Requirement of the SPS orientation architecture for synergistic interactions between the Da-N domain and NTCs. Transcription assays with co-expression of NICD and Da-N protein on the wild-type m8 promoter (m8-WT, or "FR") and an m8 promoter containing CSL binding sites with reversed orientations relative to the wild-type promoter (m8-RF). Reversal of the CSL binding site orientations in the SPS element (see insert) prevents co-expressed NICD and Da-N proteins from synergistically activating the E(spl) m8 RF promoter, even though the Da-N domain is not bound to DNA, and can contact the Su(H) protein from any direction. Asterisk in expt. 4 indicates promoter activity significantly greater than either expt. 2 or 3 (p < 0.01).