A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP.

Morphogenesis of the digestive tract is the result of several steps through development beginning with gross morphogenesis of the organ and then cytodifferentiation of the epithelium and induction of intestine-specific genes in the establishment of the functional adult epithelium (40). In the mouse small intestine, cytodifferentiation occurs between embryonic days 14 and 15 with the transition from a stratified epithelium to a columnar epithelium with nascent villi. Small intestine villi lengthen and crypts form through the first two postnatal weeks. The early development of the mouse colon shows transient similarities to that of the small intestine, with the formation of villus structures. This temporary colonic architecture regresses early during development to form the adult colonic mucosa (39). Early developmental similarities between the small intestine and colon in humans have also been described (21). The sucrase-isomaltase (SI) gene represents a useful model to elucidate the mechanisms of intestinal development. This gene encodes an enzyme that is expressed in the brush border of mature enterocytes in a complex developmental pattern (19, 20, 42). A low level of SI gene expression is first detectable in the mouse embryonic intestine, and this expression remains stable through the first 2 weeks of life after birth. A similar low and transient level of expression is also detected in the colon during early postnatal life (42). Between days 16 and 17, there is a dramatic induction of SI expression in enterocytes located at the crypt-villus junction in the small intestine (42). Similar transient expression of SI, as well as other intestinal brush border enzymes, has also been observed in the human fetal colon (48). It has been suggested that the mechanisms directing transient SI expression in the colon are recapitulated in the process of human colonic neoplasia, with the reexpression of SI in the majority of colonic adenomatous polyps and adenocarcinomas (4, 9, 45). A comparable recurrence of SI expression was also found in rodent colon tissues subjected to oncogene-mediated transformations (29). Therefore, the delineation of the molecular mechanisms involved in intestinal SI gene regulation during development could also provide insights into the molecular alterations that occur during colonic neoplasia. An evolutionarily conserved SI promoter contains three major positive regulatory elements, SIF1 (SI footprint 1), SIF2, and SIF3 (41). Caudal-related homeodomain proteins (Cdx1 and Cdx2) interact with the SIF1 element and induce gene transcription in vitro (36, 38). The SIF2 and SIF3 elements of the human SI promoter interact with hepatocyte nuclear factor 1 (HNF-1) proteins to regulate transcription (47). The SIF3 element is essential to support SI promoter activity in the mouse intestinal epithelium (7). Similar to the pattern for the endogenous gene, this short SI promoter directs transcription to mouse enterocytes in developmental and differentiation-dependent patterns and fails to support expression in colonocytes. Taken together, these studies confirm the importance of complementary activators for regulating SI gene expression. By extension, the presence of significant amounts of HNF-1 (32) and Cdx proteins (33) in the colonic epithelium supports the hypothesis that transcriptional repression rather than lack of activation is involved in the colonic regulation of SI expression. To identify regulatory elements that may repress SI expression during postnatal development, we performed a computer-based analysis of the short SI promoter (46). We identified a region between nucleotides −73 and −64 which predicted an overlapping consensus site for both the CDP (CCAAT displacement protein)/Cut repressor and GATA activators of transcription. Murine Cux/CDP and human CDP/Cut are transcriptional repressors closely related to the cut protein of Drosophila melanogaster (26, 43). CDP homologues have in common a unique homeodomain and three similar regions, designated cut repeats (CR), which function as DNA binding domains (2, 13). Several reports, mainly based on gene expression studies, suggest that CDP homologues function as transcriptional repressors by direct competition with transcriptional activators (1, 16, 18, 43) or by active repression by interacting with the HDAC1 protein to promote histone deacetylation (17, 18). Although different roles for the Cux/CDP protein have been proposed in a variety of distinct cell lineages, the potential role for this protein in the intestine still remains unexplored. In addition to a Cux/CDP binding element, a GATA consensus site overlaps within this element. GATA transcription factors are characterized by a highly conserved DNA binding domain consisting of two zinc fingers of the motif Cys-X2-Cys-X17-Cys-X2-Cys (22). GATA-4, -5, and -6 are expressed in various mesoderm- and endoderm-derived tissues such as heart, liver, lung, gonad, and gut, where they play critical roles in regulating tissue-specific gene expression (22). The main objective of this study was to evaluate the functional role of the CDP-GATA site in the regulation of SI promoter activity during intestinal development. We report that Cux/CDP and GATA-4 proteins are capable of interacting with this overlapping site, named CRESIP (colon-repressive element of the SI promoter). We present evidence that the novel CRESIP is crucial for colonic repression of the SI promoter and that Cux/CDP acts through this site to repress SI.