FIGURE 3.

Functional analysis of KLF5-binding sites in transcriptional regulation. A, schematic illustration of the cyclin D1 promoter-reporter deletion constructs that were used. Diagrams depict locations of cis-elements in the cyclin D1 promoter. CREB, cAMP-response element-binding protein; LUC, luciferase. B, schematic illustration of the cyclin D1 promoter-reporter deletion constructs that were used. wt, wild type. C, co-transfection reporter assay to map the KLF5-responsive region of the cyclin D1 promoter. Assays were done in duplicate, and data are shown as average. D, mutation alanalysis of Sp1-binding sites in the cyclin D1 promoter. Diagrams show mutated Sp1-binding site reporter constructs. Asterisks indicate the positions at which the point mutation (mut) was introduced. E, the horizontal axis shows the relative reporter activities. Assays were done in duplicate, and data are shown as average. F, EMSAs were performed using fluorescein isothiocyanate-labeled probes encoding the upstream Sp1-binding sites (Sp1-1 and Sp1-2). 100× molar excess of non-labeled double-stranded oligonucleotide encoding wild-type sites (lane 3) or mutant sites (lane 4) were used for competition experiments. Mutations to the Sp1-1 and Sp1-2 sites were introduced separately into the probe as shown in lanes 5 and 6 because of their overlapping nature. Nucleotide sequences of wild-type and mutated Sp1-binding sites are shown. DBD, KLF5-DNA binding domain. G, recruitment of KLF5 to the cyclin D1 promoter in vivo. Input samples represent 0.5% of total DNA, whereas immunoprecipitations (IP) include 5% of the resuspended DNA as shown. Note that direct recruitment of KLF5 to the cyclin D1 promoter was only seen under serum stimulation conditions (lane 6). The error bar denotes S.D.