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  • Hypoxia-induced loss of SRSF2-dependent DNA methylation promotes CTCF-mediated alternative splicing of VEGFA in breast cancer.

Hypoxia-induced loss of SRSF2-dependent DNA methylation promotes CTCF-mediated alternative splicing of VEGFA in breast cancer.

iScience (2023-05-26)
Pooja Yadav, Anchala Pandey, Parik Kakani, Srinivas Abhishek Mutnuru, Atul Samaiya, Jharna Mishra, Sanjeev Shukla
ABSTRACT

Alternative splicing of vascular endothelial growth factor A (VEGFA) generates numerous isoforms with unique roles in tumor angiogenesis, and investigating the underlying mechanism during hypoxia necessitates diligent pursuance. Our research systematically demonstrated that the splicing factor SRSF2 causes the inclusion of exon-8b, leading to the formation of the anti-angiogenic VEGFA-165b isoform under normoxic conditions. Additionally, SRSF2 interacts with DNMT3A and maintains methylation on exon-8a, inhibiting CCCTC-binding factor (CTCF) recruitment and RNA polymerase II (pol II) occupancy, causing exon-8a exclusion and decreased expression of pro-angiogenic VEGFA-165a. Conversely, SRSF2 is downregulated by HIF1α-induced miR-222-3p under hypoxic conditions, which prevents exon-8b inclusion and reduces VEGFA-165b expression. Furthermore, reduced SRSF2 under hypoxia promotes hydroxymethylation on exon-8a, increasing CTCF recruitment, pol II occupancy, exon-8a inclusion, and VEGFA-165a expression. Overall, our findings unveil a specialized dual mechanism of VEGFA-165 alternative splicing, instrumented by the cross-talk between SRSF2 and CTCF, which promotes angiogenesis under hypoxic conditions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-VEGF 165b Antibody, clone 56/1, clone 56/1, 1 mg/mL, from mouse
Sigma-Aldrich
Anti-VEGF165 antibody, Mouse monoclonal, clone VG-20, purified from hybridoma cell culture
Sigma-Aldrich
Puromycin dihydrochloride, Ready Made Solution, from Streptomyces alboniger, 10 mg/mL in H2O, suitable for cell culture