Cathy Mitchelmore

Interaction between the homeodomain proteins Cdx2 and HNF1alpha mediates expression of the lactase-phlorizin hydrolase gene.

Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1alpha (HNF1alpha), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1alpha physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1alpha. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1alpha. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.

THE HOXC11 HOMEODOMAIN PROTEIN INTERACTS WITH THE LACTASE-PHLORIZIN HYDROLASE PROMOTER AND STIMULATES HNF1ALPHA -DEPENDENT TRANSCRIPTION

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 HNF1α. The expression of HOXC11 mRNA in human fetal intestine suggests a role in early intestinal development.

Two intestinal specific nuclear factors binding to the lactase-phlorizin hydrolase and sucrase-isomaltase promoters are functionally related oligomeric molecules.

Lactase‐phlorizin hydrolase (LPH) and sucrase‐isomaltase (SI) are enterocyte‐specific gene products. The identification of regulatory cis‐elements in the promoter of these two genes has enabled us to carry out comparative studies of the corresponding intestinal‐specific nuclear factors (NF‐LPH1 and SIF1‐BP). Electrophoretic mobility shift assays demonstrated that the two nuclear factors compete for binding on the same cis‐elements. The molecular size of the DNA binding polypeptide is estimated to be approximately 50 kDa for both factors. In the native form the factors are found as 250 kDa oligomeric complexes. Based on these results NF‐LPH1 and SIF1‐BP are suggested to be either identical or closely related molecules.