Enhancing the alternative splicing of vascular endothelial growth factor (VEGF)

It is well established that transcription and splicing are processes coupled in space and time.

Therefore altering a gene’s promoter results in changes in alternative splicing. In this work, we

studied the intriguing question of whether enhancers also alter alternative splicing. We chose to

focus on the vascular endothelial growth factor ( VEGF ), whose isoforms have either pro- or

anti-angiogenic activity. In K562 cells, a chronic myeloid leukemia (CML) cell line, we demonstrated

that mutations in the enhancer of VEGF not only lower the total amount of VEGF mRNA, but also

cause a change of alternative splicing by promoting exclusion of exons 6 and 7. In order to determine

if the mechanism by which enhancers change splicing is via transcription elongation kinetic, we

conducted several studies. First we inhibited topoisomerase 1 using camptothecin (CPT) and found

exclusion of exons 6 and 7. Next we observed that transfecting the cells with slow RNAPII achieved

similar results. Our results suggest that enhancers regulate alternative splicing by increasing the

elongation rate of RNAPII. Finally, we examined RNA from 12 CML patients sampled when they were

sick and during remission. Five of the patients had upregulation of VEGF mRNA during the time they

were sick, out of which four had inclusion of exons 6 and 7. This reiteration of a connection between

expression and alternative splicing of VEGF exons 6 and 7 suggest that the upregulation in expression

could be the result of enhancer manipulation in these patients promoting a regulation of alternative

splicing.