Fig. 5. Sens directly represses Rh3 and Rh4 promoter activity. (A) Position-weighted matrix (PWM) of Gfi1 binding sites (top), table of two known Sens binding sites, R21 and S-box (Jafar-Nejad et al., 2003), and sites within the Rh3 and Rh4 promoters with >70% homology to the consensus. Corresponding PWM scores are listed. Nucleotides that are present >20% in the PWM data set are highlighted green. Rh3 and Rh4 promoter diagrams represent the conserved RCSI/Pax6 binding site present in all Rh promoters (Papatsenko et al., 2001; Sheng et al., 1997) (gray), K50 Otd-binding sites (Tahayato et al., 2003) (blue) and potential Sens binding sites (green). (B) EMSAs with Rh3 (-247 to +18) and Rh4 (-159 to +85) promoters and 0, 50 or 500 ng His-SensZF. (C) Relative luciferase activity in Drosophila S2 cells transfected with pAc5.1 or pAc-Sens and pGL3 with or without Rh3 (-247 to +18), Rh4 (-159 to +85), Rh5 (-236 to +50) or Rh6 (-555 to +121) promoters. ^**, P<0.01 compared with pAc alone. (D) Relative luciferase activity of Rh3 or Rh4-containing pGL3 reporters with mutated Sens binding sites (AATC core GGTC). Sites correspond to those in A. ^*, P<0.05 compared with pAc alone. (E,F) X-Gal staining of cryosections from transgenic lacZ reporter lines carrying wild-type or Sens mutant binding sites in the Rh3 (E) or Rh4 (F) promoters. R7 and R8 layers are bracketed.