Rescue of HIF1A protein level by E3-ligase KO
The hypoxia-inducible factor 1-a (HIF-1a) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1a are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1a under Hx are less clear. We report that ABL kinase activity protects HIF-1a from proteasomal degradation during Hx. Using a fluorescence-activated cell-sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1a as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1a for degradation in the presence of an ABL kinase inhibitor in Hx. Overall design: FACS-based CRISPR KO screen utilizing a custom E3-ligase targeted sgRNA library in conjunction witells treated with an ABL kinase inhibitor (Nilotinib) and placed into hypoxia for 24 h. Sample types: High - top 10% of HIF-1a expressing cells collected on the basis of PE signal (PC9 cells, Nil High biol rep 1/2/3) Low - bottom 10% of HIF-1a expressing cells collected on the basis of PE signal (PC9 cells, Nil Low biol rep 1/2/3) Unsorted - ungated control sample not collected on the basis of PE signal (PC9 cells, Nil Unsorted  biol rep 1/2/3)