Structural and functional analysis of amphioxus HIFα reveals ancient features of the HIFα family

Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to hypoxia. To gain insight into the structural and functional evolution of the HIF family, we characterized the HIF alpha gene from amphioxus, an invertebrate chordate, and identified several alternatively spliced HIF alpha isoforms. Whereas HIF alpha Ia, the full-length isoform, contained a complete oxygen-dependent degradation (ODD) domain, the isoforms Ib, Ic, and Id had 1 or 2 deletions in the ODD domain. When tagged with GFP and tested in mammalian cells, the amphioxus HIF alpha Ia protein level increased in response to hypoxia or CoCl2 treatment, whereas HIF alpha Ib, Ic, and Id showed reduced or no hypoxia regulation. Deletion of the ODD sequence in HIF alpha Ia up-regulated the HIF alpha Ia levels under normoxia. Gene expression analysis revealed HIF alpha Ic to be the predominant isoform in embryos and larvae, whereas isoform Ia was the most abundant form in the adult stage. The expression levels of Ib and Id were very low. Hypoxia treatment of adults had no effect on the mRNA levels of these HIF alpha isoforms. Functional analyses in mammalian cells showed all 4 HIF alpha isoforms capable of entering the nucleus and activating hypoxia response element-dependent reporter gene expression. The functional nuclear location signal (NLS) mapped to 3 clusters of basic residues. (775)KKARL functioned as the primary NLS, but (KRK)-K-737 and (KK)-K-754 also contributed to the nuclear localization. All amphioxus HIF alpha isoforms had 2 functional transactivation domains (TADs). Its C-terminal transactivation (C-TAD) shared high sequence identity with the human HIF-1 alpha and HIF-2 alpha C-TAD. This domain contained a conserved asparagine, and its mutation resulted in an increase in transcriptional activity. These findings reveal many ancient features of the HIF alpha family and provide novel insights into the evolution of the HIF alpha family.-Gao, S., Lu, L., Bai, Y., Zhang, P., Song, W., Duan, C. Structural and functional analysis of amphioxus HIF alpha reveals ancient features of the HIF alpha family.