FIGURE 3.

E4BP4 directly represses Fgf21 and is required for its circadian oscillation in mouse liver cells. A, overexpression of E4BP4 suppresses the secretion of FGF21 in culture medium of primary mouse hepatocytes. Cells were infected with either Ad-GFP or Ad-E4BP4 for 48 h and the culture medium was then collected for ELISA analysis of FGF21. Results are expressed as mean ± S.D. (error bars) of triplicates. A representative result of three individual experiments is shown. The levels of total E4BP4 protein in primary hepatocytes transduced with either Ad-GFP or Ad-E4BP4 were detected by Western blot. B, chromatin immunoprecipitation was performed to detect E4BP4 binding to the mouse Fgf21 promoter in Hepa1c1c-7 cells transfected with either GFP or E4BP4 expression plasmid. The recruitment of RNA polymerase II was examined as well. Results are expressed as mean ± S.D. of three experiments. The locations of ChIP primers are shown as well. C, Hepa1c1c-7 cells were transfected with the control or E4bp4 shRNA for 72 h. Cells were synchronized by 50% horse serum for 2 h and incubated in serum-free medium for another 16 h. Cells then were harvested for mRNA analysis of the Fgf21 gene by Q-PCR. Results are expressed as mean ± S.D. of at least three independent experiments. *, p < 0.05. Knockdown efficiency of E4BP4 protein knockdown is shown below. D, knockdown of E4BP4 alters the circadian oscillation of mouse Fgf21 in synchronized Hepa1c1c-7 cells. After synchronization treatment with both dexamethasone (100 nm) and forskolin (10 μm), the Hepa1c1c-7 cells stably expressing either control shRNA or E4bp4 shRNA were collected for mRNA analysis at the indicated time points. The value given for the amount of mRNA present at the 16 h time point was set as 1. Error bars, ±range (n = 2). Inset, circadian oscillation of Fgf21 in the shRNA control group.