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

Abstract

Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.

FIG. 1.

Functional importance of the common GATA and Cux/CDP sites for SI promoter activity in the mouse intestine. (A) Distribution and comparison of the GATA and Cux/CDP consensus sites within the mouse SI promoter. The two putative overlapping sites are aligned to consensus sites. The single nucleotide difference between the CDP consensus site and the SI promoter sequence is boxed. The GAT-to-TCG mutation is also indicated. (B) RNase protection analysis of hGH transgene expression. Total RNA (5 μg) isolated from intestinal tissues of a transgenic line with the genomic integrated −201 to +54 WT mSI-hGH construct (42) was used as a reference control in an RNase protection assay for the simultaneous detection of hGH and mSI mRNA. (C) Total RNA was isolated from intestinal tissues of three different adult founder (Fo) lines obtained with the −201 to +54 MUT mSI-hGH construct and was analyzed as described for panel B. Du, duodenum; PJ, proximal jejunum; DJ, distal jejunum; IL, ileum; Ce, cecum; PC, proximal colon; DC, distal colon.

FIG. 2.

Profile of transgene expression along the horizontal axis of the intestine during mouse postnatal (pn) development. Small intestines (A) and large intestines (B) of SI MUT transgenic mice were disposed and fixed in a Swiss roll configuration, and 5-μm-thick sections were subjected to a radioactive in situ hybridization with an antisense hGH probe (hGH) and exposed on a BIOMAX MR film for 24 h. Serial sections were counterstained with H&E to visualize the entire length of each intestinal segment. The proximal (prox.) and distal (dist.) intestinal extremities are indicated for each section.

FIG. 3.

Profile of transgene expression along the vertical axis of the small intestine during mouse postnatal development. Small intestine sections from postnatal day 17 (A) and adult (B to E) transgenic mice were subjected to radioactive (A, B, and E) or nonisotopic (C and D) in situ hybridization with an antisense hGH probe (AS hGH). Sections were counterstained with H&E when indicated. Black arrow, cytoplasm-restricted staining; red arrow, empty nucleus.

FIG. 4.

Profile of transgene expression along the vertical axis of the proximal colon during mouse postnatal development. Sections of proximal colons from postnatal (pn) days 4 and 17 and adult SI MUT (A to C) or SI WT (D) transgenic mice were subjected to radioactive (A and D) or nonisotopic (B and C) in situ hybridization with an antisense hGH probe (AS hGH). Sections were counterstained with H&E when indicated. Black arrow, cytoplasm-restricted staining; red arrow, nucleus.

FIG. 5.

Characterization of Cux/CDP and GATA interaction with CRESIP. (A) GST fusion proteins for each different CR and HD domain of the Cux/CDP protein were generated (top) and calibrated by SDS-PAGE and Coomassie staining (middle). Arrow, partial cleavage of the GST tag. GST fusion proteins (GST-fus.; 50 ng ) were used for EMSA with labeled WT-CRESIP or MUT-CRESIP oligonucleotides (bottom). Competitions (Comp) were performed with a 1,000-fold molar excess of the WT-CRESIP or MUT-CRESIP unlabeled oligonucleotides. (B) A GST-Cux/CDP mutant (mut) protein that lacked portions of the N-terminal HD- and C-terminal regions was constructed (top). Western blot analysis was performed with a rabbit anti-GST polyclonal antibody on a GST affinity-purified bacterial extract that overexpressed the GST-Cux/CDPmut protein (middle). The GST-Cux/CDPmut fusion protein was used for EMSA and was competed with a 1,000-fold molar excess of the WT-CRESIP or MUT-CRESIP unlabeled oligonucleotides (bottom). (C) Nuclear extracts of Cos-7 cells (5 μg) transfected with an empty (ctl) or GATA-4 (G4) expression vector were used for each binding reaction. Competitions (Comp) were performed with a 100-fold molar excess of WT-CRESIP or MUT-CRESIP unlabeled oligonucleotides.

FIG. 6.

Repressive effect of Cux/CDP on SI transcriptional activity in Caco-2 cells. (A) Caco-2 cells were transfected at low confluence (10 to 20%) with Lipofectamine and with 300 ng of either −201 to +54 mSI-pGL2basic (WT-SI/pGL2) or −201 + 54 GATmut mSI-pGL2basic (MUT-SI/pGL2) reporter vectors, 100 ng of simian virus 40-β-galactactosidase (βgal), and different combinations of pRC/CMV-Cdx2 (25 ng), pRC/CMV-Cux/CDP (600 ng), and pRC/CMV-Cux/CDPmut (600 ng) expression vectors, as indicated. The pRC/CMV plasmid was used as an empty control vector to calibrate the various amounts of expression vectors used in each condition. Results obtained in triplicate were reported as fold differences (means ± standard deviations) from results for transfection with the reporter construct alone and are representative of three independent experiments. (B) Western blot analysis with CDP (26) and Cdx2 antibodies was performed on protein extracts from Caco-2 cells cotransfected as described for panel A. The molecular mass for each protein is indicated. CDP/Cut, endogenous human CDP; Cux/CDP, transfected mouse CDP.

FIG. 7.

Expression profile of Cux/CDP protein along the vertical and horizontal axes of adult mouse intestine. (A to C) Immunohistochemistry was performed with a CDP polyclonal antibody on paraffin sections of adult mouse jejunum (A), ileum (B), and proximal colon (C). The CDP signal is brown (DAB). Black arrows, nuclear staining; red arrows cytoplasmic staining; black arrowheads, cellular components of the lamina propria. Magnification, ×170. (D) Western blot analysis was performed with CDP and YY1 antibodies on similar amounts of nuclear protein extracted from isolated adult mouse intestinal epithelial cells. PJ, proximal jejunum; IL, ileum; PC, proximal colon.

FIG. 8.

SI mRNA expression is altered in CDP^ΔHD/ΔHD mutant mice. (A) Western blot analysis was performed with CDP (26) and Cdx2 antibodies on similar amounts of colonic protein extracts. Arrows, Cux/CDP wild-type (wt) and truncated mutant protein (mut), respectively. (B) Total RNA (5 μg) isolated from intestinal tissues of CDP control (+/ΔHD) and mutant (ΔHD/ΔHD) mice was analyzed by an RNase protection assay for mSI and m36B4 mRNA. Sm. Int., small intestine.