(A) Schematic representation of the class C site (solid box, bp −258) in the proximal hASH1 repressor region (open box). The striped box indicates the basal hASH1 enhancer (base pairs −234 to −46). (B and C) Transient transfection assays of hASH1 promoter constructs. Fragments of the hASH1 promoter region containing 5′ flanking sequence (shaded box), the candidate class C site (open box labeled with the letter C), the generalized enhancer region (striped box), the transcription initiation site (at position +1), and a short 5′ untranslated region were subcloned into the luciferase reporter vector pGL2 and then cotransfected with CMV–β-galactosidase and either HES-1 (shaded bars) or control (solid bars) vectors into DMS53 cells [HES-1(−), hASH1(+)] (B) and NCI-H157 cells [HES-1(+), hASH1(−)] (C). Luciferase activity is normalized to CMV–β-galactosidase and expressed in relation to the most active construct (plasmid −234/+37). The letter X over the class C site represents mutation of this motif.