Estrogen receptor activation remodels TEAD1 gene expression to alleviate nonalcoholic fatty liver disease

Introduction The occurrence of obesity-related hepatic malignancies differs between sexes, suggesting the involvement of sex hormones. Female sex hormones maintain cell homeostasis through estrogen receptor (ER) signaling and protect from developing nonalcoholic fatty liver disease (NAFLD) in mice and humans. Rationale To understand recovery from high-fat diet (HFD)-induced liver disease in males upon estrogen treatment, we comprehensively characterized molecular changes in the liver upon selective activation of estrogen receptors (ERs) to identify novel therapeutic targets downstream of estrogen signaling. Methods To dissect hepatic ER isoform-driven responses, we integrated liver transcriptomes from female and male HFD mice treated with or without four different estrogen agonists, along with multiomics data, including bulk, single-cell and spatial transcriptomics, chromatin profiling, machine learning models and advanced microscopy. Patient cohorts and primary human hepatocyte spheroids datasets were included. Results Only males developed liver steatosis. We found that selective activation of either ERα or ERβ reduced HFD-induced hepatic steatosis in male mice. Systemic ER activation restored HFD-induced aberrant gene expression of cellular processes across liver cell types, including hepatocytes. Profiling of marked histones revealed that ER activation modulated promoter and enhancer sites and identified 68 estrogen-sensitive enhancer-gene pairs. Most of these genes were similarly deregulated in human nonalcoholic fatty liver disease (NAFLD) patients, including the transcription factor TEAD1 . TEAD1 expression increased in NAFLD patients, and inhibiting TEAD ameliorated steatosis in spheroids by suppressing lipogenic pathways. Conclusions Systemic activation of ERα or ERβ modulates molecular pathways in the liver to counteract NAFLD. Our study identified TEAD1 as a key ER-sensitive gene and suggested that its inhibition poses a therapeutic strategy to combat NAFLD without the undesired side effects elicited by estrogen signaling. Clinical research relevance We identified drug targets downstream of estrogen signaling, including TEAD1, and demonstrate that TEAD inhibition improves steatosis by suppressing lipogenic pathways. Basic research relevance The targeted activation of nuclear ERs recovers high-fat diet-induced molecular and physiological liver phenotypes by remodeling core pathways beyond lipid metabolism. ER-responsive enhancers regulate central metabolic genes of clinical significance in NAFLD patients, highlighting the potential impact of this research on understanding liver cell plasticity. HIGHLIGHTS ### Competing Interest Statement CP is employee of the Healthcare Business of Merck KGaA (Darmstadt, Germany). HHs institutions have received research funding from Astra Zeneca, EchoSens, Gilead, Intercept, MSD and Pfizer, all outside this study. HH has served as consultant for Astra Zeneca and has been part of hepatic events adjudication committees for KOWA and GW Pharma. VML is co-founder, CEO and shareholder of HepaPredict AB. The remaining authors declare no competing financial and non-financial interests. * AUC : area under the curve CTCF : CCCTC-binding factor CD : control diet CHi-C : promoter capture Hi-C ChIP-seq : chromatin immunoprecipitation followed by sequencing CoA: coenzyme-A CPM : counts per million DAc: differentially acetylated DEG : differentially expressed gene DIP : 4-(2-(3,5-dimethylisoxazol-4-yl)-1H-indol-3-yl)phenol DPN : diarylpropionitrile E2 : 17β-estradiol ECM : extracellular matrix ER : estrogen receptor ES-E-G : estrogen-sensitive enhancer-gene pair FCCP : carbonyl-cyanide 4-trifluoromethoxy-phenylhydrazone GEO : gene expression omnibus GO : gene ontology GPCR : G-protein coupled receptor GSEA : gene set enrichment analysis H&E : hematoxylin and eosin HFD : high-fat diet H3K27ac : histone 3 lysine 27 acetylation H3K4me1 : histone 3 lysine 4 monomethylation H3K4me3 : histone 3 lysine 4 trimethylation HSC : hepatic stellate cell KEGG : Kyoto Encyclopedia of Genes and Genomes NAFLD : nonalcoholic fatty liver disease NASH : nonalcoholic steatohepatitis NAS : NAFLD activity score NES : normalized enrichment score PCA : principal component analysis PPT : pyrazole-triol ROC : receiver operating characteristic TAZ : WW domain containing transcription regulator 1 TPM : transcripts per million tSNR : transcriptome-based signal-to-noise ratio TSS : transcription start site YAP : Yes-associated protein