FIGURE 6.

FOXO4 was directly regulated by the transcription factor HIF1α. (A) FOXO4 was significantly decreased under hypoxic cell culture induced by CoCl2 or 1% O2. (B, C) After transfection with a mutated HIF‐1α lacking the oxygen‐dependent degradation (ODD) domain, western blotting (B) and RT‐PCR (C) analysis indicated that mutated HIF‐1α decreased FOXO4 expression at both protein and transcriptional levels. (D)HIF‐1α expression was negatively correlated with FOXO4 expression in the TCGA database (P < 0.001). (E) Three HIF‐1α transcription factor binding elements were identified in FOXO4 promoter by using the JASPAR CORE database. (F, G) ChIP assays revealed that HIF‐1α mainly bound to the first HRE site 5′‐GCACATGCCT‐3′ located from ‐204 to ‐213 bp. (H) Luciferase analysis indicated that FOXO4 promoter activity was decreased in cells treated with CoCl2 and was restored in cells silencing HIF‐1α expression. Consistent with these results, when we mutated 5′‐GCACATGCCT‐3′ sequence in FOXO4 promoter to 5′‐ATGTGCTAAC‐3′, the mutated promoter completely abolished the CoCl2 responsiveness of the construct. WT: Wild type FOXO4 promote. MT: Mutated FOXO4 promote. *P < 0.05.**P > 0.05