The Chromatin Remodeling Protein BRG1 Regulates SREBP Maturation by Activating SCAP Transcription in Hepatocytes

Abstract

Sterol response element binding protein (SREBP) is a master regulator of cellular lipogenesis. One key step in the regulation of SREBP activity is its sequential cleavage and trans-location by several different proteinases including SREBP cleavage activating protein (SCAP). We have previously reported that Brahma related gene 1 (BRG1) directly interacts with SREBP1c and SREBP2 to activate pro-lipogenic transcription in hepatocytes. We report here that BRG1 deficiency resulted in reduced processing and nuclear accumulation of SREBP in the murine livers in two different models of non-alcoholic steatohepatitis (NASH). Exposure of hepatocytes to lipopolysaccharide (LPS) and palmitate (PA) promoted SREBP accumulation in the nucleus whereas BRG1 knockdown or inhibition blocked SREBP maturation. Further analysis revealed that BRG1 played an essential role in the regulation of SCAP expression. Mechanistically, BRG1 interacted with Sp1 and directly bound to the SCAP promoter to activate SCAP transcription. Forced expression of exogenous SCAP partially rescued the deficiency in the expression of SREBP target genes in BRG1-null hepatocytes. In conclusion, our data uncover a novel mechanism by which BRG1 contributes to SREBP-dependent lipid metabolism.

Keywords: chromatin remodeling protein; hepatocyte; lipid metabolism; steatosis; transcription factor; transcriptional regulation.

FIGURE 1

BRG1 regulates SREBP maturation in the liver. (A) WT and BRG1 LKO mice were fed a high-fat diet (HFD) for 16 weeks. SREBP levels were examined in whole liver lysates and liver nuclear lysates by Western blotting. (B) WT and BRG1 LKO mice were fed a methionine-and-choline deficient diet (MCD) for 4 weeks. SREBP levels were examined in whole liver lysates and liver nuclear lysates by Western blotting. N = 6 mice for each group. Data represent averages of three independent experiments and error bars represent SEM. *p < 0.05, two-tailed Student’s t-test.

FIGURE 2

BRG1 is essential for SREBP maturation in hepatocytes. (A,B) HepG2 cells were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). SREBP target genes were examined by qPCR. SREBP levels were examined in whole cell lysates and nuclear lysates by Western blotting. (C,D) HepG2 cells were treated with LPS (1 μg/ml) + PA (0.2 mM) in the presence or absence of PFI-3 (3 μM). SREBP target genes were examined by qPCR. SREBP levels were examined in whole cell lysates and nuclear lysates by Western blotting. (E,F) Primary hepatocytes isolated from WT and BRG1 LKO mice were treated with LPS (1 μg/ml) + PA (0.2 mM). SREBP levels were examined in whole cell lysates and nuclear lysates by Western blotting. Data represent averages of three independent experiments and error bars represent SEM. *p < 0.05, two-tailed Student’s t-test.

FIGURE 3

BRG1 regulates SCAP expression in vivo. (A,B) WT and BRG1 LKO mice were fed a high-fat diet (HFD) for 16 weeks. SCAP expression was examined by qPCR and Western blotting. N = 5 mice for the chow groups and N = 6 mice for the HFD groups. (C,D) WT and BRG1 LKO mice were fed a methionine-and-choline deficient diet (MCD) for 8 weeks. SCAP expression was examined by qPCR and Western blotting. N = 5 mice for the chow groups and N = 6 mice for the MCD groups. Data represent averages of three independent experiments and error bars represent SEM. *p < 0.05, two-tailed Student’s t-test.

FIGURE 4

BRG1 regulates SCAP expression in vitro. (A,B) HepG2 cells were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). SREBP target genes were examined by qPCR. SCAP expression was examined by qPCR and Western blotting. (C,D) HepG2 cells were treated with LPS (1 μg/ml) + PA (0.2 mM) in the presence or absence of PFI-3 (3 μM). SCAP expression was examined by qPCR and Western blotting. (E,F) Primary hepatocytes isolated from WT and BRG1 LKO mice were treated with LPS (1 μg/ml) + PA (0.2 mM). SCAP expression was examined by qPCR and Western blotting. Data represent averages of three independent experiments and error bars represent SEM. *p < 0.05, two-tailed Student’s t-test.

FIGURE 5

BRG1 interacts with Sp1 to activate SCAP transcription in hepatocytes. (A) An SCAP promoter-luciferase construct (–1033/+25) was transfected into HepG2 cells with or without BRG1 followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). Luciferase activities were normalized by protein concentration and GFP fluorescence. (B) An SCAP promoter-luciferase construct (–1033/+25) was transfected into primary hepatocytes isolated from WT and BRG1 LKO mice followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). Luciferase activities were normalized by protein concentration and GFP fluorescence. (C) SCAP promoter-luciferase constructs of various lengths were transfected into HepG2 cells with or without BRG1 followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). Luciferase activities were normalized by protein concentration and GFP fluorescence. (D) HepG2 cells were treated with or without LPS (1 μg/ml) + PA (0.2 mM) and harvested at indicated time points. ChIP assays were performed with anti-BRG1 or IgG. (E) HepG2 cells were treated with or without LPS (1 μg/ml) + PA (0.2 mM) for 24 h. Re-ChIP assays were performed with indicated antibodies. (F) HepG2 cells were transfected with siRNA targeting Sp1 or scrambled siRNA (SCR) followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). ChIP assays were performed with anti-Sp1 or anti-BRG1. (G) Wild type or Sp1 site mutant SCAP promoter-luciferase construct was transfected into HepG2 cells with or without BRG1 followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). Luciferase activities were normalized by protein concentration and GFP fluorescence. Data represent averages of three independent experiments and error bars represent SEM. *p < 0.05, two-tailed Student’s t-test.

FIGURE 6

SCAP re-introduction partially rescues the expression of SREBP target genes in BRG1 deficient hepatocytes. (A,B) A Myc-tagged SCAP expression construct was transfected into primary hepatocytes isolated from BRG1 LKO mice followed by treatment with LPS (1 μg/ml) + PA (0.2 mM). Gene expression levels were examined by qPCR and Western blotting. Data represent averages of three independent experiments and error bars represent SEM. *p < 0.05, two-tailed Student’s t-test. (C) A schematic model. BRG1 may contribute to pro-lipogenic transcription in hepatocytes via at least two independent mechanisms. On the one hand, BRG1 stimulates SCAP expression to promote SREBP maturation. On the other hand, BRG1 interacts with mature SREBPs in the nucleus to directly activate pro-lipogenic gene transcription. Consequently, accelerated lipogenesis in hepatocytes leads to steatosis.