Fig. 7. The Esr1 enhancer does not require E-boxes and responds to Notch through two loci. (A) Luciferase activity of Esr1/RV and Hin3 fragments co-transfected with activated Notch (ICD) plus or minus Xngnr1 (ngn). (B) Luciferase activity of Esr1/RV and Esr1/Hin3 vectors co-transfected with ICD compared with proximal elements from mouse Hes1 (Jarriault et al., 1995) and Xenopus Hairy2 (Davis et al., 2001), Esr10/Dra and a vector containing eight multimerized Su(H) sites (Ling et al., 1994). Cells were transfected simultaneously with equal levels of ICD (100 ng/well) relative to the reporter (100 ng/well). (C) Luciferase activity of Esr1/RV co-transfected with increasing (25 ng/well and 100 ng/well) levels of ICD compared with a construct in which all upstream Su(H) sites are mutant (Esr1/RVmS3-5) or the Esr1/Hin3 deletion mutant. Unlike the wild-type reporter, luciferase activity of the Su(H) and Hin3 mutant constructs saturates at low (25 ng) ICD levels. (D) An S4 mutation results in loss of transcription similar to Esr1/RVmS-35.