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 chromatin landscape, epigenetics, and importantly alternative splicing. Here, we investigated the role of CTCF, chromatin, epigenetic and alternative splicing modulator under hypoxia to promote EMT. Our result shows that hypoxia-induced epigenetic changes upregulate CTCF expression in breast cancer. We delineate that CTCF is a direct target of HIF1 and the hypoxia-induced HIF1-CTCF axis functionally contributes to promoting EMT in breast cancer cells. Finally, our work uncovers COL5A1, an EMT gene, as a direct target of CTCF. We demonstrated that hypoxia-mediated CTCF enrichment on COL5A1 promoter regulates expression as well as alternative splicing events to promote EMT. Here, we put forward an intricate mechanism of alternative splicing where CTCF-mediated promoter-exon upstream looping regulates DNA de-methylation and CTCF-mediated RNA Pol II pausing at COL5A1 exon 64A. Our global analysis of CTCF-ChIP-seq data reveals that the hypoxia-induced differential CTCF occupancy possibly regulates gene expression and alternative splicing events of many genes that are enriched in the EMT pathway, cancer cell motility and invasion, angiogenesis, and stemness under hypoxia, similarly to the proposed model. Finally, we employed the epigenetic modulator dCas9-DNMT3A system to specifically disrupt HIF1 or CTCF binding under hypoxia and hence the HIF1-CTCF-COL5A1exon64A axis that alleviates the EMT potential of breast cancer cells that may represent a novel therapeutic target in breast cancer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/539689v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e15c15org.highwire.dtl.DTLVardef@1649a71org.highwire.dtl.DTLVardef@1d77481org.highwire.dtl.DTLVardef@21f148_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG