CDX2 regulates interleukin-33 gene expression in intestinal epithelial cells (LS174T).

Dysregulation of interleukin-33 (IL-33) has been implicated in the pathogenesis of several autoimmune and inflammatory diseases, but few studies have examined transcriptional regulation of the IL33 gene. In the intestines, gene regulation is controlled by a transcription factor network of which the intestinal-specific transcription factor CDX2 is a key component. In this study, we investigated whether CDX2 regulates IL33 mRNA expression. We examined IL33 mRNA expression in primary colonic epithelial cells from healthy humans and epithelial cell lines, revealing high expression levels in primary colonic and LS174T cells. Combining genomics data (ChIP-seq, RNA-seq) and IL33 promoter analyses in LS174T cells revealed intronic enhancer activity in the IL33 gene that is dependent on CDX2 expression. Western blotting and qRT-PCR confirmed that IL33 expression is upregulated in a CDX2 concentration-dependent manner, thereby providing the first evidence that CDX2 regulates the expression of IL33.

CDX2 expression and perioperative patient serum affects the adhesion properties of cultured colon cancer cells.

BACKGROUND: Colon cancer is one of the most commonly diagnosed types of cancer with surgical resection of the tumor being the primary choice of treatment. However, the surgical stress response induced during treatment may be related to a higher risk of recurrence. The aim of this study was to examine the effect of surgery on adhesion of cultured colon cancer cells with or without expression of the tumour suppressor CDX2. METHOD: We enrolled 30 patients undergoing elective, curatively intended laparoscopic surgery for colon cancer in this study. Blood samples were drawn 1 day prior to surgery and 24 h after surgery. The samples of pre- and postoperative serum was applied to wild type colon cancer LS174T cells and CDX2 inducible LS174T cells and adhesion was measured with Real-Time Cell-Analysis iCELLigence using electrical impedance as a readout to monitor changes in the cellular adhesion. RESULTS: Adhesion abilities of wild type LS174T cells seeded in postoperative serum was significantly increased compared to cells seeded in preoperative serum. When seeding the CDX2 inducible LS174T cells without CDX2 expression in pre- and postoperative serum, no significant difference in adhesion was found. However, when inducing CDX2 expression in these cells, the adhesion abilities in pre- and postoperative serum resembled those of the LS174T wild type cell line. CONCLUSIONS: We found that the adhesion of colon cancer cells was significantly increased in postoperative versus preoperative serum, and that CDX2 expression affected the adhesive ability of cancer cells. The results of this study may help to elucidate the pro-metastatic mechanisms in the perioperative phase and the role of CDX2 in colon cancer metastasis.

HNF4α and CDX2 Regulate Intestinal YAP1 Promoter Activity.

The Hippo pathway is important for tissue homeostasis, regulation of organ size andgrowth in most tissues. The co-transcription factor yes-associated protein 1 (YAP1) serves as a maindownstream effector of the Hippo pathway and its dysregulation increases cancer development andblocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation ofYAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene andtranscription factors important for intestinal expression. Bioinformatic analysis of caudal typehomeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitatedDNA from differentiated Caco-2 cells revealed potential intragenic enhancers in the YAP1 gene.Transfection of luciferase-expressing YAP1 promoter-reporter constructs containing the potentialenhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco-2 and T84cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silicotranscription factor binding site analysis and protein-DNA binding was confirmed in vitro usingelectrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experimentsthat CDX2 and HNF4α bind to the YAP1 enhancer in Caco-2 cells. These results reveal a previouslyunknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for highexpression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding areimportant for the YAP1 enhancer activity in intestinal epithelial cells.

The VTI1A-TCF4 colon cancer fusion protein is a dominant negative regulator of Wnt signaling and is transcriptionally regulated by intestinal homeodomain factor CDX2.

Sequencing of primary colorectal tumors has identified a gene fusion in approximately 3% of colorectal cancer patients of the VTI1A and TCF7L2 genes, encoding a VTI1A-TCF4 fusion protein containing a truncated TCF4. As dysregulation of the Wnt signaling pathway is associated with colorectal cancer development and progression, the functional properties and transcriptional regulation of the VTI1A-TCF4 fusion protein may also play a role in these processes. Functional characteristics of the VTI1A-TCF4 fusion protein in Wnt signaling were analyzed in NCI-H508 and LS174T colon cancer cell lines. The NCI-H508 cell line, containing the VTI1A-TCF7L2 fusion gene, showed no active Wnt signaling, and overexpression of the VTI1A-TCF4 fusion protein in LS174T cells along with a Wnt signaling luciferase reporter plasmid showed inhibition of activity. The transcriptional regulation of the VTI1A-TCF4 fusion gene was investigated in LS174T cells where the activity of the VTI1A promoter was compared to that of the TCF7L2 promoter, and the transcription factor CDX2 was analyzed for gene regulatory activity of the VTI1A promoter through luciferase reporter gene assay using colon cancer cell lines as a model. Transfection of LS174T cells showed that the VTI1A promoter is highly active compared to the TCF7L2 promoter, and that CDX2 activates transcription of VTI1A. These results suggest that the VTI1A-TCF4 fusion protein is a dominant negative regulator of the Wnt signaling pathway, and that transcription of VTI1A is activated by CDX2.

Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression.

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.