FIGURE 4.

G9a represses Fgf21 transcription in hepatocytes. A, G9a inhibits the Fgf21 promoter activity as efficiently as E4BP4. Hepa1 cells were transfected with Fgf21-luc along with GFP, FLAG-E4bp4, or HA-G9a expression vector. 48 h later, cells were harvested for the luciferase assay and the β-gal assay as a transfection efficiency control. The data were plotted as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test. B, the inhibitory effect of G9a on Fgf21-luc activity depends on its SET domain. Hepa1 cells were transfected with Fgf21-luc along with GFP, G9a-WT, or G9a-ΔSET expression vector and lysed 48 h later for the luciferase assay. The data were plotted as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test. C, inhibition of G9a by BIX01294 causes dose-dependent activation of Fgf21-luc activity. Data were plotted as mean ± S.E. (error bars) (n = 3). *, p < 0.05. D, G9a knockdown elevates Fgf21 mRNA in PMHs. PMHs were harvested 48 h after transduction by either Ad-shLacz or Ad-shG9a, and the mRNAs were quantified by QPCR. Data were plotted as mean ± S.E. (n = 4). *, p < 0.05 by Student's t test. E, G9a knockdown enhanced circadian oscillations of Fgf21 mRNA in synchronized Hepa1 cells. After depletion of G9a expression by adenoviral knockdown, Hepa1 cells were synchronized by serum shock and then harvested at the indicated time points for RNA isolation. The expression of Fgf21 mRNA was measured by QPCR and normalized to internal control 18 S RNA. Data were plotted as mean ± S.E. (n = 3). F, occupancy of G9a on the Fgf21 promoter in the liver. G9a binding to the Fgf21 promoter in the refed mouse livers was detected by a ChIP assay with anti-G9a antibody.