Identifying Target Genes Regulated Downstream of Cdx2 by Microarray Analysis

doi:10.1016/j.jmb.2004.01.061

Journal of Molecular Biology

Volume 337, Issue 3, 26 March 2004, Pages 647-660

https://doi.org/10.1016/j.jmb.2004.01.061 Get rights and content

Abstract

The caudal-related homeobox transcription factor (Cdx2) plays an important role in intestinal development, differentiation, and homeostasis. However, only a limited number of Cdx2-regulated target genes have been elucidated. To delineate the molecular mechanism regulated downstream of Cdx2, we aimed to define Cdx2-regulated genes. We engineered a rat intestinal epithelial cell line, IEC-6, with minimal endogenous Cdx2 expression to express exogenous Cdx2. The gene expression patterns for Cdx2-inducing cells and control cells were examined using oligonucleotide arrays. In the present study, differential expression of 23 genes was confirmed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis using gene-specific primers. Increased expression of genes was involved in the Notch signaling pathway, xenobiotic metabolism, enzymes associated with tumor suppression, RNA binding protein, receptors, signal transduction, and transcription factors. The wide-ranging collection of such inducing genes suggests to the functions of Cdx2 in cell fate decision and maintenance of intestinal epithelia.

Introduction

Homeostasis of the gastrointestinal epithelium is maintained by continuous replacement from committed stem cells and subsequent high organization and rapid migration. There has long been interest in the molecular mechanisms that regulate stem cell replication or stem cell fate in normal and cancer tissue. In mammals, a member of the caudal-related homeobox transcription factors Cdx2 appears to have critical functions in intestinal development, differentiation, and maintenance.1., 2. In the early stages of the developing mouse embryo, the Cdx2 gene is broadly expressed; however, in adult tissues, this gene is expressed in the epithelium of the small intestine and colon with undetectable levels in other tissues.³ Mice null for Cdx2 die early in embryonic life (3.5–5 days postcoitum), whereas heterozygotes develop multiple intestinal polyp-like lesions in the proximal colon.⁴ The epithelial cells in the polyps do not express Cdx2. Although initially suggested to be neoplastic, thorough analysis has shown that these polyps are non-cancerous lesions, and that these lesions are gastric-like heteroplasias.5., 6. Cdx2^+/− mice do not spontaneously develop malignant tumors, but they are hypersensitive to chemically induced cancers, indicating that the reduction in Cdx2 expression facilitates tumor progression.⁷ Aoki et al. suggest that the reduced Cdx2 expression is important in colon tumorigenesis through mTOR-mediated chromosomal instability.⁸ Conversely, most differentiated types of colorectal carcinomas has shown strong Cdx2 expression,⁹ and inappropriate expression of Cdx2 has been found in intestinal metaplasia in the stomach and in some gastric carcinomas.10., 11.

In order to further understand the function of Cdx2 in regulating intestinal cell fate determination, we tried to identify the gene targets for Cdx2. A previous study revealed that Cdx2 could induce heparin binding-epidermal growth factor-like growth factor (HB-EGF) in the IEC-6 cell line.¹² The IEC-6 is an immature intestinal stem cell line that was derived from normal rat small intestine. It is well known that Cdx2 expression affects cellular proliferation and differentiation of IEC-6 cells.¹³

Here, we transfected and established the inducible expression system for Cdx2 in IEC-6 cells. Using this cell line, oligonucleotide microarray analysis of 9906 sequences was performed to characterize gene expression profiles following the induction of Cdx2 protein. Here we show that we have identified at least 23 Cdx2-induced genes involved in a broad range of transporters, metabolism enzymes, receptors, signal transduction modifiers, and transcription factors.

Section snippets

Establishment of conditional Cdx2 expression in the intestinal cell line

To investigate the induction of genes by Cdx2 in the intestinal epithelial cells, we first established an inducible system in which Cdx2 expression could be controlled. The clonal IEC-6 cell lines were co-transfected with pTRE2hyg-Cdx2 and pTet-Off and were designated IEC-6 Tet-Off/Cdx2 cells. In these transfectants, the Cdx2 gene and protein expression was turned on when 2 μg/ml of doxycycline (Dox) was removed from the culture medium (Figure 1A and B). To demonstrate that Cdx2 was functional

Conclusions

The approaches described here provide a new view of the gene regulatory network of Cdx2 in intestinal cells. Here we report the identification of novel Cdx2 target genes such as TM4SF4, 17HSD, and E2F3. Although the mechanism of function of Cdx2 to define intestinal identity is still largely unknown, accumulating data will assist to understand the mechanism of proliferation, differentiation, maintenance, and regulatory function of intestinal epithelial cells. In addition, the identification of

Cell culture

IEC-6 and HT-29 cells were obtained from the RIKEN Bioresource Center (Tsukuba, Japan) and American Type Culture Collection (Manassas, VA, USA), respectively. The stably transfected cell line that expresses Cdx2 conditionally was established with the Tet-Off Gene Expression System (BD Biosciences Clontech, Palo Alto, CA, USA). Briefly, cells from an intestinal epithelial cell line, IEC-6, were stably transfected with a pTet-Off plasmid encoding the tetracycline (Tet)-controlled transactivator

Acknowledgements

This research was supported in part by Grant-in-Aid for Young Scientists (B) (KAKENHI 14780424) from the Ministry of Education, Science, Sports and Culture, Japan.

References (56)

D.G. Silberg et al.
Cdx1 and Cdx2 expression during intestinal development
Gastroenterology
(2000)
T. Hinoi et al.
CDX2 regulates liver intestine-cadherin expression in normal and malignant colon epithelium and intestinal metaplasia
Gastroenterology
(2002)
R. James et al.
Structure of the murine homeobox gene cdx-2. Expression in embryonic and adult intestinal epithelium
J. Biol. Chem.
(1994)
T. Hinoi et al.
Loss of Cdx2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon
Am. J. Pathol.
(2001)
Y.Q. Bai et al.
Ectopic expression of homeodomain protein CDX2 in intestinal metaplasia and carcinomas of the stomach
Cancer Letters
(2002)
R. Valdes et al.
Nutritional regulation of nucleoside transporter expression in rat small intestine
Gastroenterology
(2000)
B. Ruiz-Montasell et al.
Early induction of Na⁺-dependent uridine uptake in the regenerating rat liver
FEBS Letters
(1993)
T.R. Tephly et al.
UDP-glucuronosyltransferases: a family of detroxifying enzymes
Trends Pharmacol. Sci.
(1990)
M. Nakamura et al.
Specific increase in calcium-activated neutral protease with low calcium sensitivity (m-calpain) in proerythroblastic K562 cell line cells induced to differentiation by phorbol 12-myristate 13-acetate
Expt. Cell Res.
(1992)
S. Kulkarni et al.
Calpain mediates integrin-induced signaling at a point upstream of Rho family members
J. Biol. Chem.
(1999)

Cited by (34)

The clinical significance of CDX2 in leukemia: A new perspective for leukemia research
2018, Leukemia Research
Citation Excerpt :
CDX2 functions variably depending on the cell and tissue type, so that CDX2 can target genes like insulin [31], proglucagone [32], SI [33], phospholipase A/lysophospholipase [30], carbonic anhydrase 1 [34],vitamin D receptor [30] and HOXc8 [11] (Table1). CDX target genes possessing one or more copies, represent CDX-responsive elements containing TTTAT (A/G) or TTTA (T/C) motifs [35,36]. Gene expression regulations are dependent on the interaction between CDX2 and other transcription factors and the CDX2 phosphorylation status [37–39].
CDX2 gene encodes a transcription factor involved in primary embryogenesis and hematopoietic development; however, the expression of CDX2 in adults is restricted to intestine and is not observed in blood tissues. The ectopic expression of CDX2 has been frequently observed in acute myeloid and lymphoid leukemia which in most cases is concomitant with poor prognosis. Induction of CDX2 in mice leads to hematologic complications, showing the leukemogenic origin of this gene. CDX2 plays significant role in the most critical pathways as the regulator of important transcription factors targeting cell proliferation, multi-drug resistance and survival. On the whole, the results indicate that CDX2 has the potential to be suggested as the diagnostic marker in hematologic malignancies. This review discusses the role of aberrant expression of CDX2 in the prognosis and the response to treatment in patients with different leukemia in clinical reports in the recent decades. The improvement in this regard could be of high importance in diagnosis and treatment methods.
Cdx regulates Dll1 in multiple lineages
2012, Developmental Biology
Citation Excerpt :
More recent evidence has suggested that Cdx members also function through non-Hox targets during both axis elongation and intestinal patterning (Gao et al., 2009; Savory et al., 2009a; Young et al., 2009). Furthermore, microarray and ChIP-seq studies have suggested that Cdx2 may regulate a plethora of other target genes, including Dll1 (Boyd et al., 2010; Uesaka et al., 2004). Our present finding of Dll1 as a Cdx regulated gene extends this paradigm.
Vertebrate Cdx genes encode homeodomain transcription factors related to caudal in Drosophila. The murine Cdx homologues Cdx1, Cdx2 and Cdx4 play important roles in anterior–posterior patterning of the embryonic axis and the intestine, as well as axial elongation. While our understanding of the ontogenic programs requiring Cdx function has advanced considerably, the molecular bases underlying these functions are less well understood. In this regard, Cdx1-Cdx2 conditional mutants exhibit abnormal somite formation, while loss of Cdx1-Cdx2 in the intestinal epithelium results in a shift in differentiation toward the Goblet cell lineage. The aim of the present study was to identify the Cdx-dependent mechanisms impacting on these events. Consistent with prior work implicating Notch signaling in these pathways, we found that expression of the Notch ligand Dll1 was reduced in Cdx mutants in both the intestinal epithelium and paraxial mesoderm. Cdx members occupied the Dll1 promoter both in vivo and in vitro, while genetic analysis indicated interaction between Cdx and Dll1 pathways in both somitogenesis and Goblet cell differentiation. These findings suggest that Cdx members operate upstream of Dll1 to convey different functions in two distinct lineages.
The molecular pathogenesis of Barrett's esophagus: Common signaling pathways in embryogenesis Metaplasia and Neoplasia
2010, Journal of Gastrointestinal Surgery
Although Barrett’s esophagus has been recognized for over 50 years, the cellular and molecular mechanisms leading to the replacement of squamous esophageal epithelium with a columnar type are largely unknown. Barrett’s is known to be an acquired process secondary to chronic gastroesophageal reflux disease and occurs in the presence of severe disruption of the gastroesophageal barrier and reflux of a mixture of gastric and duodenal content. Current hypothesis suggest that epithelial change occurs due to stimulation of esophageal stem cells present in the basal layers of the epithelium or submucosal glands, toward a columnar epithelial differentiation pathway. The transcription factor CDX2 seems to play a key role in promoting the cellular biology necessary for columnar differentiation, and can be induced by bile salt and acid stimulation. Several cellular signaling pathways responsible for modulation of intestinal differentiation have also been identified and include WNT, Notch, BMP, Sonic HH and TGFB. These also have been shown to respond to stimulation by bile acids, acid or both and may influence CDX2 expression. Their relative activity within the stem cell population is almost certainly responsible for the development of the esophageal columnar epithelial phenotype we know as Barrett’s esophagus.
The microenvironment controls CDX2 homeobox gene expression in colorectal cancer cells
2007, American Journal of Pathology
Citation Excerpt :
Further in vivo investigations are needed. Inversely, elements of the Notch pathway have been proposed to be under the control of Cdx2.32 Thus, the mechanism(s) of control of Cdx2 by cell interactions remain elusive.
The homeobox gene CDX2 plays a major role in development, especially in the gut, and it also acts as a tumor suppressor in the adult colon. Using orthotopic and heterotopic xenografts of human primary colorectal tumor cells and cell lines in nude mice, we addressed the effect of the microenvironment on CDX2 expression. In cells expressing CDX2 at a high level in culture, this level was maintained in subcutaneous grafts but was reduced when implanted into the cecum wall. Reciprocally, in cells with low CDX2 expression in culture, the level remained low in grafts into the cecum wall but was stimulated subcutaneously. In vitro co-cultures showed that CDX2 expression was activated in cells grown on layers of skin fibroblasts but not on intestinal fibroblasts. The stimulation was transcriptional, as assessed by transfection experiments with reporter plasmids containing the murine Cdx2 promoter. Together, these data demonstrate experimentally that CDX2 expression is adaptable and strongly dependent on the microenvironment surrounding the tumor cells. We exclude a role of the Notch pathway in this regulation. The regulation of CDX2 by the microenvironment might be relevant during the process of metastatic dissemination when the gene is transiently turned down in invasive cells.
The intestine-specific homeobox gene Cdx2 induces expression of the basic helix-loop-helix transcription factor Math1
2006, Differentiation
The basic helix–loop–helix transcription factor Math1, which is transiently expressed in proliferating neural precursors in multiple domains of the developing nervous system, is also related to the cell fate decision of enteroendocrine, goblet, and Paneth cells in the intestine. On the other hand, the transcription factor Cdx2, which is normally confined to intestinal epithelial cells, is related to the differentiation of these cells. Therefore, we investigated the relationship between Math1 and Cdx2 in intestinal epithelial cells. The Math1 and Cdx2 expressions in normal intestinal mucosa and intestinal metaplastic mucosa from mouse and human stomachs, as well as an intestinal crypt-derived cell line, were analyzed by immunohistochemistry, reverse transcription-polymerase chain reaction and Northern blotting, and Math1 enhancer element was analyzed by luciferase reporter assays. Math1-positive epithelial cells co-expressing Cdx2 were found in normal intestinal mucosa from humans and mice. Furthermore, Math1-producing epithelial cells that showed positive immunostaining for Cdx2 were also observed in intestinal metaplastic mucosa from human and Cdx2 transgenic mouse stomachs, although they were not detected in normal gastric mucosa of humans and mice. Expression of Cdx2 stimulated endogenous Math1 mRNA expression in the intestinal crypt-derived cell line IEC-6, corroborating observations in Cdx2-expressing intestinal metaplastic mucosa. Furthermore, expression of Cdx2 in IEC-6 cells conferred the ability to express a Math1 reporter gene containing a Math1 enhancer. Based on these results, we hypothesize that Cdx2 is involved in activating Math1 expression in intestinal epithelial cells.
Pbx1 is a co-factor for Cdx-2 in regulating proglucagon gene expression in pancreatic A cells
2006, Molecular and Cellular Endocrinology
Citation Excerpt :
The generation and analyses of Cdx-2−/− mice indicated that this caudal gene has multiple biological functions (Chawengsaksophak et al., 1997). Consistently, numerous in vitro studies have identified more than two dozen potential target genes of Cdx-2 (Uesaka et al., 2004). To regulate the expression of different target genes in temporal and spatial manners, Cdx-2 may utilize different co-factors.
A number of Hox and Hox-like homeodomain (HD) proteins have been previously shown to utilize members of the TALE HD protein family as co-factors in regulating gene expression. The caudal HD protein Cdx-2 is a transactivator for the proglucagon gene, expressed in pancreatic A cells and intestinal endocrine L cells. We demonstrate here that co-transfection of the TALE homeobox gene Pbx1 enhanced the activation of Cdx-2 on the proglucagon promoter in either the pancreatic A cell line InR1-G9 or BHK fibroblasts. The activation was observed for proglucagon promoter constructs with or without the binding motifs for Pbx1. Furthermore, mutating the penta-peptide motif (binding motif for TALE HD proteins) on Cdx-2 substantially attenuated its activation on proglucagon promoter, but not on the sucrase–isomaltase gene (SI) promoter, or its own (Cdx-2) promoter; suggesting that Cdx-2 utilizes Pbx1 as a co-factor for regulating the expression of selected target genes. Physical interaction between Cdx-2 and Pbx1 was demonstrated by co-immunoprecipitation as well as GST fusion protein pull-down. We suggest that this study reveals a novel function for Pbx1 in pancreatic islet physiology: regulating proglucagon expression by serving as a co-factor for Cdx-2.

View all citing articles on Scopus

^☆: Supplementary data associated with this article can be found at doi: 10.1016/j.jmb.2004.01.061

View full text

Journal of Molecular Biology

Identifying Target Genes Regulated Downstream of Cdx2 by Microarray Analysis☆

Abstract

Introduction

Section snippets

Establishment of conditional Cdx2 expression in the intestinal cell line

Conclusions

Cell culture

Acknowledgements

Gastroenterology

Gastroenterology

J. Biol. Chem.

Am. J. Pathol.

Cancer Letters

Gastroenterology

FEBS Letters

Trends Pharmacol. Sci.

Expt. Cell Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Brain Res.

J. Biol. Chem.

Brain Res. Mol. Brain Res.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Homeosis and intestinal tumours in Cdx2 mutant mice

Nature

Reprogramming of intestinal differentiation and intercalary regeneration in Cdx2 mutant mice

Proc. Natl Acad. Sci. USA

Colonic hamartoma development by anomalous duplication in Cdx2 knockout mice

Cancer Res.

The Cdx2 homeobox gene has a tumor suppressor function in the distal colon in addition to a homeotic role during gut development

Gut

Colonic polyposis caused by mTOR-mediated chromosomal instability in Apc+/Δ716 Cdx2+/− compound mutant mice

Nature Genet.

Expression of homeobox gene CDX2 precedes that of CDX1 during the progression of intestinal metaplasia

J. Gastroenterol.

Colonic polyposis caused by mTOR-mediated chromosomal instability in Apc^+/Δ716 Cdx2^+/− compound mutant mice