Multiple regulatory regions control the complex expression pattern of the mouse Cdx2 homeobox gene

Background & Aims: The Cdx2 homeobox gene exerts multiple functions including trophectoderm specification, antero-posterior patterning, and determination of intestinal identity. The aim of this study was to map genomic regions that regulate the transcription of Cdx2, with a particular interest in the gut. Methods: Genomic fragments covering 13 kilobase (kb) of the mouse Cdx2 locus were analyzed in transgenic mice and in cell assays. Results: No fragment was active in the trophectoderm. Fragments containing the first intron and extending up to -5-kb upstream of the transcription start site became active posteriorly at gastrulation and then inactive at midgestation in every tissue including the endoderm. Specific persistence of activity in the intestinal endoderm/epithelium beyond midgestation requires extending the genomic fragment up to -9 kb. We identified a 250-base pair segment around -8.5-kb binding and responding to endodermal factors, with a stimulatory effect exerted synergistically by HNF4 alpha, GATA6, Tcf4, and beta-catenin. These factors were able to activate endogenous expression of Cdx2 in nonintestinal Hela cells. Conclusions: Multiple regulatory regions control the complex developmental pattern of Cdx2, including far upstream sequences required for the persistence of gene expression specifically in the gut epithelium throughout life. Cooperation between HNF4 alpha, GATA6, beta-catenin, and Tcf4 contributes to the intestine-specific expression of Cdx2.