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IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform.

ABSTRACT: Interferon regulatory factor-1 (IRF-1) is a master regulator of IFN-? induced gene transcription. Previously we have shown that IRF-1 transcriptionally induces CEACAM1 via an ISRE (Interferon-Stimulated Response Element) in its promoter. CEACAM1 pre-mRNA undergoes extensive alternative splicing (AS) generating isoforms to produce either a short (S) cytoplasmic domain expressed primarily in epithelial cells or as an ITIM-containing long (L) isoform in immune cells.The transcriptional and molecular mechanism of CEACAM1 minigenes AS containing promoter ISREs mutations in the breast epithelial, MDA-MB-468, cell line was detected using flow cytometry. In addition, transcriptome sequencing was utilized to determine whether IRF-1 could direct the AS of other genes as well. Tumor xenografts were used to evaluate CEACAM1 isoform expression on the leading edge of breast tumor cells.In the present study, we provide evidence that CEACAM1's promoter and variable exon 7 cross-talk allowing IRF-1 to direct AS events. Transcriptome sequencing shows that IRF-1 can also induce the global AS of genes involved in regulation of growth and differentiation as well as genes of the cytokine family. Furthermore, MDA-MB-468 cells grown as tumor xenografts exhibit an AS switch to the L-isoform of CEACAM1, demonstrating that an in vivo inflammatory milieu is also capable of generating the AS switch, similar to that found in human breast cancers Mol Cancer 7:46, 2008.The novel AS regulatory activities attributed to IRF-1 indicate that the IFN-? response involves a global change in both gene transcription and AS in breast epithelial cells.

SUBMITTER: Dery KJ

PROVIDER: S-EPMC4113144 | biostudies-other | 2014 Mar

REPOSITORIES: biostudies-other

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IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform.

Dery Kenneth J KJ Kujawski Maciej M Grunert David D Wu Xiwei X Ngyuen Tung T Cheung Celeste C Yim John H JH Shively John E JE

Molecular cancer 20140321

<h4>Background</h4>Interferon regulatory factor-1 (IRF-1) is a master regulator of IFN-γ induced gene transcription. Previously we have shown that IRF-1 transcriptionally induces CEACAM1 via an ISRE (Interferon-Stimulated Response Element) in its promoter. CEACAM1 pre-mRNA undergoes extensive alternative splicing (AS) generating isoforms to produce either a short (S) cytoplasmic domain expressed primarily in epithelial cells or as an ITIM-containing long (L) isoform in immune cells.<h4>Methods</ ...[more]

PMID: 24650050

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Project description:BACKGROUND:The CEA gene family is one of the most rapidly evolving gene families in the human genome. The founder gene of the family is thought to be an ancestor of the inhibitory immune checkpoint molecule CEACAM1. Comprehensive analyses of mammalian genomes showed that the CEA gene family is subject to tremendous gene family expansion and contraction events in different mammalian species. While in some species (e.g. rabbits) less than three CEACAM1 related genes exist, were in others (certain microbat species) up to 100 CEACAM1 paralogs identified. We have recently reported that the horse has also an extended CEA gene family. Since mechanisms of gene family expansion and diversification are not well understood we aimed to analyze the equine CEA gene family in detail. RESULTS:We found that the equine CEA gene family contains 17 functional CEACAM1-related genes. Nine of them were secreted molecules and eight CEACAMs contain transmembrane and cytoplasmic domain exons, the latter being in the focus of the present report. Only one (CEACAM41) gene has exons coding for activating signaling motifs all other CEACAM1 paralogs contain cytoplasmic exons similar to that of the inhibitory receptor CEACAM1. However, cloning of cDNAs showed that only one CEACAM1 paralog contain functional immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Three receptors have acquired a stop codon in the transmembrane domain and two have lost their inhibitory motifs due to alternative splicing events. In addition, alternative splicing eliminated the transmembrane exon sequence of the putative activating receptor, rendering it to a secreted molecule. Transfection of eukaryotic cells with FLAG-tagged alternatively spliced CEACAMs indicates that they can be expressed in vivo. Thus detection of CEACAM41 mRNA in activated PBMC suggests that CEACAM41 is secreted by lymphoid cells upon activation. CONCLUSIONS:The results of our study demonstrate that alternative splicing after gene duplication is a potent mechanism to accelerate functional diversification of the equine CEA gene family members. This potent mechanism has created novel CEACAM receptors with unique signaling capacities and secreted CEACAMs which potentially enables equine lymphoid cells to control distantly located immune cells.

Project description:Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at the various developmental stages before, during, and after palate fusion using GeneChip? microarrays. Ceacam1 was one of the highly up-regulated genes during and after fusion in palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was expressed at a very low level in palatal epithelium before fusion, but highly expressed in the midline of the palate during and after fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1-/-) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1-/- mice. TGF?3 expression, apoptosis, and cell proliferation in palatal epithelium were not effected in the palate of Ceacam1-/-mice. CEACAM1 expression was down-regulated in Tgfb3-/- palate. However, exogenous TGF?3 did not induce CEACAM1 expression. These results suggest that CEACAM1 has roles in both the initiation of palate formation via epithelial cell adhesion and TGF signaling has some indirect effect on CEACAM1. Global gene expression profiling of palatal processes before, during and after fusion of palatal shelves We used microarray to investigate the gene expression of palatal tissue during palatal development. Palatal processes were microdissected at the stages of palatal development (before, during and after fusion) for RNA extraction and hybridization on Affymetrix microarrays.

Dataset Information

IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform.

Publications

IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform.

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