VEGFA's distal enhancer regulates its alternative splicing in CML

Enhancer demethylation in leukemia and lymphoma was shown to lead to overexpression of genes which promote cancer characteristics. The vascular endothelial growth factor A (VEGFA) enhancer, located 157 Kb downstream of its promoter, is demethylated in chronic myeloid leukemia (CML). VEGFA has several alternative splicing isoforms with different roles in vascularization and cancer progression. Since transcription and splicing are coupled in space and time, we hypothesized that the VEGFA enhancer can also regulate the genes alternative splicing to contribute to the pathology of CML. Our results show that mutating the VEGFA +157 enhancer promotes exclusion of exons 6b and 7 and activating the enhancer by tethering a chromatin activator has the opposite effect. In line with these results, CML patients display high enhancer activity and inclusion of VEGFA exons 6b and 7. To search for a key protein connecting transcription with alternative splicing, we analyzed 161 chromatin immunoprecipitation (ChIP)-seq experiments for DNA binding proteins and found that PML and CCNT2 bind VEGFAs promoter and enhancer. CCNT2 is a positive regulator of RNA polymerase II (RNAPII) transcription elongation and indeed its silencing promotes exclusion of exons 6b and 7. Slowing down RNAPII elongation promotes exclusion of exons 6b and 7. Thus our results suggest that VEGFAs +157 enhancer regulates its alternative splicing by increasing RNAPII elongation rate via CCNT2. Our work demonstrates the importance of the interplay between transcription and alternative splicing.