Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec;24(12):2173-2186.
doi: 10.1038/cdd.2017.140. Epub 2017 Sep 1.

Fine-tuning and Autoregulation of the Intestinal Determinant and Tumor Suppressor Homeobox Gene CDX2 by Alternative Splicing

Affiliations
Free PMC article

Fine-tuning and Autoregulation of the Intestinal Determinant and Tumor Suppressor Homeobox Gene CDX2 by Alternative Splicing

Camille Balbinot et al. Cell Death Differ. .
Free PMC article

Abstract

On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alternative splicing at the CDX2 locus. (A) CDX2 gene map. E1–3: exon-1 to -3; I1 and 2: intron-1 and -2; dotted lines represent the spliced regions to produce, respectively, the CDX2 and miniCDX2 mRNAs. The translation start codons ATG1 and ATG2, and the stop codons Stop1 and Stop2 are indicated. (B) Expression of the miniCDX2 transcript. (a) RT-PCR on intestinal cell lines with the primers CDX21F/CCR hybridizing, respectively, upstream of ATG1 in the exon-1 and downstream of Stop2 in the exon-3. The two bands correspond to the CDX2 and miniCDX2 transcripts. (b) Northern blot of polyA RNA (10 μg per lane) from Caco2TC7 cells cultured for 3 days and 14 days revealing the CDX2 and miniCDX2 mRNAs. (c) RT-qPCR quantification of CDX2 (light gray) and miniCDX2 mRNA (dark gray) in 3 days Caco2TC7 cells (semi-logarithmic scale). (d) RT-qPCR of CDX2 (light gray) and miniCDX2 mRNA (dark gray) along the murine gut (semi-logarithmic scale); CDX2 was arbitrary set at 1 in the cecum. (e) CDX2 / miniCDX2 mRNA ratio along the mouse intestine
Figure 2
Figure 2
Functionality of the conserved alternative splicing/translation motif of the CDX2 locus. (A) The conserved motif found in CDX2-type genes (see also the Supplementary Figure 2). (B) (a) Luciferase activity in human colon cancer HCT116 cells transfected with the indicated reporter plasmids; X designates the mutation of ATG1 into TAG in pATG1m and in pATG1m-ATG2; pGl3-control was set to 100 and pGl3-basic gave background activity. (b) Luciferase activity in HCT116 cells transfected with in vitro-transcribed polyadenylated RNA without (light gray) or with m7G cap (dark gray). Data are given as mean±S.D. for triplicates
Figure 3
Figure 3
Protein expression of the miniCDX2 variant. (A) Structure of the CDX2 and miniCDX2 proteins: TA, transactivation domain; HD, DNA-binding homeodomain; R, regulatory domain. The arrow points to the 12-aa Nter peptide of miniCDX2. (B) The C2T antibody raised against the 12-aa Nter peptide of miniCDX2. (a) HCT116 cells transfected with the plasmid pFlag-CDX2 (lane 1), pFlag-miniCDX2 (lane 2) or the empty vector pFlag-CMV2 (lane 3) were analyzed by western blot with Flag, CDX2 or C2T antibodies. The Flag antibody reveals both Flag-CDX2 and Flag-miniCDX2 proteins, whereas the CDX2 and C2T antibodies, respectively, detect Flag-CDX2 and Flag-miniCDX2. (b) HCT116 cells co-transfected with pFlag-miniCDX2 and pEGFP showed nuclear localization of Flag-miniCDX2 (Flag antibody, red). Bars: 10 μm. (c) Validation of the C2T antibody in mouse intestinal sections: nuclear immunostaining in the crypt epithelium of wild-type mice; loss of staining when C2T is preincubated with an excess of immunogenic peptide; absence of immunostaining in the intestinal epithelium of conditional knockout (KO) Cdx2f/f::AhCreERT mice. Bars: 50 μm. (C) Immunostaining pattern of the miniCDX2 (C2T antibody) and CDX2 proteins in the small intestine of E15 mouse embryos, and of post-natal day 2 (P2) and day 11 (P11) suckling and adult (Ad) mice. (b) Co-immunofluorescence detection of the miniCDX2 (C2T) and CDX2 proteins in the small intestine of adult mice. Bars: 100 μm. (c) Immunostaining of the miniCDX2 (C2T) and CDX2 proteins in the proximal colon of adult mice. Bars: 50 μm. Higher magnification pictures are given in the Supplementary Figure S3A. (D) MiniCDX2 expression in tumors of the proximal ileum of ApcΔ14/+ mice. (a) HE staining and C2T and CDX2 immunostaining. The region boxed in HE is shown in the immunohistochemistry pictures. Bars: 100 μm. (b) RT-qPCR quantification of CDX2 (light gray) and miniCDX2 mRNA (dark gray) (semi-logarithmic scale) in wild-type ileum (wt), in three ileal tumors of ApcΔ14/+ mice (Apc T1-3) and in the adjacent normal mucosa (Apc N1-3); data are related to the amount of CDX2 mRNA arbitrary set at 1 in the wild-type ileum. (c) Ratio between the CDX2 and miniCDX2 transcripts
Figure 4
Figure 4
Molecular functions of miniCDX2 compared to CDX2. (A) Effect of miniCDX2 on the transcriptional activity of the CDX2 protein. (a) RT-qPCR quantification of the SI RNA and (b) pSI-luc luciferase activity in HCT116 cells transfected either with pFlag-miniCDX2 or pFlag-CDX2 or pFlag-CDX2 together with increasing amounts of pFlag-miniCDX2; data are given as mean±S.D. for triplicates (***P<0.001). (c) ChIP with anti-Flag or IgG in HCT116 cells transfected with pFlag-CMV2 (lane 1), pFlag-CDX2 (lane 2) or pFlag-miniCDX2 (lane 3); co-precipitated DNA was PCR-amplified with primers of the SI, LI-cadherin and Muc2 promoters. (d) ChIP with anti-CDX2 or IgG in HCT116 cells transfected either with pFlag-CMV2 (lane 1) or pFlag-CDX2 (lane 2) or pFlag-CDX2 together with a threefold molar excess of pFlag-miniCDX2 (lane 3) or pFlag-miniCDX2 (lane 4); co-precipitated DNA was PCR-amplified with primers of the SI promoter. (B) Effect on miniCDX2 on β-catenin/Tcf4 signaling. (a) RT-qPCR quantification of the Mmp7 RNA and (b) TOP-Flash luciferase activity in HEK293 cells transfected with pS33A-β-catenin, pMyc-Tcf4, pFlag-CDX2 and/or pFlag-miniCDX2; data are given as mean±S.D. for triplicates (***P<0.001). (c) Proteins from cells transfected either with pS33A-β-catenin alone (lane 1), or pS33A-β-catenin together with pFlag-CDX2 (lane 2), or pS33A-β-catenin together with pFlag-miniCDX2 (lane 3) were immunoprecipitated with anti-β-catenin or IgG, and analyzed by western blot with anti-β-catenin and with anti-Flag to reveal Flag-CDX2 (⊳) and Flag-miniCDX2 (▸). (d) Proteins from cells transfected with pS33A-β-catenin and pMyc-Tcf4 together with increasing amounts of pFlag-CDX2 or pFlag-miniCDX2 were immunoprecipitated with anti-β-catenin, and analyzed by western blot with anti-β-catenin and anti-Myc to check the β-catenin/Tcf4 interaction. (C) Effect of miniCDX2 on P27KIP1. (a) Representative western blot and quantification and (b) RT-qPCR quantification of P27KIP1 protein and RNA in HCT116 cells transfected either with pFlag-CMV2 (ctrl) or pFlag-CDX2 or pFlag-miniCDX2. (c) Representative pictures and quantification of clonogenic assays on HCT116 cells transfected either with pFlag-CMV2 (ctrl) or pFlag-CDX2 or pFlag-miniCDX2. Data are means±S.D. for triplicates (**P<0.01). (D) Effect of miniCDX2 on double-strand break DNA repair. (a) Proteins from cells transfected either with pFlag-CMV2 (lane 1), or pFlag-CDX2 (lane 2) or pFlag-miniCDX2 (lane 3) were immunoprecipitated with anti-Flag or IgG, and analyzed by western blot with anti-Flag and anti-KU70/80 to check the interaction of Flag-CDX2 (⊳) or Flag-miniCDX2 (▸) with the KU70/80 complex. (b) For DNA repair assays, linearized plasmid pcDNA3 (lane 1) was incubated with nuclear extracts of SW480 cells transfected with pFlag-CMV2 (lane 2), pFlag-CDX2 (lane 3) or pFlag-miniCDX2 (lane 4), and then separated by electrophoresis on an agarose gel. The linear and repaired (circular and/or concatemers) plasmid forms are indicated
Figure 5
Figure 5
Overexpression of miniCDX2 in transgenic mice. (A) Map of the jojo-Flag-miniCDX2 transgene with the CAG promoter, the loxP/Cre-excisable cassette containing the GFP coding sequence followed by a transcriptional stop (pA), and the Flag-miniCDX2 coding sequence. (B) Serial sections of rare small intestinal villi of jojo-Flag-miniCDX2::VilCre mice showing Flag-miniCDX2 expression accompanied by the loss of alkaline phosphatase and the ectopic expression of Claudin-18. Bars: 50 μm. (C) (a) Histology of the Brunner’s glands of jojo-Flag-miniCDX2::VilCre mice (miniCdx2) compared to control jojo-Flag-miniCDX2 animals (ctrl); bars: 500 μm. (b) Quantification of the surface of the Brunner’s glands in 3–4-month-old control jojo-Flag-miniCDX2 (ctrl, n=12) and jojo-Flag-miniCDX2::VilCre mice (miniCdx2, n=9); **P<0.01. (D) Pdx1 immunofluorescence staining in control jojo-Flag-miniCDX2 (ctrl) and jojo-Flag-miniCDX2::VilCre (miniCdx2) mice; bars: 100 μm. (E) The Brunner’s glands of the jojo-Flag-miniCDX2::VilCre mice show no major change in cell differentiation (PAS staining) and no obvious activation of the cell proliferation (P-Hist3). Bars: 100 μm. The asterisks label the duodenal crypts overlaying the Brunner’s glands
Figure 6
Figure 6
Duodenal lesions in aged jojo-Flag-miniCDX2::VilCre mice. (A) (a) Macroscopic view of two adjacent polyps grown in the duodenal region of a 18-month-old jojo-Flag-miniCDX2::VilCre mouse. (b) Histology of a polyp near the gastric-intestinal boundary (arrow); (i), (ii) and (iii), respectively, designate the expanded territory of Brunner’s glands (encircled), the hyper-proliferating crypts and the surface area. The arrowhead locates squamous stratified epithelium. Bar: 500 μm. (B) Immunostaining of Flag-miniCDX2, phospho-histone-3, Claudin-18 and β-catenin in the regions (i), (ii) and (iii) of the duodenal polyp. Bars: 50 μm. (C) Phospho-EGFR immunodetection in the regions (i) and (ii) of the polyps, and in normal adjacent duodenal crypts. Bars: 50 μm
Figure 7
Figure 7
Autoregulation of the CDX2 pre-mRNA splicing. (a) Immunodetection of the CDX2 and miniCDX2 (C2T antibody) proteins at the border between normal cecal epithelium and heteroplasia (asterisk) in Cdx2+/- mice. Bars: 50 μm. (b) Map of the eC2I1 reporter plasmid. (c) Production of the two protein variants CDX2-Flag (⊳) and miniCDX2-Flag (▸) from the reporter plasmid eC2I1. HCT116 cells were transfected either with the plasmids eC2I1, pFlag-CDX2 or pFlag-miniCDX2, and the proteins were revealed using anti-Flag, anti-CDX2 and anti-miniCDX2 (C2T) antibodies, respectively. (d) HCT116 cells transfected with the plasmid eC2I1 or with the mutant form eC2I1m. (e) Inverse correlation between the production of miniCDX2-Flag and the endogenous level of CDX2 in HCT116 and SW480 colon cancer cell lines. Upper panel: HCT116 and SW480 transfected with eC2I1. Lower panel: endogenous level of expression of CDX2 protein in HCT116 and SW480 cells related to actin. (f) HCT116 cells transfected with eC2I1 and either the control empty plasmid, or the plasmids encoding pHA-CDX2 or pHA-miniCDX2. The lower panel confirms the expression of HA-CDX2 and HA-miniCDX2 in these cells, using anti-HA antibody
Figure 8
Figure 8
Mechanism of regulation of the CDX2 pre-mRNA splicing. (A) Expression of the splicing factors ASF/SF2 and SRp30c in HCT116 and SW480 colon cancer cells. (B) Impact of ASF/SF2, SRp30c and CDX2 on the reporter plasmid eC2I1. HCT116 cells were transfected with eC2I1 and the expression plasmids for the indicated proteins, and the two splicing variants CDX2-Flag (⊳) and miniCDX2-Flag (▸) were detected with anti-Flag antibody. (C) Interaction between ASF/SF2 and SRp30c. (a) HCT116 cells were transfected with the plasmids encoding His-ASF/SF2 and His-SRp30c. After immunoprecipitation with anti-ASF/SF2 antibody or with IgG, the proteins were revealed with anti-ASF/SF2 and anti-SRp30c antibodies. (b) Endogenous proteins of HCT116 cells were immunoprecipitated with IgG or with anti-ASF/SF2 antibody without or with pre-treatment with RNase, and revealed with anti-SRp30c antibody. (D) Interaction between ASF/SF2 and CDX2 or miniCX2. (a) HCT116 cells were transfected with the control plasmids pCMV2-Flag (lane 1) or pHA-CDX2 (lane 2). Protein extracts immunoprecipitated with anti-HA beads, and revealed with anti-HA and anti-ASF/SF2 antibodies. (b) Same as (a) with pHA-miniCDX2 instead of pHA-CDX2. (E) Interaction between SRp30c and CDX2 or miniCDX2. Same as (D) except that protein detection used anti-SRp30c instead of anti-ASF2/SF2 antibody. (F) Effect of CDX2 and miniCDX2 on the interaction between ASF/SF2 and SRp30c. (a) HCT116 cells were transfected with the control plasmid pCMV2-Flag (lane 1), with pHA-CDX2 (lane 2) or pHA-miniCDX2 (lane 3). Cell extracts were immunoprecipitated with anti-ASF/SF2 antibody, and revealed with anti-ASF/SF2 and anti-SRp30c antibodies. (b) Quantification of immunoprecipitated SRp30c relative to ASF/SF2

Similar articles

See all similar articles

Cited by 3 articles

Feedback