As we had observed HIF-1-dependent increases in mRNA and protein of KDM2A in hypoxia, we next investigated the functional significance of HIF-1 presence at the KDM2A promoter. To this end, HIF-1 subunits were depleted by siRNA from cells and levels of RNA polymerase II present at the KDM2A promoter were analysed by ChIP-qPCR. This analysis was performed following 24 h of hypoxia, where we had observed the highest mRNA induction, in a HIF-dependent manner. Levels of RNA polymerase II were robustly detected in control cells exposed to hypoxia, when the KDM2A promoter was analysed (Figure 5B). However, when either HIF-1α or HIF-1β were depleted by siRNA from cells, levels of RNA polymerase II present at the KDM2A promoter dropped significantly and to almost background levels (Figure 5B). These results demonstrate the importance of HIF-1 subunits in the regulation of the KDM2A promoter, indicating that HIF-1 is required for transcriptional activation of this gene. To further determine the functionality of the HRE sites present at the KDM2A promoter, we created luciferase constructs expressing the two putative HRE sites in tandem, as well as mutant versions of the HRE sites. Analysis of luciferase activity in cells revealed that indeed these HREs confirm promoter activity (Figure 5D). Furthermore, this analysis revealed that both HREs are required for full luciferase activity, since, when mutated, luciferase output reverts to background level, while mutation of the −186 HRE site gives an intermediate activity level (Figure 5D).