Fig. 3

CArG sites are required for InvINH1 promoter activity. To delete the four putative CArG sites (denoted by open squares) located within the 189 bp enhancer 1 region (CArG1: -426 bp, CArG2: -374 bp, CArG3: -333 bp, and CArG4: -269 bp), a series of internal promoter deletion constructs were generated by making 20 bp sequential deletions from the 3′ end of enhancer 1 (D5-d1 to D5-d7), and by deleting an 80 bp region encompassing the CArG1 and CArG2 sites (D5-d8) from the D5 promoter fragment. D5-ΔCArG-3,4 and D5-ΔCArG-1,2,3,4 represent constructs in which only the CArG sites were specifically deleted. Promoter activity in the presence of AGL40-AGL90 dimer was measured as the ratio of promoter-GUS activity over luciferase activity (control for transfection efficiency). All promoter activities were normalized as the percentage of the full-length (FL) promoter activity in the presence of AGL40-AGL90 dimer. Averages and standard deviations were calculated from 4 to 32 biological replicates and two technical replicates for each biological sample