Figure 6.

Amphioxus HIFα contained 2 functional TADs. A) Sequence alignment of human HIF-1α C-TAD with the corresponding region of amphioxus HIFα. Asterisks indicate identical residues. Arrowheads mark the conserved leucine residues that are known to be important for human HIF-1α binding to its coactivators. The conserved asparagine residue is marked in a larger, bold letter. B) Deletion of C-TAD in amphioxus HIFα decreased its transactivation activity. HEK 293T cells were transfected with pCS2-EGFP, pCS2-AmHIFIa (Ia), or pCS2-AmHIFIaΔCTAD, together with p2.1 and pRL-SV40T (Renilla). Luciferase activity was measured 24 h after transfection. C) Amphioxus HIFα C-TAD had strong transactivation activity, and mutation of N856 further increased its activity. HEK 293T cells were transfected with pBIND, pBIND-CTAD, or pBIND-CTAD-N856A, together with pG5luc and pRL-SV40T (Renilla). At 24 h after transfection, luciferase activity was measured. Transactivation activity is expressed as fold increase over the control group in all panels. Values are means ± sd (n=3). Groups with different letters differ significantly from each other (P<0.05). D) Mutation of N856 increased full-length amphioxus HIFα activity. HEK 293T cells were transfected with pCS2-AmHIFIa (Ia) or pCS2-AmHIFIa-N856A (Ia-N856A), together with p2.1 and pRL-SV40T. E–G) Identification and mapping of an additional TAD. E) The 7 truncation mutants used to map the additional TAD. Numbers above the gray boxes indicate amino acid positions according to isoform Ia. F) Results of the measurement of their activities. G) Successful expression of the indicated Gal4-fusion proteins was validated by immunoblot analysis. H, I) Both C- and N-TAD are necessary for full transcriptional activity. H) Schematic diagram of the amphioxus HIFα mutants. I) Mutant activities were measured as described above and are shown relative to that of amphioxus HIFα Ia. Values are means ± sd (n=3). Groups with different letters differ significantly from each other (P<0.05).