Exploring the role of SWI/SNF complex subunit BAF60c in lipid metabolism and inflammation in fish

Abstract

Chromatin remodeling plays an important role in regulating gene transcription, in which chromatin remodeling complex is a crucial aspect. Brg1/Brm-associated factor 60c (BAF60c) subunit forms a bridge between chromatin remodeling complexes and transcription factors in mammals; hence, it has received extensive attention. However, the roles of BAF60c in fish remain largely unexplored. In this study, we identified BAF60c-interacting proteins by using HIS-pull-down and LC-MS/MS analysis in fish. Subsequently, the RNA-seq analysis was performed to identify the overall effects of BAF60c. Then, the function of BAF60c was verified through BAF60c knockdown and overexpression experiments. We demonstrated for the first time that BAF60c interacts with glucose-regulated protein 78 (GRP78) and regulates lipid metabolism, endoplasmic reticulum (ER) stress, and inflammation. Knockdown of BAF60c reduces fatty acid biosynthesis, ER stress, and inflammation. In conclusion, the results enriched BAF60c-interacting protein network and explored the function of BAF60c in lipid metabolism and inflammation in fish.

Keywords: Biological sciences; Molecular biology; Physiology; Zoology.

Graphical abstract

Figure 1

Identification of BAF60c-interacting proteins (A) KEGG enrichment analysis. (B) GO enrichment analysis. (C) Venn diagram. BP: biological process; CC: cellular component; MF: molecular function. See also Figures S1 and S2.

Figure 2

BAF60c interacts with GRP78 (A) Co-IP of HEK293T cells overexpressing BAF60c-HA and GRP78-FLAG. (B) HEK293T cells were co-transfected with BAF60c-GFP and GRP78-RFP plasmids for 24 h and followed by laser scanning confocal microscopy to observe the colocalization of BAF60c and GRP78. Scale bars, 20 μm.

Figure 3

BAF60c regulates GRP78 expression in fish (A) Relative mRNA expression of BAF60c and GRP78 after BAF60c-siRNA treatment for 36 h in macrophage. (B) Relative mRNA expression of BAF60c and GRP78 after BAF60c-siRNA treatment for 36 h in hepatocyte. (C) Relative mRNA expression of BAF60c and GRP78 after overexpression of BAF60c for 24 h in hepatocyte. (D) Relative mRNA expression of BAF60c and GRP78 after BiP treatment for 4 h with different concentrations in macrophages. (E) Protein level of BAF60c after 10 μM BiP treatment for 4 and 8 h in macrophages. (F) Relative mRNA expression of BAF60c and GRP78 after HM03 treatment for 4 h with different concentrations in macrophages. (G) Protein level of BAF60c after 10 μM HM03 treatment for 3 and 5 h in macrophages. Data are represented as mean ± SEM (n = 3) and were analyzed using independent t test. The “∗” means a significant difference (p < 0.05), and “∗∗” means a highly significant difference (p < 0.01).

Figure 4

Analysis of differentially expressed genes in transcriptome (A and B) The differential expressed genes of BAF60c-siRNA vs. control were shown as volcano plot (A) and heatmap (B). (C) GO enrichment analysis of differentially expressed genes obtained by transcriptomic sequencing analysis of hepatocytes treated with BAF60c-siRNA (n = 3). (D) KEGG enrichment analysis of differentially expressed genes obtained by transcriptomic sequencing analysis of hepatocytes treated with BAF60c-siRNA (n = 3). See also Figures S3 and S4.

Figure 5

Knockdown of BAF60c in macrophages and hepatocytes can reduce ER stress, inflammation, and fatty acid biosynthesis (A) Relative mRNA expression of ER stress-related genes after BAF60c-siRNA treatment for 36 h in macrophages. (B) Relative mRNA expression of inflammatory genes after BAF60c-siRNA treatment for 36 h in macrophages. (C) Relative mRNA expression of ER stress-related genes after BAF60c-siRNA treatment for 36 h in hepatocytes. (D) Relative mRNA expression of inflammatory genes after BAF60c-siRNA treatment for 36 h in hepatocytes. (E) Relative mRNA expression of lipid metabolism-related genes after BAF60c-siRNA treatment for 36 h in hepatocytes. (F) Protein levels after BAF60c-siRNA treatment for 36 h in hepatocytes. Data are represented as mean ± SEM (n = 3) and were analyzed using independent t test. The “∗” means a significant difference (p < 0.05), and “∗∗” means a highly significant difference (p < 0.01).

Figure 6

Overexpression of BAF60c in hepatocytes can induce ER stress and inflammation (A) Relative mRNA expression of ER stress related genes after BAF60c-overexpressing treatment for 24 h in hepatocytes. (B) Protein levels after BAF60c-overexpressing treatment for 24 h in hepatocytes. Data are represented as mean ± SEM (n = 3) and were analyzed using independent t test. The “∗” means a significant difference (p < 0.05), and “∗∗” means a highly significant difference (p < 0.01).

Figure 7

Knockdown of BAF60c can reduce lipogenesis, ER stress, and inflammation in vivo (A) Relative mRNA expression of lipid metabolism-related genes and inflammatory genes in fish liver after injecting BAF60c-dsRNA for 36 h. (B) Hepatic triglyceride (TG) levels after injecting BAF60c-dsRNA for 36 h. (C) Relative mRNA expression of ER stress-related genes in fish head kidney after injecting BAF60c-dsRNA for 36 h. (D) Relative mRNA expression of inflammatory genes in fish head kidney after injecting BAF60c-dsRNA for 36 h. (E) Hepatic transmission electron microscopy (TEM) observation after injecting BAF60c-dsRNA for 36 h. Scale bars, 4000×: 5 μm; 10000×: 2 μm; 30000×: 1 μm. Data are represented as mean ± SEM (n = 3) and were analyzed using independent t test. The “∗” means a significant difference (p < 0.05), and “∗∗” means a highly significant difference (p < 0.01).

Figure 8

Knockdown of GRP78 in macrophages and hepatocytes can reduce ER stress, inflammation, and fatty acid biosynthesis (A) Relative mRNA expression of grp78 after GRP78-siRNA treatment for 36 h in macrophages. (B) Relative mRNA expression of ER stress-related genes after GRP78-siRNA treatment for 36 h in macrophages. (C) Relative mRNA expression of inflammatory genes after GRP78-siRNA treatment for 36 h in macrophages. (D) Relative mRNA expression of grp78 after GRP78-siRNA treatment for 36 h in hepatocytes. (E) Relative mRNA expression of ER stress-related genes after GRP78-siRNA treatment for 36 h in hepatocytes. (F) Relative mRNA expression of inflammatory genes after GRP78-siRNA treatment for 36 h in hepatocytes. (G) Relative mRNA expression of lipid metabolism-related genes after GRP78-siRNA treatment for 36 h in hepatocytes. Data are represented as mean ± SEM (n = 3) and were analyzed using independent t test. The “∗” means a significant difference (p < 0.05), and “∗∗” means a highly significant difference (p < 0.01).