The homeodomain protein Cdx2 regulates lactase gene promoter activity during enterocyte differentiation

doi:10.1016/S0016-5085(00)70420-3

Gastroenterology

Volume 118, Issue 1, January 2000, Pages 115-127

https://doi.org/10.1016/S0016-5085(00)70420-3 Get rights and content

Abstract

Background & Aims: Lactase is the intestinal disaccharidase responsible for digestion of lactose, the predominant carbohydrate in milk. Transcription of the lactase gene is activated during enterocyte differentiation. We have characterized the interaction between the lactase promoter and Cdx2, a homeodomain protein involved in regulating intestinal development and differentiation. Methods: Nuclear protein bound to the lactase gene cis element, CE-LPH1, was analyzed by electrophoretic mobility shift assays and supershifts with Cdx2 antibody. Lactase promoter activities were assayed in cells transfected with luciferase reporter constructs and a Cdx2 expression construct. Results: Electrophoretic mobility shift assay with CE-LPH1 yields a specific DNA/protein complex that requires the caudal-related protein binding site, TTTAC. The complex is recognized by Cdx2 antibody and is more abundant in differentiated enterocytes. A Cdx2 expression construct is able to activate transcription driven by the wild-type, but not a mutated, promoter and results in increased endogenous lactase messenger RNA. Conclusions: The homeodomain protein Cdx2 interacts with the lactase promoter and is capable of activating transcription of the endogenous gene. In contrast to a previous report, Cdx2 interaction with the lactase promoter correlates with enterocyte differentiation. These conclusions are consistent with the role of Cdx2 in regulating intestinal cell differentiation.

GASTROENTEROLOGY 2000;118:115-127

Section snippets

Materials and reagents

Restriction endonucleases and modifying enzymes were purchased from New England Biolaboratories Inc. (Beverly, MA). Radioisotopes were purchased from DuPont NEN (Boston, MA). Oligonucleotides were synthesized in the Molecular Biology Core Laboratory of the Stanford Digestive Disease Center.

Subcloning of rat lactase promoter-reporter constructs and rat Cdx2 expression construct

A rat genomic DNA library (Fisher strain liver in Lambda Dash; Stratagene, La Jolla, CA) was screened by plaque hybridization with a lactase complementary DNA (cDNA) HindIII subfragment corresponding to the

Regulatory regions of lactase gene 5'-flanking DNA mapped by deletional analysis of reporter constructs

To identify regions of the lactase gene capable of mediating regulation of gene transcription, fragments of the 5'-flanking region of the gene were cloned upstream of the firefly luciferase cDNA in the reporter plasmid pGL3 and transfected into preconfluent Caco-2 cells. The −3 kb, −2 kb, −800 bp, and −200 bp lactase reporter constructs, shown in Figure 2, were transiently transfected into Caco-2 cells, a human adenocarcinoma-derived cell line that mimics a small intestinal enterocyte phenotype

Discussion

Expression of the intestinal brush border disaccharidase LPH is restricted to the small intestinal enterocyte and is activated during cell differentiation. Intestine-specific lactase expression is regulated at the level of gene transcription, as evidenced by the absence of lactase mRNA production in nonintestinal cells with the exception of fetal colon. In addition, the increase in lactase mRNA levels in preweaned animals followed by a decrease in the maturing adult is also consistent with

Acknowledgements

The authors thank Drs. G. M. Gray and M. P. Scott for helpful discussion and suggestions.

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^☆: Address requests for reprints to: Eric Sibley, M.D., Ph.D., Department of Pediatrics, Stanford University Medical Center, 750 Welch Road, Suite 116, Palo Alto, California 94305. Fax: (650) 498-5608; e-mail: [email protected].

^☆☆: Supported by the Robert Wood Johnson Foundation Minority Medical Faculty Development Program and the Stanford University Child Health Research Center under National Institutes of Health grant P30-HD33698 (to E.S.).

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Alimentary TractThe homeodomain protein Cdx2 regulates lactase gene promoter activity during enterocyte differentiation☆,☆☆

Abstract

Section snippets

Materials and reagents

Subcloning of rat lactase promoter-reporter constructs and rat Cdx2 expression construct

Regulatory regions of lactase gene 5'-flanking DNA mapped by deletional analysis of reporter constructs

Discussion

Acknowledgements

Gastroenterology

J Biol Chem

Gastroenterology

FEBS Lett

FEBS Lett

FEBS Lett

J Biol Chem

FEBS Lett

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

Anal Biochem

Gastroenterology

J Biol Chem

J Biol Chem

J Biol Chem

Ontogeny of the intestinal mucosa

Alimentary Tract
The homeodomain protein Cdx2 regulates lactase gene promoter activity during enterocyte differentiation☆,☆☆