Recruitment of histone methyltransferase G9a mediates transcriptional repression of Fgf21 gene by E4BP4 protein

Abstract

The liver responds to fasting-refeeding cycles by reprogramming expression of metabolic genes. Fasting potently induces one of the key hepatic hormones, fibroblast growth factor 21 (FGF21), to promote lipolysis, fatty acid oxidation, and ketogenesis, whereas refeeding suppresses its expression. We previously reported that the basic leucine zipper transcription factor E4BP4 (E4 binding protein 4) represses Fgf21 expression and disrupts its circadian oscillations in cultured hepatocytes. However, the epigenetic mechanism for E4BP4-dependent suppression of Fgf21 has not yet been addressed. Here we present evidence that histone methyltransferase G9a mediates E4BP4-dependent repression of Fgf21 during refeeding by promoting repressive histone modification. We find that Fgf21 expression is up-regulated in E4bp4 knock-out mouse liver. We demonstrate that the G9a-specific inhibitor BIX01294 abolishes suppression of the Fgf21 promoter activity by E4BP4, whereas overexpression of E4bp4 leads to increased levels of dimethylation of histone 3 lysine 9 (H3K9me2) around the Fgf21 promoter region. Furthermore, we also show that E4BP4 interacts with G9a, and knockdown of G9a blocks repression of Fgf21 promoter activity and expression in cells overexpressing E4bp4. A G9a mutant lacking catalytic activity, due to deletion of the SET domain, fails to inhibit the Fgf21 promoter activity. Importantly, acute hepatic knockdown by adenoviral shRNA targeting G9a abolishes Fgf21 repression by refeeding, concomitant with decreased levels of H3K9me2 around the Fgf21 promoter region. In summary, we show that G9a mediates E4BP4-dependent suppression of hepatic Fgf21 by enhancing histone methylation (H3K9me2) of the Fgf21 promoter.

FIGURE 1.

E4BP4 suppresses Fgf21 gene expression in vivo. A, loss of E4BP4 protein expression in the liver of E4pb4^−/− mice. Pooled protein lysates (about 500 μg) from three mice (WT or E4pb4^−/−) were immunoprecipitated by anti-E4BP4 (A9 from Santa Cruz Biotechnology, Inc.). The presence of E4BP4 protein was detected by immunoblotting with anti-E4BP4 (H300 from Santa Cruz Biotechnology, Inc.). HC, heavy chain. B, serum levels of FGF21 are elevated in E4pb4^−/− mice. Serum samples of 3-month E4pb4^−/− male mice fed regular chow were collected for FGF21 measurement. *, p < 0.05 by Student's t test. C, mRNA levels of Fgf21 are increased in the liver of E4bp4 knock-out mice. Liver tissues from the same cohort of mice shown in B were harvested for mRNA measurement. Data were plotted as mean ± S.E. (error bars) (n = 4–5). *, p < 0.05 by Student's t test.

FIGURE 2.

Effects of inhibitors of histone-modifying enzymes on E4BP4-mediated suppression of the Fgf21 promoter. A, trichostatin A (TSA; class I and II HDAC inhibitor) does not affect E4BP4-dependent suppression of Fgf21 promoter activity. 24 h postcotransfection with Fgf21-luc plus E4bp4 expression vector versus GFP control vector, Hepa1 cells were exposed to 100 μm trichostatin A overnight prior to the luciferase assay. The luciferase levels were normalized to an internal control of β-galactosidase, and the value of the GFP control group was set as 1. Data were plotted as mean ± S.E. (error bars) (n = 3). *, p < 0.05 by Student's t test. B, nicotinamide (NAM; class III HDAC inhibitor) shows no impact on repression of the Fgf21 promoter activity by E4BP4. Hepa1 cells were similarly transfected as in A and then subjected to 20 mm nicotinamide overnight before luciferase assay. Data were plotted as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test. C, BIX01294, a specific inhibitor of histone methyltransferase G9a, abolishes E4BP4-dependent suppression of the Fgf21 promoter activity. Transfected Hepa1 cells were subjected to 10 μm BIX01294 treatment overnight before the luciferase assay. Data were plotted as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test. D, overexpression of E4BP4 increases the level of H3K9me2 on the 5′-UTR and TSS region around the mouse Fgf21 promoter. 48 h after transduction by either Ad-GFP or Ad-E4bp4, Hepa1 cells were harvested for a ChIP assay with antibody against H3K9me2. The -fold increase was calculated with the level of the Ad-GFP control group set as 1. Primers used for the ChIP assay are indicated. The overexpression of E4BP4 was determined by immunoblotting with anti-E4BP4.

FIGURE 3.

E4BP4 interacts with the histone methyltransferase G9a. A, E4BP4 and G9a interact with each other in vitro. 293T cells were transfected with FLAG-E4bp4 expression vector in the presence or absence of full-length HA-G9a. 48 h post-transfection, protein lysates were used for IP with anti-FLAG antibody, and the presence of G9a was detected with immunoblotting with anti-HA antibody. B, E4BP4 does not interact with LSD1 in vitro. The potential interaction between FLAG-E4BP4 and HA-LSD1 was similarly assayed in transiently transfected 293T cells. IP was performed with anti-FLAG antibody. The presence of HA-LSD1 was detected by immunoblotting with anti-HA antibody. NS, nonspecific signal. C, the C-terminal repression domain of E4BP4 is required for interaction with G9a. The HA-G9a expression vector was co-transfected with GFP, FLAG-E4bp4-WT, or FLAG-E4bp4-Δ-R (amino acids 1–380) expression vector. IP was performed as usual with anti-FLAG antibody. The presence of HA-G9a was detected by immunoblotting with anti-HA antibody. D, insulin has no effect on interaction between E4BP4 and G9a. Interactions between FLAG-E4BP4 and HA-G9a were tested in the presence of insulin treatment. 293T cells were first transfected with FLAG-E4bp4 and HA-G9a vectors. 24 h later, cells were switched to serum-free medium and treated with 100 nm insulin for 16 h before IP with anti-FLAG antibody. The presence of HA-G9a was detected by immunoblotting with anti-HA.

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.

FIGURE 5.

E4BP4 represses Fgf21 expression and promoter activity via G9a. A, G9a repression of Fgf21 requires a functional E4BP4-binding site. Hepa1 cells were transfected with either Fgf21-luc-WT or Fgf21-luc-ΔD1 mutant (D-box 1 deletion) along with G9a expression vector. 48 h later, cells were harvested for the luciferase assay and the β-gal assay as a transfection efficiency control. Data were plotted as mean ± S.E. (error bars) (n = 3). *, p < 0.05 by Student's t test. B, knockdown of E4bp4 abrogates G9a-dependent suppression of the Fgf21 promoter activity in hepatocytes. Hepa1 cells were cotransfected with Fgf21-WT-luc and G9a expression vectors after transduction by either Ad-shLacz or Ad-shE4bp4. 48 h later, cells were harvested for the luciferase assay and the β-gal assay as a transfection efficiency control. Data were plotted as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test. C, knockdown of G9a abrogates E4BP4-dependent suppression of Fgf21 promoter activity in hepatocytes. Hepa1 cells were cotransfected with Fgf21-WT-luc and E4bp4 expression vector after transduction of either Ad-shLacz or Ad-shG9a. 48 h later, cells were harvested for the luciferase assay and the β-gal assay as a transfection efficiency control. Data were plotted as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test. D, depletion of G9a blocks E4BP4-dependent repression of Fgf21 mRNA expression in hepatocytes. Hepa1 cells were transduced with the following paired adenoviruses: Ad-GFP plus Ad-shLacz versus Ad-E4bp4 plus Ad-shLacz; Ad-GFP plus Ad-shG9a versus Ad-E4bp4 plus Ad-shG9a. Cells were then synchronized by serum shock and harvested 36 h postsynchronization. Data were plotted as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test.

FIGURE 6.

In vivo role of G9a in regulating hepatic Fgf21 expression. A, depletion of G9a in mouse liver tissues injected with either Ad-shLacz or Ad-shG9a. The mRNA levels of G9a were detected by QPCR. Data were plotted as mean ± S.E. (error bars) (n = 5). **, p < 0.01 by Student's t test. B, G9a knockdown elevated the serum level of the ketone body in mice during refeeding. Shown is the serum β-hydroxybutyrate level in mice injected with Ad-shLacz or Ad-shG9a and subjected to a fasting-refeeding regimen. Levels of serum β-hydroxybutyrate are shown as mean ± S.E. (n = 5). *, p < 0.05 by Student's t test. C, abolishment of refeeding-induced suppression of Fgf21 by G9a knockdown in the mouse liver. Liver tissues after injection of either Ad-shLacz or Ad-shG9a were harvested during a fasting and refeeding cycle. mRNA of Fgf21 was extracted and assayed by QPCR. Data are plotted as mean ± S.E. (n = 5). **, p < 0.01 by Student's t test. D, G9a knockdown reduced dimethylation of H3K9 of the Fgf21 promoter in the refed mouse liver. Levels of H3K9me2 around the Fgf21 promoter after adenoviral knockdown of G9a were measured by a ChIP assay in refed mouse liver tissues. PCR primers amplifying various regions of the Fgf21 promoter are indicated. Data were shown as mean ± S.E. (n = 5). *, p < 0.05 by Student's t test.

FIGURE 7.

The working model of G9a-dependent repression of Fgf21 by E4BP4. E4BP4 is a potent repressor for the Fgf21 promoter in response to insulin stimulation and refeeding. Such potent repression is achieved through the recruitment of E4BP4-associated histone methyltransferase G9a to the Fgf21 promoter. Enzymatic activity of G9a enhances the repressive transcription mark of H3K9me2 around the Fgf21 promoter. During the fasting state, PPARα activates Fgf21 transcription while the E4BP4-G9a complex is disrupted and dissociated from the Fgf21 promoter.