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

ABSTRACT: Cancer cells experiencing hypoxic stress employ epithelial-mesenchymal transition (EMT) to undergo metastasis through rewiring of the chromatin landscape, epigenetics and importantly alternative splicing. However, how hypoxia influences expression and genome-wide redistribution of CTCF is still not known. Here, we performed CTCF-ChIP-seq and CUT&RUN assay to analyze differential occupancy of CTCF in normoxic and hypoxic breast cancer cell line. We discovered that hypoxia-induced gain of CTCF occupancy across the genome regulates EMT in hypoxic cells. Specifically, we did 4C-seq experiments on COL5A1 (EMT associated gene) gene loci and showed that CTCF-mediated promoter-exon upstream looping regulates epigenetic mark mainly DNA demethylation at distal alternatively spliced exon 64 and regulates CTCF-mediated RNA Pol II pausing at COL5A1exon64A that decides the outcome of splicing to favor EMT under hypoxia. Overall, we uncover that hypoxia-induced differential CTCF occupancy across the genome is associated with gene expression and alternative splicing events related to cancer progression, similarly to the proposed model.

ORGANISM(S): Homo sapiens

PROVIDER: GSE216843 | GEO | 2024/12/31

REPOSITORIES: GEO

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Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells

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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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