Characterization of regulatory transcriptional mechanisms in hepatocyte lipotoxicity

Abstract

Non-alcoholic fatty liver disease is a continuum of disorders among which non-alcoholic steatohepatitis (NASH) is particularly associated with a negative prognosis. Hepatocyte lipotoxicity is one of the main pathogenic factors of liver fibrosis and NASH. However, the molecular mechanisms regulating this process are poorly understood. The main aim of this study was to dissect transcriptional mechanisms regulated by lipotoxicity in hepatocytes. We achieved this aim by combining transcriptomic, proteomic and chromatin accessibility analyses from human liver and mouse hepatocytes. This integrative approach revealed several transcription factor networks deregulated by NASH and lipotoxicity. To validate these predictions, genetic deletion of the transcription factors MAFK and TCF4 was performed, resulting in hepatocytes that were better protected against saturated fatty acid oversupply. MAFK- and TCF4-regulated gene expression profiles suggest a mitigating effect against cell stress, while promoting cell survival and growth. Moreover, in the context of lipotoxicity, some MAFK and TCF4 target genes were to the corresponding differentially regulated transcripts in human liver fibrosis. Collectively, our findings comprehensively profile the transcriptional response to lipotoxicity in hepatocytes, revealing new molecular insights and providing a valuable resource for future endeavours to tackle the molecular mechanisms of NASH.

Figure 1

Shared transcriptional response in NASH and lipotoxicity. (A) Characteristics of human subjects. (B,C) Mouse hepatocytes were stimulated with BSA (control) or PAL for 24 h, and (B) cytotoxicity and (C) intracellular lipid content were measured (n = 3 independent experiments; values are mean ± SD; **p < 0.01, ***p < 0.001). (D,E) Volcano plots showing differentially regulated genes in (D) human liver from NASH patients (n = 4 per group) and (E) mouse hepatocytes stimulated with PAL for 24 h (n = 3 per group). Blue and red dots denote significantly down- and up-regulated genes, respectively. (F,G) Circular plots showing the gene (purple lines) and functional (blue lines) overlap between (F) up- and (G) down-regulated genes in human NASH and mouse hepatocyte lipotoxicity. (H,I) Gene ontology analysis showing biological processes significantly regulated in both human NASH and mouse hepatocyte lipotoxicity (H) up- and (I) down-regulated genes.

Figure 2

Shared proteome remodelling in NASH and lipotoxicity. (A,B) Volcano plots showing differentially regulated proteins in (A) human liver from NASH patients (n = 4 per group) and (B) mouse hepatocytes stimulated with PAL for 24 h (n = 3 per group). Blue and red dots denote significantly down- and up-regulated proteins, respectively. (C,D) Circular plots showing the protein (purple lines) and functional (blue lines) overlap between (C) up- and (D) down-regulated proteins in human NASH and mouse hepatocyte lipotoxicity. (E,F) Gene ontology analysis showing biological processes significantly regulated in both human NASH and mouse hepatocyte lipotoxicity (E) up- and (F) down-regulated proteins.

Figure 3

Chromatin accessibility is altered by NASH and lipotoxicity. (A,B) Annotation of ATAC-seq peaks in (A) human liver (n = 4 per group) and (B) mouse hepatocytes (n = 3 per group). (C) Heat maps of ATAC-seq peaks aligned to their centre ± 1 kb. (D) Density plots showing averaged normalized signal of ATAC-seq peaks aligned to their centre ± 1 kb. (E,F) Overlap of ATAC-seq peaks between (E) CON and NASH liver or (F) BSA and PAL hepatocytes. (G,H) Genome browser views of representative (G) CON- or NASH-specific peaks in human liver and (H) BSA- or PAL-specific peaks in mouse hepatocytes (marked yellow). (I,J) Gene ontology analysis showing biological processes regulated by genes linked to (I) CON- or NASH-specific peak in human liver and (J) BSA- or PAL-specific peaks in mouse hepatocytes.

Figure 4

Conserved transcription factor networks at regulatory elements in NASH and lipotoxicity. (A–D) Transcription factor motif enrichment analysis in chromatin regions showing a loss (CON/BSA-specific) or gain (NASH/PAL-specific) in accessibility at (A,B) distal intergenic regions and (C,D) promoters in the context of NASH or PAL treatment (red line denotes significance cut-off of E-value < 0.05). (E,F). Circular plots showing conserved transcription factors (purple lines) shared between CON/BSA- and NASH/PAL-specific chromatin regions at (E) distal intergenic regions and (F) promoters. (G,H) Protein–protein interaction (PPI) network analysis of common and CON/BSA-specific transcription factors at (G) distal intergenic regions and (H) promoters.

Figure 5

Omics data integration revealed candidate lipotoxicity-sensitive transcription factors at proximal promoters. (A) Overlap between all ISMARA (RNA-seq) and motif enrichment analysis (ATAC-seq) predicted transcription factors (TFs). (B) Heat map with ISMARA-predicted activity (left panel; clustering based of predicted TF activity) and gene expression changes (right panel; asterisk denotes a significant change) of TFs regulated in both systems. (C) Cytotoxicity and (D) intracellular lipid content measurement following BSA or PAL stimulation for 24 h in mouse hepatocytes with either overexpression (OE) or knockout (KO) of candidate TFs. Data are expressed as percentage of control cells (CON; corresponding to 100% denoted by the horizontal dashed line) undergoing the same treatment (n = 4 independent experiments; values are mean ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001). (E,F) ISMARA-predicted activity of MAFK and TCF4 in (E) human liver from CON subjects or NAFL and NASH patients (GEO accession: GSE126848), and (F) hepatocytes isolated from a mouse model of diet-induced NASH (GEO accession: GSE162876).

Figure 6

TCF4 and MAFK target genes are linked to hepatocyte lipotoxicity. (A,C) Overlap between DEG of CON and (A) TCF4-KO or (C) MAFK-KO hepatocytes stimulated with PAL for 24 h (n = 3 per group). (B,D) Gene ontology terms exclusively regulated in CON and (B) TCF4-KO or (D) MAFK-KO cells.

Figure 7

Overlap between TCF4 and MAFK target genes and human liver fibrosis. (A) Schematic representation of liver fibrosis stages from which samples used for downstream data integration were obtained (prepared with Biorender). (B,D) Overlap between DEG in human liver from NASH patients with cirrhosis (F4; GEO accession: GSE135251) and PAL-stimulate CON and (B) TCF4-KO or (D) MAFK-KO mouse hepatocytes. (C,E) Heat maps and clustering analysis of fold changes of (C) TCF4- or (E) MAFK-sensitive genes regulated both in human NASH F4 and mouse hepatocyte lipotoxicity, including data from human liver from NASH patients with moderate (F2) and severe (F3) fibrosis (grey denotes genes not significantly regulated in human NASH).