The transcriptional regulation of intestinal epithelial development and adenomatous polyposis coli tumour suppressor gene expression by Dlx homeobox genes
Fonseca, Mario Alberto
Introduction: Colorectal cancer (CRC) is the fourth-most common cancer in Canada with a high mortality rate. Familial adenomatous polyposis (FAP) is a hereditary form of CRC; FAP patients carry germline mutations of the tumour suppressor gene adenomatous polyposis coli (APC). The function of Dlx genes in the gastrointestinal tract (GIT) has not been previously explored. Methods: Immunofluorescence (IF) was performed to identify Dlx2+ intestinal cells. Chromatin immunoprecipitation (ChIP) was performed to identify DLX2-Apc promoter interaction. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on mouse small and large intestines (normal and Dlx1/Dlx2 mutant mice). Electrophoretic mobility shift assays (EMSA) and reporter assays were carried out to investigate direct binding and activity, respectively, of DLX2 on the Apc promoter in-vitro. Dlx2 expression was explored in ApcMIN mice and human CRC tumor specimens. Results: Dlx2 is highly expressed in mouse embryonic and adult intestinal epithelia. Moreover, Dlx2 is expressed in the ApcMIN mice GIT as well as in some human CRC tumor specimens. ChIP, EMSA and reporter gene assays demonstrated that DLX2 protein specifically interacts with the Apc promoter in-situ and activates its expression in vitro. In-vivo and in-vitro, β-catenin protein levels are increased when DLX2 is absent or reduced by shRNA to Dlx2. Conclusions: Regulation of APC expression during development is poorly understood. We have evidence that DLX2 interacts with the Apc promoter in-vivo. We have shown that DLX2 induces Apc transcription by directly binding to the Apc promoter in-vitro. We also showed that β-catenin expression is altered in the Dlx1/Dlx2 mutant GIT. This finding implicates the involvement of DLX2 in the canonical Wnt signalling pathway. Ultimately, restoring APC expression may be a novel strategy towards preventing progression of intestinal polyps to adenocarcinoma. This research will contribute to our knowledge of the genetic and epigenetic regulatory pathways that control intestinal development, mucosal self-renewal and CRC.
transcription, intestine, homeobox, Distal-less, regulation, development