Fig. 8. Esr1 enhancer activity requires upstream Su(H) sites in vivo. (A,B) GFP expression in frogs transgenic with enhancer elements containing mutant E-boxes (Esr1/RVmE1E2E3) versus wild-type controls. Wild-type (C) and E1E2 mutant (D) embryos were injected with mRNA encoding Xngnr1 (ngn) and stained for GFP. GFP expression in frogs with mutant enhancers is unchanged relative to controls. (E,F) Transgenic frogs bearing Esr1 enhancer elements mutant in upstream Su(H) sites (Esr1/RVmS3-5) show greatly attenuated GFP activity (E) relative to controls (A), and activity is not inducible following Xngnr1 injection (F). (G) Within the S3-5 cluster, mutations within S4 (H), which is conserved in sequence and position in numerous Esr1 homologs, greatly attenuate enhancer activity.