Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer

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Hypoxia plays a critical role in tumor progression, including invasion and metastasis. Epithelial-mesenchymal transition (EMT) is one of the first steps in a metastatic cascade that hypoxic cells achieve through modulating the chromatin structure, epigenetics, and, importantly, alternative splicing. Recently, we reported hypoxia-driven CTCF induction through HIF1α that promotes EMT in hypoxic breast cancer cells. Nevertheless, it is still unknown how the HIF1α-CTCF axis promotes the invasiveness of hypoxic breast cancer cells. In the present study, we investigated the molecular mechanism of CTCF-mediated EMT under hypoxia. We observed a significant gain in CTCF occupancy throughout the genome, correlating with the increased CTCF expression under hypoxia. Many of these CTCF gain sites are observed at the promoter of the genes associated with cancer and hypoxia. Here, we put forward an intricate mechanism regulated by CTCF to promote EMT where CTCF modulates both gene expression and alternative splicing of an EMT gene, COL5A1. Mechanistically, the hypoxia-mediated CTCF binding to the COL5A1 promoter is an integral part of the mechanism of CTCF-HIF1α mediated transcriptional upregulation of COL5A1. Our data further demonstrates hypoxia-driven inclusion of exon 64A in the mutually exclusive alternative splicing event of COL5A1exon 64 (exon 64A or 64B). Notably, in the COL5A1 gene, CTCF mediates promoter-exon upstream looping, regulates DNA demethylation at distal alternatively spliced exon 64, and regulates RNA Pol II pausing at COL5A1 exon64A that decides the outcome of splicing to favor EMT under hypoxia. Further, we observed that hypoxia-induced differential CTCF occupancy across the genome is associated with gene expression and alternative splicing of other cancer-related genes, similar to the proposed model. Finally, specifically disrupting the HIF1α-CTCF-COL5A1 exon64A axis through the dCas9-DNMT3A system alleviates the EMT potential of hypoxic breast cancer cells and may represent a novel therapeutic target in breast cancer.

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