Cells (2×10^5) were seeded in 6-well plates and transfected after 24 h with 30 nM siRNA duplexes using INTERFERin. siRNA and DNA co-transfections were performed with 30 nM siRNA and 1 μg of plasmid, using jetPRIME.

dnMechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFalpha subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFalpha by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2alpha (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2alpha mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2alpha gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2alpha transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2alpha, demonstrating that the HIF2alpha promoter is regulated by E2F1 directly and that Cezanne regulates HIF2alpha expression through control of E2F1 levels. Our results thus suggest that HIF2alpha is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling.