Fig. 5. Hesx1 represses transcription induced by paired class activator proteins. (A) Hesx1 but not Hesx1(50-185) can repress transcription stimulated by Bix, Mix-1, and Mixer. Expression vectors for these paired class activators were co-transfected along with the indicated reporter, (P3)6E4, which contains six dimeric paired class binding sites upstream of the minimal E4 promoter and expression vectors for either GAL4(1-147), GAL4(1-147)-Hesx1 or GAL4(1-147)-Hesx1(50-185). Increasing concentrations of expression vectors (25 and 100 ng) for Bix and Mix and 100 ng for Mixer were co-transfected with 25 ng of the indicated Hesx1 derivative. (B) Cooperative repression by Hesx1 but not Hesx1(50-185). A Bix expression vector was co-transfected with the (P3)6E4 as in A. Increasing amounts of GAL4-Hesx1 or GAL4-Hesx1(50-185) were co-transfected with the Bix reporter. Titrations of the Bix expression vector were used to determine the optimal levels of induction of the (P3)6E4 reporter. Optimal levels of induction were found to be between 220- and 380-fold depending on the experiment. The addition of expression vector encoding either full length Hesx1 or Hesx1(50-185) always produced the same repressed level of transcription (i.e. 20-fold when Bix was co-transfected with 25 ng of vector encoding full length GAL4-Hesx1 compared to 225-fold when Bix was co-transfected with vector encoding GAL4-Hesx1(50-185)).
