Histone methyltransferase G9a protects against acute liver injury through GSTP1

Abstract

Acute liver injury is commonly caused by bacterial endotoxin/lipopolysaccharide (LPS), and by drug overdose such as acetaminophen (APAP). The exact role of epigenetic modification in acute liver injury remains elusive. Here, we investigated the role of histone methyltransferase G9a in LPS- or APAP overdose-induced acute liver injury. Under D-galactosamine sensitization, liver-specific G9a-deficient mice (L-G9a^-/-) exhibited 100% mortality after LPS injection, while the control and L-G9a^+/- littermates showed very mild mortality. Moreover, abrogation of hepatic G9a or inhibiting the methyltransferase activity of G9a aggravated LPS-induced liver damage. Similarly, under sublethal APAP overdose, L-G9a^-/- mice displayed more severe liver injury. Mechanistically, ablation of G9a inhibited H3K9me1 levels at the promoters of Gstp1/2, two liver detoxifying enzymes, and consequently suppressed their transcription. Notably, treating L-G9a^-/- mice with recombinant mouse GSTP1 reversed the LPS- or APAP overdose-induced liver damage. Taken together, we identify a novel beneficial role of G9a-GSTP1 axis in protecting against acute liver injury.

Fig. 1

Hepatic G9a deletion aggravates LPS-induced liver damage. a Relative mRNA levels of Ehmt2 and Ehmt1 in the liver, muscle and kidney of Control, L-G9a^+/− and L-G9a^−/− mice. b Representative western blots for G9a, GLP, H3K9me1, H3K9me2, H3, and HSP70 in the liver and muscle of Control, L-G9a^+/− and L-G9a^−/− mice. c Survival rate (n = 21 for Controls; n = 16 for L-G9a^+/− mice; n = 11 for L-G9a^−/− mice) and d H&E staining (n = 6–8 per group; scale bar, 50 μm) after LPS/d-Gal co-injection. e Serum levels of ALT and AST with or without LPS/d-Gal co-injection. f Representative pictures for F4/80 staining in livers (left) and quantitative results (right) after LPS/d-Gal co-injection (scale bar, 50 μm). All data were obtained from male mice. n = 3–4 per group; n.s., not significant; ^*p < 0.05

Fig. 2

Hepatic G9a deletion augments LPS-induced inflammation, RNS/ROS production, DNA damage and hepatocyte apoptosis. a Representative pictures for F4/80, Ly-6G and CD3 staining in the liver (left) and quantitative results (right). b qPCR results of the indicated genes in the liver of different groups. c, d Representative western blots (left) and quantitative results (right) for VCAM-1, ICAM-1, and MCP-1 (c), iNOS, eNOS and 3-NT (d) in livers of different groups. HSP70 serves as the loading control. e Representative pictures for 3-NT staining in the liver (left) and quantitative results (right). f Relative mRNA level of Atf3 in the liver at 1 h after LPS injection. g Representative pictures for 8-oxoG staining in the liver (left) and quantitative results (right). n = 3–4 per group; 3-NT, 3-Nitrotyrosine; n.s., not significant; ^*p < 0.05; scale bar, 50 μm

Fig. 3

Hepatic G9a deletion augments DNA damage and hepatocyte apoptosis upon LPS injection. a, b Representative western blots (up) and quantitative results (down) for markers of DNA damage response signaling (a) and apoptosis (b) in livers of different groups. HSP70 serves as the loading control. c Representative pictures for TUNEL staining in the liver (left) and quantitative results (right). All data were obtained from male mice. n = 3–4 per group; n.s., not significant; ^*p < 0.05; scale bar, 50 μm

Fig. 4

Inhibiting G9a methyltransferase activity deteriorates LPS-induced liver damage. a, b Representative pictures for H&E staining (a; scale bar, 100 μm); and F4/80 staining (b, left; scale bar, 50 μm) with quantitative results (b, right) in the liver. c, d Representative western blots (c) and quantitative results (d) of indicated proteins in livers of different groups. HSP90 serves as the loading control. e Relative mRNA level of Atf3 in the liver at 24 h after LPS injection. n = 3–4 per group; ^*p < 0.05

Fig. 5

Deletion of G9a exacerbates APAP overdose-induced acute liver injury. a Representative pictures of livers from different groups at 24 h after APAP injection. b Representative pictures for H&E staining in the liver; scale bar, 200 μm. c Serum levels of ALT and AST. d Levels of GSH in the liver. e Relative mRNA levels of Atf3 and Cox-2 in the liver. f Representative western blots (left) and quantitative results (right) for the indicated proteins in the liver of different groups. HSP70 serves as the loading control. g Representative pictures for Ly-6G staining in the liver (left) and quantitative results (right); scale bar, 50 μm. n = 4 per group; ^*p < 0.05

Fig. 6

G9a mediates H3K9me1 at the promoters of Gstp. a Coomassie brilliant blue staining of whole liver extracts from Control, L-G9a^+/− and L-G9a^−/− mice. b Relative mRNA levels of Gstp1 and Gstp2 in the liver of different groups at 24 h after LPS injection. c, d Representative western blots of GSTP1/2 (left) and quantitative results (right) in livers of different groups. HSP70 serves as the loading control. e ChIP-qPCR analysis of H3K9me1 levels at the promoters of Gstp1 and Gstp2 in livers of different groups at 24 h after LPS injection. f ChIP-qPCR analysis of G9a levels at the promoter of Gstp1 in livers of control and L-G9a^−/− mice (error bars indicate SEM). Above data (a–f) were obtained from mice with n = 3–4 per group. g, h Representative western blots of GSTP1/2, G9a, H3K9me1, H3K9me2, or GLP after overexpression of pCAGGS-mG9a in primary hepatocytes obtained from WT (g) or G9a^−/− (h). HSP70 serves as the loading control. Similar results were observed from the Ad-G9a transfection. i ChIP-qPCR analysis of H3K9me1 levels at the promoters of Gstp1 in control, G9a^−/− or adenovirus-mediated G9a transfected G9a^−/− primary hepatocytes. ^*p < 0.05

Fig. 7

rGSTP1 attenuates LPS-induced liver damage in L-G9a^−/− mice. a Representative western blots (left) and quantitative results (right) for GSTP1/2, H3K9me1/me2 in the livers of different groups. H3 or HSP90 serves as the loading control. b Serum levels of ALT and AST. c Representative pictures for H&E staining in the liver; scale bar, 100 μm. d Representative pictures for F4/80 and 3-NT staining (left) and quantitative results (right); scale bar, 50 μm. e, f Representative western blots (up) and quantitative results (down) of the indicated proteins in livers of different groups. HSP90 serves as the loading control. All these data were obtained from male L-G9a^−/− mice. n = 4–6 per group; ^*p < 0.05

Fig. 8

rGSTP1 attenuates APAP-induced liver damage in L-G9a^−/− mice. a Representative pictures of livers from different groups at 24 h after APAP injection. b Representative pictures for H&E staining in the liver; scale bar, 200 μm. c Serum levels of ALT and AST. d Levels of GSH in the liver. e Relative mRNA level of Atf3 in the liver. f Representative western blots (left) and quantitative results (right) for the indicated proteins in livers of different groups. HSP70 serves as the loading control. g Representative pictures for Ly-6G staining in the liver (up) and quantitative results (down); scale bar, 50 μm. All these data were obtained from male L-G9a^−/− mice. n = 4–6 per group; n.s., not significant; ^*p < 0.05. h Working model for how G9a regulates acute liver injury