Fig. 7. Repression of Hb9 by homeodomain transcription factors. (A) Plasmids encoding progenitor factors, Nkx2.2, Nkx6.1, Pax6 and Irx3, were co-transfected with the 2.5 kb distal+TK:luciferase reporter into 293 cells and luciferase activity was used to monitor the fold repression relative to vector-only transfections. (B) Hb9 and EnR-Hb9 (Hb9 homeodomain linked to eh1 engrailed repressor domain) repressed the synergistic activation of M250 by Isl1/Lhx3/NeuroM in transfected P19 cells. By contrast, Hb9-HD and Hb9-HD fused VP16 activation domain (VP16-Hb9) did not. (C,D) Gel retardation analysis reveals that Hb9 binds to the M50 and M100 (not shown) portions of its enhancer. Antibodies against Hb9 supershifted or disrupted the DNA:protein complex, whereas IgG control serum had no effect on DNA binding. Mutation of the ATTA sequences within M50 (ATTAm) disrupted Hb9 binding. The protein complex formed by Isl1/Lhx3 relies on the same ATTA elements for binding (Lee and Pfaff, 2003), suggesting Hb9 may compete with the LIM factors for access to the enhancer.