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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 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.
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pone-0104293-g001: 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.

Mentions: We sequenced full-length CDX2 cDNA clones generated by RT-PCR using RNA isolated from normal human colonic mucosa and identified two transcripts, wild type CDX2 and a variant transcript (CDX2/AS) lacking the four bases CAGG (Fig. 1A) (Genbank ID: KJ081251). Sequence analysis of the genomic CDX2 region corresponding to the four base pair deletion revealed an intact gene (data not shown). Given these findings, we attributed the generation of CDX2/AS to alternative splicing in which the non-canonical 5′ splice donor sequence, GC, located at the distal segment of exon 2 is utilized rather than the wild type GU donor sequence of intron 2 (Fig. 1B). The alternative splicing-induced frameshift occurs in the distal region of the homeobox binding domain and encodes a protein in which the 85 carboxyl terminal residues in the wild type protein are replaced with a novel 45 residue domain enriched in serine and arginine amino acids (Fig. 1C). Western blot analysis of whole cell lysates from normal colonic mucosa and from the HT29 colorectal cancer cell line using polyclonal antisera to the unique carboxy-terminal domain of CDX2/AS revealed a 38 kD band representing the alternatively spliced protein (Fig. 1D). Using an antibody recognizing the common amino-terminal activation domain, proteins at 42 kD (CDX2) and 38 kD (CDX2/AS) were identified in whole cell lysates from human colonic tumors, normal adjacent colonic mucosa, and several colon cancer cell lines (Fig. 1E).


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125279&req=5

pone-0104293-g001: 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.
Mentions: We sequenced full-length CDX2 cDNA clones generated by RT-PCR using RNA isolated from normal human colonic mucosa and identified two transcripts, wild type CDX2 and a variant transcript (CDX2/AS) lacking the four bases CAGG (Fig. 1A) (Genbank ID: KJ081251). Sequence analysis of the genomic CDX2 region corresponding to the four base pair deletion revealed an intact gene (data not shown). Given these findings, we attributed the generation of CDX2/AS to alternative splicing in which the non-canonical 5′ splice donor sequence, GC, located at the distal segment of exon 2 is utilized rather than the wild type GU donor sequence of intron 2 (Fig. 1B). The alternative splicing-induced frameshift occurs in the distal region of the homeobox binding domain and encodes a protein in which the 85 carboxyl terminal residues in the wild type protein are replaced with a novel 45 residue domain enriched in serine and arginine amino acids (Fig. 1C). Western blot analysis of whole cell lysates from normal colonic mucosa and from the HT29 colorectal cancer cell line using polyclonal antisera to the unique carboxy-terminal domain of CDX2/AS revealed a 38 kD band representing the alternatively spliced protein (Fig. 1D). Using an antibody recognizing the common amino-terminal activation domain, proteins at 42 kD (CDX2) and 38 kD (CDX2/AS) were identified in whole cell lysates from human colonic tumors, normal adjacent colonic mucosa, and several colon cancer cell lines (Fig. 1E).

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