Genome-wide analysis of CDX2 binding in intestinal epithelial cells (Caco-2)

Abstract

The CDX2 transcription factor is known to play a crucial role in inhibiting proliferation, promoting differentiation and the expression of intestinal specific genes in intestinal cells. The overall effect of CDX2 in intestinal cells has previously been investigated in conditional knock-out mice, revealing a critical role of CDX2 in the formation of the normal intestinal identity. The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. The ChIP-seq technique combines chromatin immunoprecipitation (ChIP) with next generation sequencing resulting in a high throughput experimental method of identifying direct targets of specific transcription factors. The method was applied to CDX2, leading to the identification of the direct binding of CDX2 to several known and novel target genes in the intestinal cell. Examination of the transcript levels of selected genes verified the regulatory role of CDX2 binding. The results place CDX2 as a key node in a transcription factor network controlling the proliferation and differentiation of intestinal cells.

FIGURE 1.

CDX2 binding is dependent on the level of differentiation. A–R, regulatory regions for 13 different genes (HEPH, CDX2, HNF4α, CDX1, HNF1β, ETS1, TCF7L2, Axin2, HBP1, LGALS8, CDKN2D, CD9, and MEP1A) were selected for investigation based on the CisGenome list of potential CDX2 target genes (supplemental Data S3) and manual inspection of ChIP-seq tags (supplemental data S2). Shown are real-time quantitative PCR with CDX2- and HA (negative control)-ChIP DNA from Caco-2 cells at different stages during differentiation. Day 0 = undifferentiated, Day 4 = initial differentiation, Day 10 = fully differentiated. Data are expressed as mean values ± S.E. (n = 3). CDX2 versus HA ChIP DNA; *, p < 0.05. Day 0 versus Day 4, p < 0.05 in (A–R). Day 0 versus Day 10, p < 0.05 for (A–R, except L and N). Results are representative of at least three independent experiments.

FIGURE 2.

Analysis of CDX2 expression in Caco-2 cells. A, the binding activity of CDX2 protein was determined by EMSA during differentiation (days 0, 4, and 10); lanes 1, 2, and 3, respectively. Lanes 4, 5, and 6 corresponds to days 0, 4, and 10, where the CDX2-DNA complex was supershifted with specific CDX2 antibody. B, the binding activity of CDX2 protein in the normal (lane 1) and two CDX2 knockdown Caco-2 strains (lane 2 and 3) was determined by EMSA. The CDX2 knockdown strain 2 (lane 3) was used in this study.

FIGURE 3.

CDX2 knockdown affects expression of selected genes. A–R, real-time quantitative PCR with CDX2- and HA (negative control)-ChIP DNA from differentiated Caco-2 cells with either normal CDX2 (control) or reduced CDX2 (shRNA) expression. The same regions as in Fig. 2 were analyzed. Results are representative of at least three independent experiments.

FIGURE 4.

CDX2 influences the transcription of promoter/enhancer constructs. The promoters of HEPH, MEP1A, HBP1, HNF4α, and CDX2 were used to create promoter reporter gene constructs. For HNF4α and CDX2, the corresponding enhancer element was cloned into the promoter reporter gene constructs. A–E, Caco-2 cells with normal CDX2 (Control) or reduced CDX2 (shRNA) were transfected with reporter plasmid and the expression vector CMV-βGal. After 48 h luciferase were quantified and normalized to the β-galactosidase activity. -Fold induction was calculated against the basic reading from the pGL4.10. Data are expressed as mean values ± S.E. (n = 4). The difference between control and shRNA for each expression construct is significant, p < 0.01. The results are representative of at least two independent experiments.

FIGURE 5.

The activity of the promoter/enhancer constructs increases during differentiation. The promoter/enhancer reporter gene constructs for HEPH, MEP1A, HBP1, CDKN2D, HNF4α, and CDX2 were transfected into Caco-2 cells with the expression vector CMV-βGal. After 2 and 10 days luciferase were quantified and normalized to the β-galactosidase activity. -Fold induction was calculated against the reading from day 2. Data are expressed as mean values +S.E. (n = 4).

FIGURE 6.

Expression of selected genes in the CDX2 knockdown Caco-2 cells. The levels of mRNA were measured by real-time PCR and normalized to the expression in Caco-2 cells transfected with a negative control shRNA plasmid. n = 3.