Abstract
The adhesion protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is widely expressed in epithelial cells as a short cytoplasmic isoform (S-iso) and in leukocytes as a long cytoplasmic isoform (L-iso) and is frequently silenced in cancer by unknown mechanisms. Previously, we reported that interferon response factor 1 (IRF1) biases alternative splicing (AS) to include the variable exon 7 (E7) in CEACAM1, generating long cytoplasmic isoforms. We now show that IRF1 and a variant of heterogeneous nuclear ribonucleoprotein L (Lv1) coordinately silence the CEACAM1 gene. RNAi-mediated Lv1 depletion in IRF1-treated HeLa and melanoma cells induced significant CEACAM1 protein expression, reversed by ectopic Lv1 expression. The Lv1-mediated CEACAM1 repression resided in residues Gly71-Gly89 and Ala38-Gly89 in Lv1's N-terminal extension. ChIP analysis of IRF1- and FLAG-tagged Lv1-treated HeLa cells and global treatment with the global epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A indicated that IRF1 and Lv1 together induce chromatin remodeling, restricting IRF1 access to the CEACAM1 promoter. In interferon γ-treated HeLa cells, the transcription factor SP1 did not associate with the CEACAM1 promoter, but binding by upstream transcription factor 1 (USF1), a known CEACAM1 regulator, was greatly enhanced. ChIP-sequencing revealed that Lv1 overexpression in IRF1-treated cells induces transcriptional silencing across many genes, including DCC (deleted in colorectal carcinoma), associated with CEACAM5 in colon cancer. Notably, IRF1, but not IRF3 and IRF7, affected CEACAM1 expression via translational repression. We conclude that IRF1 and Lv1 coordinately regulate CEACAM1 transcription, alternative splicing, and translation and may significantly contribute to CEACAM1 silencing in cancer.
Highlights
The adhesion protein carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) is widely expressed in epithelial cells as a short cytoplasmic isoform (S-iso) and in leukocytes as a long cytoplasmic isoform (L-iso) and is frequently silenced in cancer by unknown mechanisms
We showed that RNA splicing regulators heterogeneous nuclear ribonucleoprotein (hnRNP) L and hnRNP A1 interact directly with CEACAM1 exon 7 (E7) to mediate production of short isoform (S-iso) whereas hnRNP M is essential for L-iso production [14]
We established that interferon response factor 1 (IRF1) biases the alternative splicing (AS) of CEACAM1 mRNA to generate the L-iso via the inclusion of E7 [21], we did not address the possibility that the RNAbinding proteins would coordinately affect transcription, splicing, and translation
Summary
The adhesion protein carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) is widely expressed in epithelial cells as a short cytoplasmic isoform (S-iso) and in leukocytes as a long cytoplasmic isoform (L-iso) and is frequently silenced in cancer by unknown mechanisms. The molecular mechanism of how CEACAM1 is systematically down-regulated in breast, and other cancers, is still not understood To address this problem, we have considered the possibility that the normal coordination of RNA transcription and splicing is disrupted in cancer, leading to chromatin remodeling as a mechanism of gene silencing. We have considered the possibility that the normal coordination of RNA transcription and splicing is disrupted in cancer, leading to chromatin remodeling as a mechanism of gene silencing In this regard, the dependence of inclusion (or not) of E7 in CEACAM1 AS by the transcription factor IRF1 and splicing factor hnRNP L, occurred to us a logical starting point to investigate this possibility. This study may help explain why CEACAM1 expression is down-regulated in a subset of cancers
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