Functional interaction between the homeoprotein CDX1 and the transcriptional machinery containing the TATA-binding protein

We have previously reported that the CDX1 homeoprotein interacts with the TATA-box binding protein (TBP) on the promoter of the glucose-6-phosphatase (G6Pase) gene. We show here that CDX1 interacts with TBP via the homeodomain and that the transcriptional activity additionally requires the N-terminal domain upstream of the homeodomain. CDX1 interacting with TBP is connected to members of the TFIID and Mediator complexes, two major elements of the general transcriptional machinery. Transcription luciferase assays performed using an altered-specificity mutant of TBP provide evidence for the functionality of the interaction between CDX1 and TBP. Unlike CDX1, CDX2 does not interact with TBP nor does it transactivate the G6Pase promoter. Swapping experiments between the domains of CDX1 and CDX2 indicate that, despite opposite functional effects of the homeoproteins on the G6Pase promoter, the N-terminal domains and homeodomains of both CDX1 and CDX2 have the intrinsic ability to activate transcription and to interact with TBP. However, the carboxy domains define the specificity of CDX1 and CDX2. Thus, intra-molecular interactions control the activity and partner recruitment of CDX1 and CDX2, leading to different molecular functions

CDX2 autoregulation in human intestinal metaplasia of the stomach: impact on the stability of the phenotype

Background and aims Intestinal metaplasia (IM) is a gastric preneoplastic lesion that appears following Helicobacter pylori infection and confers an increased risk for development of cancer. It is induced by gastric expression of the intestine-specific transcription factor CDX2. The regulatory mechanisms involved in triggering and maintaining gastric CDX2 expression have not been fully elucidated. The Cdx2(+/-) mouse develops intestinal polyps with gastric differentiation and total loss of Cdx2 expression in the absence of structural loss of the second allele, suggesting a regulatory defect. This putative haplo-insufficiency, together with the apparent stability of IM, led to the hypothesis that CDX2 regulates its own expression through an autoregulatory loop in both contexts.Methods Gastrointestinal cell lines were co-transfected with wild-type or mutated Cdx2 promoter constructs and CDX2 expression vector for luciferase assays. Transfection experiments were also used to assess endogenous CDX2 autoregulation, evaluated by RT-PCR, qPCR and western blotting. Chromatin immunoprecipitation was performed in a cell line, mouse ileum and human IM. Results CDX2 binds to and transactivates its own promoter and positively regulates its expression in gastrointestinal human carcinoma cell lines. Furthermore, CDX2 is bound to its promoter in the mouse ileum and in human gastric IM, providing a major contribution to understanding the relevance of this autoregulatory pathway in vivo.Conclusion The results of this study demonstrate another layer of complexity in CDX2 regulation by an effective autoregulatory loop which may have a major impact on the stability of human IM, possibly resulting in the inevitable progression of the gastric carcinogenesis pathway

CDX2 expression in the hematopoietic lineage promotes leukemogenesis via TGFβ inhibition

The intestine-specific caudal-related homeobox gene-2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane-bound inhibitor (Bambi), an inhibitor of transforming growth factor-β (TGF-β) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2-expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF-β-dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF-β signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment

The Cdx2 homeobox gene suppresses intestinal tumorigenesis through non-cell-autonomous mechanisms

Developmental genes contribute to cancer, as reported for the homeobox gene Cdx2 playing a tumor suppressor role in the gut. In this study, we show that human colon cancers exhibiting the highest reduction in CDX2 expression belong to the serrated subtype with the worst evolution. In mice, mosaic knockout of Cdx2 in the adult intestinal epithelium induces the formation of imperfect gastric-type metaplastic lesions. The metaplastic knockout cells do not spontaneously become tumorigenic. However, they induce profound modifications of the microenvironment that facilitate the tumorigenic evolution of adjacent Cdx2-intact tumor-prone cells at the surface of the lesions through NF-κB activation, induction of inducible nitric oxide synthase, and stochastic loss of function of Apc. This study presents a novel paradigm in that metaplastic cells, generally considered as precancerous, can induce tumorigenesis from neighboring nonmetaplastic cells without themselves becoming cancerous. It unveils the novel property of non-cell-autonomous tumor suppressor gene for the Cdx2 gene in the gut

Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells

Cdx2, a gene of the paraHox cluster, encodes a homeodomain transcription factor that plays numerous roles in embryonic development and in homeostasis of the adult intestine. Whereas Cdx2 exerts a tumor suppressor function in the gut, its abnormal ectopic expression in acute leukemia is associated to a pro-oncogenic function. To try to understand this duality, we have hypothesized that Cdx2 may interact with different protein partners in the two tissues and set up experiments to identify them by tandem affinity purification. We show here that Cdx2 interacts with the Ku heterodimer specifically in intestinal cells, but not in leukemia cells, via its homeodomain. Ku proteins do not affect Cdx2 transcriptional activity. However, Cdx2 inhibits in vivo and in vitro the DNA repair activity mediated by Ku proteins in intestinal cells. Whereas Cdx2 does not affect the recruitment of Ku proteins and DNA-PKcs into the DNA repair complex, it inhibits DNA-PKcs activity. Thus, we report here a new function of Cdx2, acting as an inhibitor of the DNA repair machinery, that may contribute to its tumor suppressor function specifically in the gut

Cdx2 Controls Expression of the Protocadherin Mucdhl, an Inhibitor of Growth and beta-Catenin Activity in Colon Cancer Cells

BACKGROUND & AIMS: The intestine-specific homeobox transcription factor Cdx2 is an important determinant of intestinal identity in the embryonic endoderm and regulates the balance between proliferation and differentiation in the adult intestinal epithelium. Human colon tumors often lose Cdx2 expression, and heterozygous inactivation of Cdx2 in mice increases colon tumorigenesis. We sought to identify Cdx2 target genes to determine how it contributes to intestinal homeostasis. METHODS: We used expression profiling analysis to identify genes that are regulated by Cdx2 in colon cancer cells lines. Regulation and function of a potential target gene were further investigated using various cell assays. RESULTS: In colon cancer cell lines, Cdx2 directly regulated the transcription of the gene that encodes the protocadherin Mucdhl. Mucdhl localized to the apex of differentiated cells in the intestinal epithelium, and its expression was reduced in most human colon tumors. Overexpression of Mucdhl inhibited low-density proliferation of colon cancer cells and reduced tumor formation in nude mice. One isoform of Mucdhl interacted with beta-catenin and inhibited its transcriptional activity. CONCLUSIONS: The transcription factor Cdx2 activates expression of the protocadherin Mucdhl, which interacts with beta-catenin and regulates activities of intestinal cells. Loss of Cdx2 expression in colon cancer cells might reduce expression of Mucdhl and thereby lead to tumor formation

Different expression of the mRNA detected by the cDNA for fetal rat lactase in the jejunum and colon

International audienceTo study the genetical expression of lactase-phlorizin hydrolase in the jejunum and colon of developing rats we cloned the corresponding cDNA in a cDNA library constructed from the intestinal RNA of fetal rats. The structure of this 6.1 kb-long cDNA was similar to that of the cDNA of adult rabbits and humans, indicating that the enzyme was synthesized as a precursor organized into repeated homologous domains. The mRNA for lactase-phlorizin hydrolase accumulated at a constant level throughout the development in the jejunum despite the decline in lactase activity occurring at weaning. In the colon, the cDNA for lactase-phlorizin hydrolase hybridized to a transcript that was similar in size to the jejunal mRNA. This mRNA was transiently found during the first week after birth. Its rate of accumulation defined the level of lactase activity measured at this stage. We conclude that the expression of lactase-phlorizin hydrolase during the development is essentially regulated at the post-transcriptional level in the jejunum, in contrast to the colon where it is regulated at the pretranslational level

Extending the functions of the homeotic transcription factor Cdx2 in the digestive system through nontranscriptional activities

The homeoprotein encoded by the intestinal-specific Cdx2 gene is a major regulator of gut development and homeostasis, also involved in colon cancer as well as in intestinal-type metaplasias when it is abnormally expressed outside the gut. At the molecular level, structure/function studies have demonstrated that the Cdx2 protein is a transcription factor containing a conserved homeotic DNA-binding domain made of three alpha helixes arranged in a helix-turn-helix motif, preceded by a transcriptional domain and followed by a regulatory domain. The protein interacts with several thousand sites on the chromatin and widely regulates intestinal functions in stem/progenitor cells as well as in mature differentiated cells. Yet, this transcription factor also acts trough original nontranscriptional mechanisms. Indeed, the identification of novel protein partners of Cdx2 and also of a splicing variant revealed unexpected functions in the control of signaling pathways like the Wnt and NF-kappa B pathways, in double-strand break DNA repair and in premessenger RNA splicing. These novel functions of Cdx2 must be considered to fully understand the complexity of the role of Cdx2 in the healthy intestine and in diseases

Ultrastructural study of intestinal lactase gene expression

The distribution of the mRNA encoding rat intestinal lactase-phlorizin hydrolase (LPH) was analyzed by ultrastructural in situ hybridization, and compared to the distribution of the cognate protein. Different pictures were obtained depending on the epithelial cell position along the crypt-villus axis: i) cells localized at the crypt base were devoid of LPH mRNA and protein; ii) cells of the crypt-villus junction and of the villus base showed a high level of mRNA but a low amount of enzyme; and iii) enterocytes of the middle and upper part of the villi exhibited an intense protein immunolabeling, but a low content of LPH mRNA. The analysis of the intracellular distribution of the LPH mRNA revealed a gradient of concentration along the cellular axis, as the transcripts were the most abundant in the apical and subapical domains of the enterocytes. In addition, LPH mRNA labeling was often paired or arranged in a circular shape, suggesting that the transcripts may be located in closed vicinity to rounded structures within the cytoplasm. The results indicate that distinct patterns of mRNA and protein occur along the villus height during the continuous process of cell differentiation of the intestinal epithelium. In addition, the emergence of the functional polarity of the enterocytes comes together with the establishment of an intracellular gradient of concentration of the mRNA encoding a brush border enzyme. A link between the intracellular distribution of LPH mRNA molecules and the membranous flow is proposed