Lactase-phlorizin Hydrolase Promoter Research Articles

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4.

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.

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.

An upstream polymorphism associated with lactase persistence has increased enhancer activity

Background & Aims : Intestinal lactase activity declines during childhood in some humans. This phenotypic polymorphism of lactase persistence or nonpersistence into adult life has been shown in a recent study to be 100% associated with a T/C nucleotide polymorphism at position −13910 and approximately 97% with an A/G nucleotide polymorphism at position −22018. The aim of this study was to investigate the role of these nucleotide polymorphisms for lactase-phlorizin hydrolase (LPH) gene expression. Methods : The −13910 and −22018 regions were cloned from lactase-persistent and -nonpersistent individuals, and the regions were analyzed for gene regulatory activity of a luciferase reporter gene by transfection experiments using the intestinal cell line Caco-2. Electrophoretic mobility shift assays (EMSAs) were used to investigate protein/DNA interactions with the −13910 sequence. Results : We show that the −13910 region contains a strong enhancer. The −13910 regions from both lactase persistent (−13910T variant) and lactase nonpersistent (−13910C variant) have enhancer activity. However, the −13910T variant enhances the LPH promoter approximately 4 times more than the −13910C variant when analyzed in differentiated Caco-2 cells. A nuclear factor from both an intestinal and a nonintestinal extract binds strongly to the −13910T variant whereas the binding to the −13910C variant is much weaker. Conclusions : The discovery of a functional difference between the 2 alleles at position −13910 supports the notion that the molecular difference between lactase persistence and nonpersistence is caused by the mutation at position −13910.

Differential HNF-1α-independent activation of the human lactase-phlorizin hydrolase promoter between GATA-4 and GATA-5 is due to the zinc finger and basic regions

Differential HNF-1α-independent activation of the human lactase-phlorizin hydrolase promoter between GATA-4 and GATA-5 is due to the zinc finger and basic regions

Physical Interaction between GATA-5 and Hepatocyte Nuclear Factor-1α Results in Synergistic Activation of the Human Lactase-Phlorizin Hydrolase Promoter

GATA-4, -5, and -6 zinc finger and hepatocyte nuclear factor-1alpha (HNF-1alpha) homeodomain transcription factors are expressed in the intestinal epithelium and synergistically activate the promoter of intestinal genes. Here, we demonstrate that GATA-5 and HNF-1alpha physically associate both in vivo and in vitro and that this interaction is necessary for cooperative activation of the lactase-phlorizin hydrolase promoter. Furthermore, physical association is mediated by the C-terminal zinc finger of GATA factors and the homeodomain of HNF-1alpha. Deletion of HNF-1alpha activation domains or interruption of HNF-1-binding sites in the lactase-phlorizin hydrolase promoter resulted in a complete loss of cooperativity, whereas deletion of GATA-5 activation domains or interruption of GATA-binding sites resulted in a reduction, but not an elimination, of cooperativity. We hypothesize that GATA/HNF-1alpha cooperativity is mediated by HNF-1alpha through its activation domains, which are oriented for high levels of activation through binding to DNA and physical association with GATA factors. These data suggest a paradigm whereby intestine-specific gene expression is regulated by unique interactions among tissue-restricted transcription factors coexpressed in the intestine. Parallel mechanisms in other tissues as well as in Drosophila suggest that zinc finger/homeodomain interactions are an efficient pathway of cooperative activation of gene transcription that has been conserved throughout evolution.

Differential activation of intestinal gene promoters: functional interactions between GATA-5 and HNF-1 alpha.

The effects of GATA-4, -5, and -6, hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and -beta, and Cdx-2 on the rat and human lactase-phlorizin hydrolase (LPH) and human sucrase-isomaltase (SI) promoters were studied using transient cotransfection assays in Caco-2 cells. GATA factors and HNF-1 alpha were strong activators of the LPH promoters, whereas HNF-1 alpha and Cdx-2 were strong activators of the SI promoter, although GATA factors were also necessary for maximal activation of the SI gene. Cotransfection of GATA-5 and HNF-1 alpha together resulted in a higher activation of all three promoters than the sum of the activation by either factor alone, demonstrating functional cooperativity. In the human LPH promoter, an intact HNF-1 binding site was required for functional synergy. This study is the first to demonstrate 1) differential activation of the LPH and SI promoters by multiple transcription factors cotransfected singly and in combination and 2) that GATA and HNF-1 transcription factors cooperatively activate intestinal gene promoters. Synergistic activation is a mechanism by which higher levels of tissue-specific expression might be attained by overlapping expression of specific transcription factors.

Physical interaction between GATA-5 and HNF-1α results in synergistic activation of the human lactase-phlorizin hydrolase promoter

Physical interaction between GATA-5 and HNF-1α results in synergistic activation of the human lactase-phlorizin hydrolase promoter

Physical interaction between GATA-5 and HNF-1α results in synergistic activation of the human lactase-phlorizin hydrolase promoter

Physical interaction between GATA-5 and HNF-1α results in synergistic activation of the human lactase-phlorizin hydrolase promoter

Differentiation-dependent activation of the lactase-phlorizin hydrolase promoter by PDX-1

Differentiation-dependent activation of the lactase-phlorizin hydrolase promoter by PDX-1

Interaction between the homeodomain proteins Cdx2 and HNF1α 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 1α (HNF1α), 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 HNF1α 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 HNF1α. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1α. 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.

Interaction between the homeodomain proteins Cdx2 and HNF1α 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.

Transcriptional regulation of pig lactase-phlorizin hydrolase: Involvement of HNF-1 and FREACs

Background & Aims: One-kilobase sequence of the upstream fragment of the pig lactase-phlorizin hydrolase gene has been shown to control small intestinal–specific expression and postweaning decline of lactase-phlorizin hydrolase in transgenic mice. The aim of this study was to identify the regulatory DNA elements and transcription factors controlling lactase-phlorizin hydrolase expression. Methods: The activity of different lactase-phlorizin hydrolase promoter fragments was investigated by transfection experiments using Caco-2 cells. Electrophoretic mobility shift assays and supershift analyses were used to characterize the interaction between intestinal transcription factors and the identified regulatory elements. Results: Functional analysis revealed three previously undescribed regulatory regions in the lactase-phlorizin hydrolase promoter: a putative enhancer between −894 and −798 binding hepatocyte nuclear factor (HNF)-1 at position −894 to −880; a repressor-binding element between −278 to −264 to which an HNF-3–like factor is able to bind; and an element between −178 to −164 that binds an activating transcription factor. Conclusions: Identification of three new regulatory regions and HNF-1 and HNF-3–like transcription factor as players in the regulation of lactase-phlorizin hydrolase gene transcription has an impact on the understanding of the molecular mechanisms behind age-dependent, tissue-specific, differentiation-dependent, and regional regulation of expression in the intestine. GASTROENTEROLOGY 1999;116:842-854

The HOXC11 Homeodomain Protein Interacts with the Lactase-Phlorizin Hydrolase Promoter and Stimulates HNF1α-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 HNF1alpha. The expression of HOXC11 mRNA in human fetal intestine suggests a role in early intestinal development.

GATA-6 stimulates a cell line-specific activation element in the human lactase promoter

Lactase-phlorizin hydrolase (LPH) synthesis is restricted to differentiated small intestinal enterocytes and is highly regulated during development. Analysis of expression of LPH promoter segments fused with luciferase transfected in Caco-2 cells, a line that uniquely expresses LPH mRNA, mapped an 18-base pair (bp) segment 100 bp upstream of the transcription start site that is required for transactivation. Remarkably, the LPH upstream element (LUE) has no stimulatory activity in both human intestinal and nonintestinal lines in which LPH mRNA is absent. Electrophoretic analysis of sequence-specific DNA-nuclear protein complexes demonstrated the presence of a Caco-2 cell-specific protein(s) (CCP), which is uniformly absent in LPH nonproducer cell lines. Mutational analysis of the LUE demonstrated that bases contained within a GATA consensus motif are critical for both CCP binding and transcription from the LPH promoter. Caco-2 cells express high levels of GATA-6 mRNA in a cell line-specific manner, suggesting that GATA-6 is a CCP that complexes with the LUE. When expressed by a plasmid, GATA-6 transactivated the LPH promoter. The stimulation was abrogated with mutations in the GATA consensus motif as well as mutations in a flanking downstream element. These studies are consistent with an important role of an intestinal GATA binding protein in cell type-specific transactivation of the LPH promoter.

Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2.

Lactase-phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase-phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.

1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice

Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity to hydrolyze lactose. We here demonstrate that the stretch −17 to −994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit β-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due to a transcriptional down-regulation.

A novel intestinal trans-factor (NF-LPH1) interacts with the lactase-phlorizin hydrolase promoter and co-varies with the enzymatic activity.

The promoter of the pig lactase-phlorizin hydrolase was cloned and showed to be functional in the human intestinal cell line Caco2. The proximal promoter was analyzed for binding of nuclear proteins from small intestine and liver. DNase I footprinting and electrophoretic mobility shift assays show, that an intestinal nuclear factor (NF-LPH1) binds to a sequence (-40 to -54) located close to the TATA-box. Enterocytes from newborn pigs with high lactase activity contain high amounts of NF-LPH1, whereas enterocytes from adult pigs with low lactase activity contain low amounts of NF-LPH1. The liver does not contain lactase activity, and NF-LPH1 is not present in liver nuclear extracts in detectable amounts. This indicates that NF-LPH1 is involved in the decline of lactase at weaning and may be of importance for the molecular explanation of hypolactasia in humans. It was demonstrated by transfection of two different promoter-reporter gene constructs into Caco2 cells, that there are additional cis-element(s) in the region -142 to approximately -980, which are important for the transcription of the lactase-phlorizin hydrolase gene.