FIG. 3.

CLOCK-BMAL1 heterodimer activates the promoter activity of ERβ gene via the E box. (A) Cotransfection of CLOCK and BMAL1 activates expression of a luciferase reporter construct flanked by the ERβ promoter region of human and mouse in HeLa cells. (B) The minimal 2×ERβ E box (schematically presented) luciferase construct (pGL3 luc) is transcriptionally activated in the presence of both CLOCK and BMAL1 in HeLa cells. (C) Mutation of the ERβ E-box promoter construct impairs the ability of CLOCK and BMAL1 to activate transcription of the luciferase reporter gene construct. All results presented here are compared to reporter luciferase activity of controls (pGL3-hERβ, pGL3-mERβ, pGL3-2×ERβ E box, or pGL3-ERβmut alone), which was arbitrarily set to be 1, and expressed as means ± standard deviations of three independent experiments performed in triplicate. Data were analyzed by using two-way ANOVA. *, the contribution of CLOCK or BMAL1 is significant with a P value of <0.01; **, statistically significant interaction between CLOCK and BMAL1 with a P value of <0.01.