TY - JOUR
T1 - FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs
AU - Ramos Pittol, Jose Miguel
AU - Milona, Alexandra
AU - Morris, Imogen
AU - Willemsen, Ellen C L
AU - van der Veen, Suzanne W
AU - Kalkhoven, Eric
AU - van Mil, Saskia W C
N1 - Funding Information:
Funding This work was financially supported by Marie Skłodowska-Curie Actions of the European Commission (PIAPP-GA-2013-611979) and the European Union’s Horizon 2020 Marie Skłodowska-Curie Innovative Training Network, Tribbles Research and Innovation Network (TRAIN) (project no. 721532).
Funding Information:
Funding This work was financially supported by Marie Sk?odowska-Curie Actions of the European Commission (PIAPP-GA-2013-611979) and the European Union's Horizon 2020 Marie Sk?odowska-Curie Innovative Training Network, Tribbles Research and Innovation Network (TRAIN) (project no. 721532).
Publisher Copyright:
© 2020 The Authors
Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2020/11
Y1 - 2020/11
N2 - BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR.METHODS: We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus.RESULTS: In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif.CONCLUSIONS: Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.
AB - BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR.METHODS: We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus.RESULTS: In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif.CONCLUSIONS: Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.
KW - Fatty Liver
KW - NASH
KW - OCA
KW - Steatosis
UR - http://www.scopus.com/inward/record.url?scp=85096164716&partnerID=8YFLogxK
U2 - 10.1053/j.gastro.2020.07.036
DO - 10.1053/j.gastro.2020.07.036
M3 - Article
C2 - 32712104
SN - 0016-5085
VL - 159
SP - 1853-1865.e10
JO - Gastroenterology
JF - Gastroenterology
IS - 5
ER -