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A novel CDX2 isoform regulates alternative splicing.

Witek ME, Snook AE, Lin JE, Blomain ES, Xiang B, Magee MS, Magee M, Waldman SA - PLoS ONE (2014)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kimmel Cancer Center & Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

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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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.
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pone-0104293-g003: 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.

Mentions: Although the homology of CDX2 and CDX2/AS diverges distal to the alternative splicing-induced frameshift, the considerable structural similarities between their entire amino-terminal activation and proximal homeobox binding domains suggest that they might possess analogous functions. To explore this possibility, we compared the transcriptional activity of CDX2 with CDX2/AS in 293T cells using the CDX2-dependent luciferase reporter construct GUCY2C-Luc [15]. Cells transiently transfected with pMSCV-CDX2-Flag (1.88±0.72; p0.05) significantly increased activity of the GUCY2C-Luc reporter compared to control pMSCV-EGFP (0.79±0.15) while pMSCV-CDX2/AS-His (1.0±0.31; p = ns) was similar to control suggesting CDX2/AS does not possess the transcriptional activity inherent to CDX2 (Fig. 3A). Given the lack of transcriptional activity of CDX2/AS and the well-established role of splice variants in regulating the function of their wild type counterparts [20–[22], we next investigated the potential for CDX2/AS to influence the transcriptional activity of CDX2. We compared activation of GUCY2C-Luc in 293T cells by transiently transfecting with equal amounts of pMSCV-EGFP, pMSCV-CDX2-Flag and pMSCV-EGFP, or pMSCV-CDX2-Flag with pMSCV-CDX2/AS-His. We used the GUCY2C-CDX2mut-Luc promoter, which simulates complete inhibition of the CDX2 contribution to activation of the GUCY2C promoter, as a negative control. Here, co-transfection of pMSCV-CDX2-Flag with pMSCV-CDX2/AS-His did not significantly alter the activation of GUCY2C-Luc compared to pMSCV-CDX2-Flag alone (1.59±0.71 versus 1.75±0.69; p = ns) whereas mutation of the CDX2 binding site significantly decreased activity of the GUCY2C promoter for CDX2 alone (1.83±0.29 versus 0.52±0.07; p0.05) or in combination with CDX2/AS (1.75±0.69 versus 0.65±0.41; p0.05) suggesting that CDX2/AS does not alter the putative transcriptional activity of CDX2 (Fig. 3B).


A novel CDX2 isoform regulates alternative splicing.

Witek ME, Snook AE, Lin JE, Blomain ES, Xiang B, Magee MS, Magee M, Waldman SA - PLoS ONE (2014)

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.
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pone-0104293-g003: 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.
Mentions: Although the homology of CDX2 and CDX2/AS diverges distal to the alternative splicing-induced frameshift, the considerable structural similarities between their entire amino-terminal activation and proximal homeobox binding domains suggest that they might possess analogous functions. To explore this possibility, we compared the transcriptional activity of CDX2 with CDX2/AS in 293T cells using the CDX2-dependent luciferase reporter construct GUCY2C-Luc [15]. Cells transiently transfected with pMSCV-CDX2-Flag (1.88±0.72; p0.05) significantly increased activity of the GUCY2C-Luc reporter compared to control pMSCV-EGFP (0.79±0.15) while pMSCV-CDX2/AS-His (1.0±0.31; p = ns) was similar to control suggesting CDX2/AS does not possess the transcriptional activity inherent to CDX2 (Fig. 3A). Given the lack of transcriptional activity of CDX2/AS and the well-established role of splice variants in regulating the function of their wild type counterparts [20–[22], we next investigated the potential for CDX2/AS to influence the transcriptional activity of CDX2. We compared activation of GUCY2C-Luc in 293T cells by transiently transfecting with equal amounts of pMSCV-EGFP, pMSCV-CDX2-Flag and pMSCV-EGFP, or pMSCV-CDX2-Flag with pMSCV-CDX2/AS-His. We used the GUCY2C-CDX2mut-Luc promoter, which simulates complete inhibition of the CDX2 contribution to activation of the GUCY2C promoter, as a negative control. Here, co-transfection of pMSCV-CDX2-Flag with pMSCV-CDX2/AS-His did not significantly alter the activation of GUCY2C-Luc compared to pMSCV-CDX2-Flag alone (1.59±0.71 versus 1.75±0.69; p = ns) whereas mutation of the CDX2 binding site significantly decreased activity of the GUCY2C promoter for CDX2 alone (1.83±0.29 versus 0.52±0.07; p0.05) or in combination with CDX2/AS (1.75±0.69 versus 0.65±0.41; p0.05) suggesting that CDX2/AS does not alter the putative transcriptional activity of CDX2 (Fig. 3B).

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kimmel Cancer Center & Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

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).

Show MeSH
Related in: MedlinePlus