Fig. 3. Structure-function mapping of the transactivation domain of Sox17ß. (A) The Gal4 DNA-binding domain (Gal4DBD) was fused to various parts of the Sox17ß open reading frame. COS-1 cells were co-transfected with the indicated Gal4-fusion constructs (300 ng), UAS:luciferase reporter (100 ng) containing five Gal4 binding sites and a pTK-Renilla Luciferase plasmid (50 ng). The average relative luciferase activity normalized to renilla activity, from a triplicate experiment is shown. The Gal4 DNA-binding domain alone (Gal4DBD) is a negative control and Gal4:VP16 is a positive control containing the viral VP16 transactivation domain. (B) 200 pg of RNA encoding the indicated HA-tagged Sox17ß deletion constructs was injected into two-cell stage embryos, animal cap tissue was isolated at blastula stage, cultured until gastrula stage and assayed by real-time RT-PCR for the expression of Sox17 target genes. The histogram shows the relative gene expression normalized to the loading control ODC. (C) The schematic shows the transactivation domain of Sox17ß contains a sequence motif conserved in all members of the SoxF subfamily of Sox proteins. A sequence alignment of this conserved motif from representative proteins is shown. Identical amino acids are white on black, conserved resides are in bold. Below the schematic the ‘3G’ and ‘TA’ mutations generated in both Sox17 and Sox17ß are shown. (D) 200 pg of RNA encoding either wild-type or mutant versions of Sox17ß (top three constructs) or Sox17 (bottom three constructs) were injected into two-cell stage embryos, animal cap tissue was isolated at blastula stage, cultured until gastrula stage and assayed by real-time RT-PCR for the expression of Sox17 target genes. The histogram shows the relative gene expression normalized to the loading control ODC.