher levels in newborn pigs than in adult animals [5]. By using competition electrophoretic mobility shift assays (EMSAs) and photoaffinity-labeling, it was suggested that NF-LPH1 in Caco-2 nuclear extracts is functionally related to a nuclear factor binding to the SIF1 element of the sucrase-isomaltase gene. Both factors recognize a TTTA(T/C) core sequence [6]. We have suggested that a repressor binds to the other half-site because a mutation here, which does not affect Cdx-2 binding, leads to higher transcriptional activity of a linked reporter gene [10]
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