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. 2014 Aug 7;9(8):e104293.
doi: 10.1371/journal.pone.0104293. eCollection 2014.

A novel CDX2 isoform regulates alternative splicing

Matthew E Witek  1 , Adam E Snook  2 , Jieru E Lin  2 , Erik S Blomain  2 , Bo Xiang  2 , Michael S Magee, Scott A Waldman  2
Affiliations

A novel CDX2 isoform regulates alternative splicing

Matthew E Witek et al. PLoS One. .

Erratum in

  • PLoS One. 2014;9(10):e112164. Magee, Michael [corrected to Magee, Michael S]

Abstract

Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS) that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain). CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS).

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Conflict of interest statement

Competing Interests: SAW is the Chair of the Data Safety Monitoring Board for the C-Cure Trial™ sponsored by Cardio Biosciences, and the Chair (uncompensated) of the Scientific Advisory Board of Targeted Diagnostics and Therapeutics, Inc., which provided research funding that, in part, supported this work and has a license to commercialize inventions related to this work. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. CDX2 is alternatively spliced.
(A) Sequence analysis of CDX2 mRNA isolated from normal colonic epithelial cells revealed wild type mRNA and a variant transcript (CDX2/AS) lacking the bases CAGG. (B) Alternative splicing of CDX2 occurs via utilization of the non-canonical splice donor sequence GC located at the 3′ region of exon 2. (C) The alternatively induced frameshift results in deletion of the carboxy-terminal 85 bases in the wild type protein that are replaced by a novel 45 domain enriched in serine and arginine residues. (D) Western blot analysis of whole cell lysates from a normal colonic epithelium and from the HT29 colorectal cancer cell line blotted with anti-CDX2/AS revealed a band at 38 kD. (E) Western blot analysis of whole cell lysates from normal adjacent colonic epithelium (N) and colon adenocarcinomas (T) as well as whole cell lysates from various colon cancer cell lines blotted with antibody CDX2ACT that recognizes the amino-terminal domain of CDX2 and CDX2/AS revealed two bands at 42 and 38 kD representing CDX2 and CDX2 kD representing CDX2 and CDX2/AS, respectively.
Figure 2
Figure 2. Focal nuclear localization of CDX2/AS is RS-domain dependent.
(A) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag or MSCV-CDX2/AS-His revealed diffuse and focal nuclear localization patterns for CDX2 and CDX2/AS, respectively, with minimal areas of overlap (dotted circles). (B) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag, MSCV-CDX2/AS-His revealed nuclear localization whereas MSCV-CDX2/ASΔRS-His, a mutant CDX2/AS protein lacking bases distal to the alternatively induced frameshift, resulted in peri-nuclear localization.
Figure 3
Figure 3. CDX2/AS lacks transcriptional activity and does not possess dominant-negative activity against CDX2.
(A) 293T cells were transiently transfected with the CDX2-dependent promoter of GUCY2C (GUCY2C-Luc) along with MSCV-EGFP, MSCV-CDX2-Flag, or MSCV-CDX2/AS-His. Cells transfected with CDX2 exhibited increased luciferase activity compared to control EGFP or CDX2/AS transfected cells (p0.05). Luciferase activity between control EGFP and CDX2/AS transfected cells was similar (p = ns). (B) 293T cells were transiently transfected with either GUCY2C-Luc or GUCY2C-CDX2mut-Luc, a GUCY2C promoter with a TTT→CCC mutation in the CDX2 binding domain. Cells transfected with either GUCY2C-Luc or GUCY2C-CDX2mut-Luc plasmids were co-transfected with either 1 µg MSCV-EGFP, 0.5 µg of MSCV-EGFP and 0.5 µg of MSCV-CDX2-Flag, or 0.5 µg of MSCV-CDX2-Flag and 0.5 µg of MSCV-CDX2/AS-His. There was no difference between CDX2-induced GUCY2C-Luc activity in cells transfected with CDX2 alone or CDX2 with CDX2/AS. GUCY2C-CDX2mut-Luc was significantly different in both CDX2 alone and CDX2 and CDX2/AS transfected cells (p0.05). (C) CDX2 significantly increased expression of GUCY2C mRNA in HT29 and SW480 cells compared to EGFP expressing controls (p0.05) while GUCY2C mRNA in cells over-expressing either EGFP or CDX2/AS was similar.
Figure 4
Figure 4. Amino-acid alignment of the RS-like domain of CDX2/AS and that of SR and SR-like proteins revealed 31% homology.
Figure 5
Figure 5. Co-localization of CDX2/AS with putative splicing factors.
(A) F-IHC analysis of 293T and RKO cells transiently transfected with CDX2/AS-His and co-stained for CDX2/AS-His and either, SC35 or ASF/SF2. All proteins localized to the nucleus and merged images revealed co-localization of CDX2/AS with both SC35 and ASF/SF2. CDX2-Flag did not co-localize with either protein.
Figure 6
Figure 6. CDX2/AS influences alternative splice site selection of minigenes in 293T and LovoCDX2−/− cells.
CDX2/AS regulates alternative splicing of Tra2β-1 and CD44v5 minigenes in Lovo minigenes in LovoCDX2−/− cells independent of re-introduction of CDX2 expression (p0.05). Reverse images of four independent experiments.
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