Animals, quality and the pursuit of relevance.

In 2021, the National Institutes of Health Advisory Committee to the Director (ACD) announced recommendations to improve the reproducibility of biomedical research using animals. In response, The Jackson Laboratory faculty and institutional leaders identified key strategies to further address this important issue. Taking inspiration from the evolution of clinical trials over recent decades in response to similar challenges, we identified opportunities for improvement, including establishment of common standards, use of genetically diverse populations, requirement for robust study design with appropriate statistical methods, and improvement in public databases to facilitate meta-analyses. In this Perspective, we share our response to ACD recommendations, with a specific focus on mouse models, with the aim of promoting continued active dialogue among researchers, using any animal system, worldwide. Such discussion will help to inform the biomedical community about these recommendations and further support their much-needed implementation.

The Jackson Laboratory Nathan Shock Center: impact of genetic diversity on aging.

Healthspan is a complex trait, influenced by many genes and environmental factors that accelerate or delay aging, reduce or increase disease risk, and extend or reduce lifespan. Thus, assessing the role of genetic variation in aging requires an experimental strategy capable of modeling the genetic and biological complexity of human populations. The goal of the The Jackson Laboratory Nathan Shock Center (JAX NSC) is to provide research resources and training for geroscience investigators that seek to understand the role of genetics and genetic diversity on the fundamental process of aging and diseases of human aging using the laboratory mouse as a model system. The JAX NSC has available novel, deeply characterized populations of aged mice, performs state-of-the-art phenotyping of age-relevant traits, provides systems genetics analysis of complex data sets, and provides all of these resources to the geroscience community. The aged animal resources, phenotyping capacity, and genetic expertise available through the JAX NSC benefit the geroscience community by fostering cutting-edge, novel lines of research that otherwise would not be possible. Over the past 15 years, the JAX NSC has transformed aging research across the geroscience community, providing aging mouse resources and tissues to researchers. All JAX NSC data and tools are publicly disseminated on the Mouse Phenome Database and the JAX NSC website, thus ensuring that the resources generated and expertise acquired through the Center are readily available to the aging research community. The JAX NSC will continue to enhance its ability to perform innovative research using a mammalian model to illuminate novel genotype-phenotype relationships and provide a rational basis for designing effective risk assessments and therapeutic interventions to boost longevity and disease-free healthspan.

Transcription factors GATA4 and HNF4A control distinct aspects of intestinal homeostasis in conjunction with transcription factor CDX2.

Distinct groups of transcription factors (TFs) assemble at tissue-specific cis-regulatory sites, implying that different TF combinations may control different genes and cellular functions. Within such combinations, TFs that specify or maintain a lineage and are therefore considered master regulators may play a key role. Gene enhancers often attract these tissue-restricted TFs, as well as TFs that are expressed more broadly. However, the contributions of the individual TFs to combinatorial regulatory activity have not been examined critically in many cases in vivo. We address this question using a genetic approach in mice to inactivate the intestine-specifying and intestine-restricted factor CDX2 alone or in combination with its more broadly expressed partner factors, GATA4 and HNF4A. Compared with single mutants, each combination produced significantly greater defects and rapid lethality through distinct anomalies. Intestines lacking Gata4 and Cdx2 were deficient in crypt cell replication, whereas combined loss of Hnf4a and Cdx2 specifically impaired viability and maturation of villus enterocytes. Integrated analysis of TF binding and of transcripts affected in Hnf4a;Cdx2 compound-mutant intestines indicated that this TF pair controls genes required to construct the apical brush border and absorb nutrients, including dietary lipids. This study thus defines combinatorial TF activities, their specific requirements during tissue homeostasis, and modules of transcriptional targets in intestinal epithelial cells in vivo.

Spdef deletion rescues the crypt cell proliferation defect in conditional Gata6 null mouse small intestine.

BACKGROUND: GATA transcription factors are essential for self-renewal of the small intestinal epithelium. Gata4 is expressed in the proximal 85% of small intestine while Gata6 is expressed throughout the length of small intestine. Deletion of intestinal Gata4 and Gata6 results in an altered proliferation/differentiation phenotype, and an up-regulation of SAM pointed domain containing ETS transcription factor (Spdef), a transcription factor recently shown to act as a tumor suppressor. The goal of this study is to determine to what extent SPDEF mediates the downstream functions of GATA4/GATA6 in the small intestine. The hypothesis to be tested is that intestinal GATA4/GATA6 functions through SPDEF by repressing Spdef gene expression. To test this hypothesis, we defined the functions most likely regulated by the overlapping GATA6/SPDEF target gene set in mouse intestine, delineated the relationship between GATA6 chromatin occupancy and Spdef gene regulation in Caco-2 cells, and determined the extent to which prevention of Spdef up-regulation by Spdef knockout rescues the GATA6 phenotype in conditional Gata6 knockout mouse ileum. RESULTS: Using publicly available profiling data, we found that 83% of GATA6-regulated genes are also regulated by SPDEF, and that proliferation/cancer is the function most likely to be modulated by this overlapping gene set. In human Caco-2 cells, GATA6 knockdown results in an up-regulation of Spdef gene expression, modeling our mouse Gata6 knockout data. GATA6 occupies a genetic locus located 40 kb upstream of the Spdef transcription start site, consistent with direct regulation of Spdef gene expression by GATA6. Prevention of Spdef up-regulation in conditional Gata6 knockout mouse ileum by the additional deletion of Spdef rescued the crypt cell proliferation defect, but had little effect on altered lineage differentiation or absorptive enterocytes gene expression. CONCLUSION: SPDEF is a key, immediate downstream effecter of the crypt cell proliferation function of GATA4/GATA6 in the small intestine.

Role of GATA factors in development, differentiation, and homeostasis of the small intestinal epithelium.

The small intestinal epithelium develops from embryonic endoderm into a highly specialized layer of cells perfectly suited for the digestion and absorption of nutrients. The development, differentiation, and regeneration of the small intestinal epithelium require complex gene regulatory networks involving multiple context-specific transcription factors. The evolutionarily conserved GATA family of transcription factors, well known for its role in hematopoiesis, is essential for the development of endoderm during embryogenesis and the renewal of the differentiated epithelium in the mature gut. We review the role of GATA factors in the evolution and development of endoderm and summarize our current understanding of the function of GATA factors in the mature small intestine. We offer perspective on the application of epigenetics approaches to define the mechanisms underlying context-specific GATA gene regulation during intestinal development.

GATA6 is required for proliferation, migration, secretory cell maturation, and gene expression in the mature mouse colon.

Controlled renewal of the epithelium with precise cell distribution and gene expression patterns is essential for colonic function. GATA6 is expressed in the colonic epithelium, but its function in the colon is currently unknown. To define GATA6 function in the colon, we conditionally deleted Gata6 throughout the epithelium of small and large intestines of adult mice. In the colon, Gata6 deletion resulted in shorter, wider crypts, a decrease in proliferation, and a delayed crypt-to-surface epithelial migration rate. Staining techniques and electron microscopy indicated deficient maturation of goblet cells, and coimmunofluorescence demonstrated alterations in specific hormones produced by the endocrine L cells and serotonin-producing cells. Specific colonocyte genes were significantly downregulated. In LS174T, the colonic adenocarcinoma cell line, Gata6 knockdown resulted in a significant downregulation of a similar subset of goblet cell and colonocyte genes, and GATA6 was found to occupy active loci in enhancers and promoters of some of these genes, suggesting that they are direct targets of GATA6. These data demonstrate that GATA6 is necessary for proliferation, migration, lineage maturation, and gene expression in the mature colonic epithelium.

Blimp1 regulates the transition of neonatal to adult intestinal epithelium.

In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mother's milk to the adult diet at the end of the suckling period. These complex developmental changes are the result of a genetic programme intrinsic to the gut tube, but its regulators have not been identified. Here we show that transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) is highly expressed in the developing and postnatal intestinal epithelium until the suckling to weaning transition. Intestine-specific deletion of Blimp1 results in growth retardation and excessive neonatal mortality. Mutant mice lack all of the typical epithelial features of the suckling period and are born with features of an adult-like intestine. We conclude that the suckling to weaning transition is regulated by a single transcriptional repressor that delays epithelial maturation.

GATA factors regulate proliferation, differentiation, and gene expression in small intestine of mature mice.

BACKGROUND & AIMS: GATA transcription factors regulate proliferation, differentiation, and gene expression in multiple organs. GATA4 is expressed in the proximal 85% of the small intestine and regulates the jejunal-ileal gradient in absorptive enterocyte gene expression. GATA6 is co-expressed with GATA4 but also is expressed in the ileum; its function in the mature small intestine is unknown. METHODS: We investigated the function of GATA6 in small intestine using adult mice with conditional, inducible deletion of Gata6, or Gata6 and Gata4, specifically in the intestine. RESULTS: In ileum, deletion of Gata6 caused a decrease in crypt cell proliferation and numbers of enteroendocrine and Paneth cells, an increase in numbers of goblet-like cells in crypts, and altered expression of genes specific to absorptive enterocytes. In contrast to ileum, deletion of Gata6 caused an increase in numbers of Paneth cells in jejunum and ileum. Deletion of Gata6 and Gata4 resulted in a jejunal and duodenal phenotype that was nearly identical to that in the ileum after deletion of Gata6 alone, revealing common functions for GATA6 and GATA4. CONCLUSIONS: GATA transcription factors are required for crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene expression in the small intestinal epithelium.

Characterization of expression in mice of a transgene containing 3.3 kb of the human lactase-phlorizin hydrolase (LPH) 5' flanking sequence.

BACKGROUND AND AIM: The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator. METHODS: Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22. RESULTS: In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28 days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7 days and declines significantly by 28 days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells. CONCLUSIONS: Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.

Conditional Gata4 deletion in mice induces bile acid absorption in the proximal small intestine.

BACKGROUND AND AIMS: The transcription factor GATA4 is expressed throughout most of the small intestine except distal ileum, and restricts expression of the apical sodium-dependent bile acid transporter (ASBT), the rate-limiting intestinal bile acid transporter, to distal ileum. The hypothesis was tested that reduction of GATA4 activity in mouse small intestine results in an induction of bile acid transport in proximal small intestine sufficient to restore bile acid absorption and homeostasis after ileocaecal resection (ICR). METHODS: Bile acid homeostasis was characterised in non-surgical, sham or ICR mice using two recombinant Gata4 models in which Asbt expression is induced to different levels. RESULTS: Reduction of intestinal GATA4 activity resulted in an induction of ASBT expression, bile acid absorption and expression of bile acid-responsive genes in proximal small intestine, and a reduction of luminal bile acids in distal small intestine. While faecal bile acid excretion and bile acid pool size remained unchanged, the bile acid pool became more hydrophilic due to a relative increase in tauro-beta-muricholate absorption. Furthermore, proximal induction of Asbt in both Gata4 mutant models corrected ICR-associated bile acid malabsorption, reversing the decrease in bile acid pool size and increase in faecal bile acid excretion and hepatic cholesterol 7alpha-hydroxylase expression. CONCLUSIONS: Reduction of intestinal GATA4 activity induces bile acid absorption in proximal small intestine without inducing major changes in bile acid homeostasis. This induction is sufficient to correct bile acid malabsorption caused by ICR in mice.

GATA4 mediates gene repression in the mature mouse small intestine through interactions with friend of GATA (FOG) cofactors.

GATA4, a transcription factor expressed in the proximal small intestine but not in the distal ileum, maintains proximal-distal distinctions by multiple processes involving gene repression, gene activation, and cell fate determination. Friend of GATA (FOG) is an evolutionarily conserved family of cofactors whose members physically associate with GATA factors and mediate GATA-regulated repression in multiple tissues. Using a novel, inducible, intestine-specific Gata4 knock-in model in mice, in which wild-type GATA4 is specifically inactivated in the small intestine, but a GATA4 mutant that does not bind FOG cofactors (GATA4ki) continues to be expressed, we found that ileal-specific genes were significantly induced in the proximal small intestine (P<0.01); in contrast, genes restricted to proximal small intestine and cell lineage markers were unaffected, indicating that GATA4-FOG interactions contribute specifically to the repression function of GATA4 within this organ. Fog1 mRNA displayed a proximal-distal pattern that parallels that of Gata4, and FOG1 protein was co-expressed with GATA4 in intestinal epithelial cells, implicating FOG1 as the likely mediator of GATA4 function in the small intestine. Our data are the first to indicate FOG function and expression in the mammalian small intestine.

Gata4 and Hnf1alpha are partially required for the expression of specific intestinal genes during development.

The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1alpha (Hnf1alpha) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1alpha regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1alpha resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1alpha had a partial effect ( approximately 50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1alpha protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1alpha mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1alpha that are dependent on the developmental time frame in which they are expressed.

Lactose and lactase--who is lactose intolerant and why?

Lactase-phlorizin hydrolase (LPH) is expressed only in the small intestine and is confined to absorptive enterocytes on the villi with a tightly controlled pattern of expression along the proximal to distal and crypt-villus axes of the intestine. LPH expression is regulated mainly at the level of lactase (LCT) gene transcription that directs 2 phenotypes: a decline in LCT activity (LCT nonpersistence) in mid-childhood in the majority of the world's population, and maintenance of the lactase levels found in infancy (LCT persistence) in people of northern European extraction and scattered populations elsewhere. The molecular mechanisms that regulate these phenotypes are not completely understood. A population genetic association of lactase persistence with 2 single nucleotide polymorphisms in the distal 5'-flanking region of LCT (-13.9T and -22A) has been confirmed in northern Europeans, but this fails to explain lactase persistence found in some African groups. Any hypothesis for the control of lactase expression must reconcile the presence of high levels of activity in early life in all humans and the characteristic loss of activity found subsequently in many but not all people.

Gata4 is essential for the maintenance of jejunal-ileal identities in the adult mouse small intestine.

Gata4, a member of the zinc finger family of GATA transcription factors, is highly expressed in duodenum and jejunum but is nearly undetectable in distal ileum of adult mice. We show here that the caudal reduction of Gata4 is conserved in humans. To test the hypothesis that the regional expression of Gata4 is critical for the maintenance of jejunal-ileal homeostasis in the adult small intestine in vivo, we established an inducible, intestine-specific model that results in the synthesis of a transcriptionally inactive Gata4 mutant. Synthesis of mutant Gata4 in jejuna of 6- to 8-week-old mice resulted in an attenuation of absorptive enterocyte genes normally expressed in jejunum but not in ileum, including those for the anticipated targets liver fatty acid binding protein (Fabp1) and lactase-phlorizin hydrolase (LPH), and a surprising induction of genes normally silent in jejunum but highly expressed in ileum, specifically those involved in bile acid transport. Inactivation of Gata4 resulted in an increase in the goblet cell population and a redistribution of the enteroendocrine subpopulations, all toward an ileal phenotype. The gene encoding Math1, a known activator of the secretory cell fate, was induced approximately 75% (P < 0.05). Gata4 is thus an important positional signal required for the maintenance of jejunal-ileal identities in the adult mouse small intestine.

Hepatocyte nuclear factor-1alpha is required for expression but dispensable for histone acetylation of the lactase-phlorizin hydrolase gene in vivo.

Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1alpha for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1alpha targets lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) in hnf1alpha null mice. We found that in adult jejunum, LPH mRNA in hnf1alpha(-/-) mice was reduced 95% compared with wild-type controls (P < 0.01, n = 4), whereas SI mRNA was virtually identical to that in wild-type mice. Furthermore, SI mRNA abundance was unchanged in the absence of HNF-1alpha along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1alpha occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas, where HNF-1alpha regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1alpha. Finally, we showed that a subset of hypothesized intestinal target genes is regulated by HNF-1alpha in vivo and that this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1alpha is an activator of a subset of intestinal genes and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling.

Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression.

OBJECTIVES: Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb of the 5'-flanking region of the rat LPH gene control the correct tissue, cell, and crypt-villus expression in transgenic animals. METHODS: To examine further the regulation conferred by this region, protein-DNA interactions were studied using DNase I footprint analyses in LPH-expressing and nonexpressing cell lines. Functional delineation of this 5'-flanking sequence was performed using deletion analysis in transient transfection assays. RESULTS: Studies revealed a generally positive activity between -74 and -37 bp, a cell-specific negative region between -210 and -95 bp, and additional elements further toward the 5'-terminus that conferred a highly cell-specific response in reporter activity. Computer analysis of distal regions encompassing identified footprints revealed potential binding sites for various intestinal transcription factors. Co-transfection and electromobility shift assay experiments indicated binding of HNF3beta at three sites relevant to LPH expression. CONCLUSIONS: The data demonstrate that the cell specificity of LPH gene expression depends upon both positive and negative interactions among elements in the first 2 kb of the LPH 5'-flanking region.

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.

Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1alpha.

Calbindin D9k (CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH)2 vitamin D (<2-fold) in the Caco-2 clone TC7. Deletion-mutation studies revealed that conserved promoter sequences for cdx-2 (at -3158 bp) and hepatocyte nuclear factor (HNF)-1 (at -3131 and at -98 bp) combined to control CaBP expression during differentiation. Other putative response elements were not important for CaBP regulation in TC7 cells (CCAAT enhancer binding protein, pancreatic duodenal homebox-1 (pdx-1), a proximal cdx-2 element). Mutation of the distal HNF-1 site had the greatest impact on CaBP gene expression through disruption of HNF-1alpha binding; both basal and differentiation-mediated CaBP expression was reduced by 80%. In contrast, mutation of the distal cdx-2 element reduced only basal CaBP expression. Whereas a 60% reduction of CaBP mRNA in the duodenum of HNF-1alpha null mice confirmed the physiological importance of HNF-1alpha for CaBP gene regulation, additional studies showed that maximal CaBP expression requires the presence of both HNF-1alpha and cdx-2. Our data suggest that cdx-2 is a permissive factor that influences basal CaBP expression in enterocytes and that HNF-1alpha modulates CaBP gene expression during cellular differentiation.

Novel interaction at the Cdx-2 binding sites of the lactase-phlorizin hydrolase promoter.

Cdx-2 is an intestine-specific homeodomain-containing transcription factor that activates the promoters of intestinal genes through specific interactions with the consensus, TTTAT/C. Here, we demonstrate that Cdx-2 interacts with the lactase-phlorizin hydrolase (LPH) promoter at cis-element (CE)-LPH1a (-54 to -40 bp) as well as the LPH TATA-box. Affinity comparisons between SIF-1, CE-LPH1a, and the LPH TATA-box revealed that the TATA-box has the lowest affinity for Cdx-2. Characterization of CE-LPH1a using EMSAs revealed binding of a novel, non-Cdx-2 complex in multiple cell lines that bind to sequence that is different from that of the Cdx-2 binding site. Heterologous promoter analysis in transient transfection assays revealed a repressor function for this protein, and thus, it was designated as nuclear factor-LPH1/repressor (NF-LPH1/R). These data are consistent with the hypothesis that NF-LPH1/R represses LPH gene expression in non-Cdx-2-producing cells, and that this repression is released in cells that synthesize Cdx-2, such as those in the intestinal epithelium.

Hepatocyte nuclear factor-1 alpha, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription. Implication for the developmental regulation of the sucrase-isomaltase gene.

Sucrase-isomaltase (SI), an intestine-specific gene, is induced in the differentiated small intestinal villous epithelium during the suckling-weaning transition in mice. We have previously identified cis-acting elements within a short evolutionarily conserved SI promoter. However, the nature and profile of expression of the interacting proteins have not been fully characterized during this developmental transition. Herein, we show that hepatocyte nuclear factor-1 alpha (HNF-1 alpha), GATA-4, and caudal related homeodomain proteins Cdx2 and Cdx1 are the primary transcription factors from the adult mouse intestinal epithelium to interact with the SIF3, GATA, and SIF1 elements of the SI promoter. We wanted to study whether HNF-1 alpha, GATA-4, and Cdx2 can cooperate in the regulation of SI gene expression. Immunolocalization experiments revealed that HNF-1 alpha is detected in rare epithelial cells of suckling mice and becomes progressively more expressed in the villous epithelial cells during the suckling-weaning transition. GATA-4 protein is expressed exclusively in villous differentiated epithelial cells of the proximal small intestine, decreases in expression in the ileum, and becomes undetectable in the colon. HNF-1 alpha, GATA-4, and Cdx2 interact in vitro and in vivo. These factors activate SI promoter activity in cotransfection experiments where GATA-4 requires the presence of both HNF-1 alpha and Cdx2. These findings imply a combinatory role of HNF-1 alpha, Cdx2, and GATA-4 for the time- and position-dependent regulation of SI transcription during development.