Figure 4. The heterodimer controls leptin promoter activity in vitro.

A. Circadian heterodimers marginally stimulate a leptin promoter-driven luciferase (lep₂₇₆₂-Luc) reporter at a low concentration but inhibit the reporter activity at higher concentrations independent of CRY1. Numbers indicate the amounts of reporter and expression vector DNA (ng) used in each transfection (±SEM, *: stimulation and ^#: suppression). B. The C/EBPα binding sites (green) overlaps with consensus E-boxes (red boxes) in both human and mouse leptin promoters. C. The strategy for generating mutant leptin promoter-driven luciferase reporters with mutated bases shown in blue. D. The heterodimer potentiates C/EBPα-mediated lep₄₅₀-Luc reporter activation at a low concentration but inhibits C/EBPα activity at higher concentrations. Mutation of E1 and 2 abolishes the inhibitory effect of heterodimer. Mutation of E3 abolishes the stimulatory effect of heterodimer (±SEM, *: stimulation and #: suppression of by the heterodimers). *^/#p < 0.05, **^/##p < 0.01 and ***p < 0.001. See also Figure S6A–D.