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. 2020 May 13:8:340.
doi: 10.3389/fcell.2020.00340. eCollection 2020.

Activation of TWIST Transcription by Chromatin Remodeling Protein BRG1 Contributes to Liver Fibrosis in Mice

Wenhui Dong  1 , Ming Kong  1 , Yuwen Zhu  1 , Yang Shao  2 , Dongmei Wu  3 , Jun Lu  3 , Junli Guo  2 , Yong Xu  1   4
Affiliations

Activation of TWIST Transcription by Chromatin Remodeling Protein BRG1 Contributes to Liver Fibrosis in Mice

Wenhui Dong et al. Front Cell Dev Biol. .

Abstract

Liver fibrosis is a complex pathophysiological process to which many different cell types contribute. Endothelial cells play versatile roles in the regulation of liver fibrosis. The underlying epigenetic mechanism is not fully appreciated. In the present study, we investigated the role of BRG1, a chromatin remodeling protein, in the modulation of endothelial cells in response to pro-fibrogenic stimuli in vitro and liver fibrosis in mice. We report that depletion of BRG1 by siRNA abrogated TGF-β or hypoxia induced down-regulation of endothelial marker genes and up-regulation of mesenchymal marker genes in cultured endothelial cells. Importantly, endothelial-specific BRG1 deletion attenuated CCl4 induced liver fibrosis in mice. BRG1 knockdown in vitro or BRG1 knockout in vivo was accompanied by the down-regulation of TWIST, a key regulator of endothelial phenotype. Mechanistically, BRG1 interacted with and was recruited to the TWIST promoter by HIF-1α to activate TWIST transcription. BRG1 silencing rendered a more repressive chromatin structure surrounding the TWIST promoter likely contributing to TWIST down-regulation. Inhibition of HIF-1α activity dampened liver fibrosis in mice. Similarly, pharmaceutical inhibition of TWIST alleviated liver fibrosis in mice. In conclusion, our data suggest that epigenetic activation of TWIST by BRG1 contributes to the modulation of endothelial phenotype and liver fibrosis. Therefore, targeting the HIF1α-BRG1-TWIST axis may yield novel therapeutic solutions to treat liver fibrosis.

Keywords: Brg1; endothelial cell; epigenetics; liver fibrosis; transcriptional regulation.

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Figures

FIGURE 1
FIGURE 1
BRG1 is essential for endothelial-mesenchymal transition in vitro. (A) HUVECs were transfected with siRNA targeting BRG1 (siBRG1) or scrambled siRNA (SCR). BRG1 expression was examined by qPCR and Western. (B,C) HUVECs were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with TGF-β. Gene expression levels were examined by qPCR and Western. (D,E) HUVECs were transfected with siRNA targeting BRG1 or scrambled siRNA (SCR) followed by treatment with hypoxia. Gene expression levels were examined by qPCR and Western. Error bars represent SEM (*p < 0.05, two-way ANOVA with post-hoc Scheffe test). All experiments were repeated three times and data represent averages of three independent experiments.
FIGURE 2
FIGURE 2
Endothelial BRG1 deficiency attenuates liver fibrosis in mice. Liver fibrosis was induced in endothelial-specific BRG1 knockout mice (ecKO) and wild type (WT) mice by CCl4 injection. (A) Plasma ALT levels. (B) Plasma AST levels. (C,D) Expression levels of pro-fibrogenic genes were examined by qPCR and Western. (E) Paraffin sections were stained with picrosirius red and Masson’s trichrome. (F) Hepatic hydroxylproline levels. N = 3∼4 mice for the corn oil groups and N = 6∼8 mice for the CCl4 groups. Error bars represent SD (*p < 0.05, two-way ANOVA with post-hoc Scheffe test).
FIGURE 3
FIGURE 3
BRG1 regulates TWIST transcription. (A,B) Liver fibrosis was induced in WT and ecKO mice by CCl4 injection. Primary liver sinusoidal endothelial cells were isolated and gene expression levels were examined by qPCR. N = 6 mice for each group. (C,D) Human primary vascular endothelial cells were transfected with siRNA targeting BRG1 or SCR followed by treatment with TGF-β or hypoxia. TWIST expression was examined by qPCR and Western. (E) Wild type and HIF-1α mutant TWIST promoter-luciferase constructs were transfected into HEK293 cells or EAhy926 cells with or without BRG1. Luciferase activities were normalized by both protein concentration and GFP fluorescence. (F,G) Human primary vascular endothelial cells were transfected with siRNA targeting HIF-1α or SCR followed by treatment with TGF-β. HIF-1α expression was examined by qPCR and Western. ChIP assays were performed with indicated antibodies. (H) Human primary vascular endothelial cells were treated with or without TGF-β. Re-ChIP assays were performed with indicated antibodies. Error bars represent SEM (*p < 0.05, two-way ANOVA with post-hoc Scheffe test). All experiments were repeated three times and data represent averages of three independent experiments.
FIGURE 4
FIGURE 4
BRG1 regulates TWIST transcription by modulating histone modifications. (A–F) HUVECs were transfected with siRNA targeting BRG1 or SCR followed by treatment with TGF-β. ChIP assays were performed with anti-acetyl H3 (A), anti-trimethyl H3K4 (B), anti-dimethyl H3K9 (C), anti-p300 (D), anti-KMT2F (E), and anti-KDM3A (F). Error bars represent SEM (*p < 0.05, two-way ANOVA with post-hoc Scheffe test). All experiments were repeated three times and data represent averages of three independent experiments.
FIGURE 5
FIGURE 5
HIF-1α inhibition attenuates liver fibrosis in mice. Liver fibrosis was induced in by CCl4 injection. The mice were injected with two different HIF-1α inhibitors every other day. (A) Plasma ALT levels. (B) Plasma AST levels. (C,D) Expression levels of pro-fibrogenic genes were examined by qPCR and Western. (E) Paraffin sections were stained with picrosirius red and Masson’s trichrome. (F) Hepatic hydroxylproline levels. N = 8 mice for each group. Error bars represent SD (*p < 0.05, two-way ANOVA with post-hoc Scheffe test).
FIGURE 6
FIGURE 6
TWIST inhibition attenuates liver fibrosis in mice. Liver fibrosis was induced in by CCl4 injection. The mice were injected with the TWIST inhibitor harmine every other day. (A) Plasma ALT levels. (B) Plasma AST levels. (C,D) Expression levels of pro-fibrogenic genes were examined by qPCR and Western. (E) Paraffin sections were stained with picrosirius red and Masson’s trichrome. (F) Hepatic hydroxylproline levels. N = 8 mice for each group. Error bars represent SD (*p < 0.05, two-way ANOVA with post-hoc Scheffe test).
FIGURE 7
FIGURE 7
A schematic model. In response to liver injuries, liver sinusoidal endothelial cells undergo EndMT contributing to myofibroblast activation and liver fibrosis. Specifically, HIF-1α recruits BRG1, which in turn recruits histone modifying enzymes (histone acetyltransferase, HAT, and histone methyltransferase, HMT), to the TWIST promoter to activate TWIST transcription. TWIST represses endothelial gene expression and activates mesenchymal gene expression to promote EndMT and liver fibrosis. Pharmaceutical targeting of various factors involved in the proposed model, some of which (harmine for instance) have been tested in this paper, will hopefully provide novel interventional strategies against liver fibrosis.
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