Figure 5.

Slow elongation of RNAPII promotes exclusion of VEGFA exons 6a and 7. (A and B) K562 cells were treated with 6 μM CPT for 6 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of VEGF relative to CycloA and hTBP reference genes (A) and for VEGFA₁₂₁ and VEGFA₁₈₉ relative to VEGFA total mRNA amount (B). PSI was calculated by VEGFA₁₈₉ relative to VEGFA₁₂₁. (C) Nascent mRNA production in WT K562 cells and enhancer mutated C9 and C26 cells in different regions of the VEGFA gene after release from DRB-inhibition. (D)K562 cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the VEGFA promoter or +157 enhancer for 30 h. ChIP was performed of H3 pan-acetyl at the VEGFA enhancer, promoter and intron 7 following tethering to the +157 enhancer. Values represent averages of three independent experiments ± SD and are expressed as dCas9-p300 core (WT) relative to dCas9-p300 core (mut) (**P< 0.01, ***P< 0.001).