Activation of FXR by GW4064 induces FincoR, a novel eRNA, in mouse liver.

(A) Volcano plot from RNA-Seq showing significantly induced eRNAs (FincoR is highlighed) in the livers of C57BL/6 male mice treated with GW4064 (i.p. injection, 30 mg/kg, 1 h) or vehicle. The x axis denotes log2 fold change (GW4064/Veh) of eRNAs and the y axis denotes -log10 FDR of eRNAs. (B) A Bar plot showing the reads per million (RPMs) of up regulated eRNAs by GW4064 treatment. (C) IGV genome browser track showing RNA-seq signals from vehicle or GW4064 treated samples around the FincoR locus and its neighboring regions. A zoom-in view of FincoR is shown below. Veh, vehicle; GW, GW4064; pos, positive strand; neg, negative strand; Refseq, reference sequence. (D) RT-qPCR data showing GW4064 induction of FincoR eRNA and Shp mRNA in the liver (n=4/group). Data are presented as mean ± SEM. Statistical significance was determined by the two-way ANOVA Sidak’s multiple comparisons test with **p < 0.01 and ***p < 0.001.

Ligand-activated FXR directly activates transcription of eRNAs including FincoR in the liver.

(A) Left: experimental outline. FXR-Flox and LKO male mice were fasted overnight and treated with vehicle or GW4064, and livers were collected 1 h later with nuclei isolated for Gro-seq (n=2/group). Right: a boxplot shows the Gro-Seq signals for GW4064 up-regulated eRNAs in different conditions. RPKM: reads per kbp per million. (B) IGV genome browser track showing RNA-seq, Gro-seq and ChIP-seq signals in the FincoR locus. An arrow at the bottom points to the main enhancer FXR ChIP-seq peak that overlaps the start site of FincoR. (C) ChIP assays were performed in the same liver samples described in Fig. 1A to detect FXR, RXR𝛂, and BRD4 occupancy at the FXR binding peak region close to the transcription start site of FincoR (black arrow in Fig.2B). (D) HepG2 cells were transfected with luciferase reporter expressing wild type FXRE or mutant FXRE (see materials and methods) for 24 h before treatment with GW4064 for an additional 6 h. Relative luciferase activities are shown. (C-D) Data are presented as mean ± SEM. Statistical significance was determined by the Student’s t test with *p < 0.05 and ***p < 0.001.

FincoR is a liver-specific nucleus-enriched eRNA.

(A) Expression levels of FincoR in various tissues after GW4064 treatment. Data from C57BL/6 male mice fasted overnight and i.p. injected with vehicle or GW4064 (30 mg/kg) for 1 h (n=2/group). (B) C57BL/6 male mice were fasted overnight and injected i.p. with vehicle or GW4064 (30 mg/kg) for 1 h. One small piece of liver was snap-frozen for later RNA isolation and the remaining part was used for immediate primary hepatocyte isolation. Then, RNAs were extracted from liver or primary hepatocytes and FincoR expression was measured (n=3/group). (C) Agarose gel electrophoresis of PCR products generated in 5′ (left) and 3′ (right) RACE of FincoR in liver samples. Primer locations are shown. RACE, rapid amplification of cDNA ends. GSP: gene specific primer. (D) A schematic diagram showing location of FincoR relative to nearby genes in the mice genome. (E) PhyloCSF analysis of the coding potential of FincoR. (F) In vitro translation of FincoR using the Promega Transcend Non-Radioactive Translation Detection Systems. Luciferase is used as a control for coding RNA. (G) qPCR analysis of FincoR, 36b4 in Poly(A)+ and Poly(A)-RNA fractions from GW4064 treated mouse liver. (H) FincoR identified in the subcellular fractions using cellular fractionation assays. Primary hepatocytes were isolated from GW4064 or DMSO treated mice and the cytoplasm and nucleus fractions of these hepatocytes were separated and both fractions were subjected to RNA extraction and qPCR.

FincoR is induced by the hammerhead class of non-steroidal FXR agonists, including GW4064 and tropifexor.

(A) The chemical structures of the FXR agonists including the hammerhead class of synthetic FXR agonists, non-hammerhead-type synthetic agonists, semi-synthetic BA and natural BA. BA: bile acid. (B) qPCR data showing FincoR expression levels in C57BL/6 mice liver respectively treated with GW4064 (30 mg/kg), Cilofexor (30 mg/kg), Tropifexor (0.5 mg/kg), Fexaramine (100 mg/kg), or OCA (20 mg/kg) for 1 h (n=3∼4/group). (C) FincoR expression levels in C57BL/6 mice liver treated with OCA (20 mg/kg) or Fexaramine (100 mg/kg) for 4 hrs (n=3/group). (D) Expression of FincoR in C57BL/6 mice liver after daily treatment with OCA (20 mg/kg) for 7 days (n=5/group). Shp gene mRNA was measured as a positive control. (E) Expression of FincoR in C57BL/6 mice fed with 0.5% cholic acid (CA) diet for 6 hrs (n=3∼4/group). In panels B-E, all mice underwent over-night fasting. (B-E) Data are presented as mean ± SEM. Statistical significance was determined by the Student’s t test with *p < 0.05, **p < 0.01 and ***p < 0.001.

Generation of CRISPR/Cas9-mediated FincoR liver-specific knockdown mice.

(A) Experimental scheme: male Cas9 mice were infected with adenovirus expressing sgRNA for FincoR or a control for 1 week. Then the liver and serum were collected from these mice after 4∼5 hrs of fasting. (B) The expression of FincoR in the liver was measured by qPCR (n=4/group). (C) Hepatic triglyceride, cholesterol, bile acid, glycogen and serum NEFA were measured (n=5∼7/group) (D) Male Cas9 mice were infected with adenovirus expressing sgRNA for FincoR or control for 1 week. Then these mice were fasted overnight and treated with GW4064 for 3 h before tissue collection. RNA-seq profiles of expression of hepatic FincoR and the adjacent genes were shown (n=2/group). (E) Genome-wide changes in mRNA expression shown in a volcano plot. The numbers refer to the number of genes up- or down-regulated by 2-fold or more with an adjusted p-value < 0.01. (F) Gene ontology analysis of biological pathways using DAVID Tools for genes downregulated after FincoR knockdown. (B) Data are presented as mean ± SEM. Statistical significance was determined by the Student’s t test with ***p < 0.001.

In diet-induced NASH mice, tropifexor-mediated beneficial effects on reducing hepatic steatosis are largely independent of FincoR.

(A-E) Male Cas9 mice were fed a NASH diet for 12 weeks. The mice were randomly assigned to 3 groups and infected with adenovirus expressing sgRNA for FincoR or control. Three days later, the mice were treated with tropifexor (0.3 mg/kg) or vehicle for 12 days. The mice were administered tropifexor or vehicle and fasted for 4 h before tissues were collected. (A) Experimental scheme. (B) Hepatic FincoR expression was measured (n=6∼7/group). (C) Oil Red O staining of liver sections. Scale bar (50 μm). Image analyses were done using Image J and the areas of stained field were quantified (n=5/group) (D) Hepatic TG, hepatic cholesterol, gallbladder BA and hepatic BA levels were measured (n=6∼7/group). (E) mRNA levels in the liver of the indicated genes involved in bile acid regulation and lipid regulation (n=6∼7/group). (B, D and E) Data are presented as mean ± SEM. Statistical significance was determined by the one-way ANOVA (Sidak’s multiple comparisons test) with *p < 0.05, **p < 0.01 and ***p < 0.001. Ad, adenovirus; H & E, hematoxylin and eosin; TG, triglyceride; Veh, vehicle; ns, not significant.

In diet-induced NASH mice, tropifexor-mediated beneficial effects on reducing liver fibrosis and inflammation are diminished by FincoR downregulation.

(A) Representative images from H & E, F4/80, Sirius Red and TUNEL staining of liver sections from the same cohort of mice described in Figure 6. Scale bar (50 μm). Image analyses were done using Image J and the area of collagen staining, TUNEL and F4/80 levels were quantified (n=5/group). (B) Serum ALT and AST levels were measured (n=5/group). (C) IL-1β and CCL2 levels in the liver tissues were determined by ELISA (n=5/group). (D) mRNA levels in the liver of the indicated genes involved in inflammation, fibrosis and cell death (n=5/group). (E) Model: FincoR is a liver-enriched eRNA that is induced specifically by hammerhead-type FXR agonists (top). In diet-induced NASH mice, FincoR is required for tropifexor-mediated beneficial effects on reducing hepatic inflammation, fibrosis, and cell death with the mechanisms to be determined (bottom). (A-D) Data are presented as mean ± SEM. Statistical significance was determined by the one-way ANOVA (Sidak’s multiple comparisons test) with *p < 0.05, **p < 0.01 and ***p < 0.001.

(A, B) Examples of FXR-regulated eRNAs produced near the genes Hes1 (A) and Slc35g1 (B) were shown. (C) Time course expression of FincoR. C57BL/6 male mice were fasted overnight and injected i.p. with vehicle or GW4064 (30 mg/kg) for 1, 3, 6,12 h. Livers were collected at the indicated time points (n=3/group) and FincoR and mRNA levels of Shp and Gcnt1 were measured. Data are presented as mean ± SEM. Statistical significance was determined by the two-way ANOVA Sidak’s multiple comparisons test with *p < 0.05 and ***p < 0.001.

(A) Volcano plot showing the differential expressed genes (DEGs) after GW4064 treatment (DESeq2 FDR<0.05). The numbers refer to the number of genes up- or down-regulated. (B) Bar plot showing the enriched Gene Ontology in terms of biological processes for up-regulated genes.

(A) FXR protein levels in the livers isolated from FXR-Flox and FXR-LKO mice are shown. (B) Validation of hepatic FXR-dependent induction of FincoR by qPCR (n = 3 mice). The Shp gene was used as a control. Data are presented as mean ± SEM. Statistical significance was determined by the two-way ANOVA Tukey’s multiple comparisons test with *p < 0.05 and **p < 0.01. (C) Metagene plots showing H3K27ac, H3K4me1, FXR, and RXR𝛂 ChIP-Seq profiles centered on up-regulated eRNAs.

(A) Left: experimental scheme for the FincoR loss of function experiments. Right: illustration demonstrating the sequence targeted by the sgRNA in relation to the transcriptional and epigenetic profile. (B) The genomic DNAs from liver, spleen, intestine, brain, heart, muscle, kidney, lung and adipose tissue were isolated and PCR was performed using the primers (Supplementary Table S6) to verify tissue-specific knock-out. (C-G) RNA-seq profiles of expression of hepatic Prune2 (C), PPP1r3g (D), Igfbp2 (E), Eda2r (F) and Fndc1 (G) are shown (n=2/group).

The effects of FincoR downregulation on NASH pathologies.

Male Cas9 mice were fed with a NASH diet for 12 weeks. Then these mice were randomly assigned to 2 groups and infected with adenovirus expressing sgRNA for FincoR or control. The tissues were collected 2 weeks later. Liver histology analysis was performed and representative images were shown. Scale bar (50 μm).

Hepatic expression of FincoR is elevated in liver disease associated with inflammation and fibrosis.

(A) C57BL/6 mice were fed with a high fat diet for 12 weeks. The liver RNAs were extracted and FincoR expression was measured (n=5/group). (B) C57BL/6 mice were fed with a high fat diet with high fructose water for 12 weeks. The liver RNAs were extracted and FincoR expression was measured (n=7/group). (C) C57BL/6 mice were treated with ANIT (75 mg/kg) for 48 h and then sacrificed after 5 h of fasting. The liver RNAs were extracted, and FincoR expression was measured (n=4∼5/group). (D) C57BL/6 mice were bile duct ligated for 1 day or 3 days. They were then sacrificed after 5 h of fasting. The liver RNAs were extracted and FincoR expression was measured (n=4∼5/group). (E) FincoR conservation between mice and human as displayed in the UCSC Genome Browser. Red arrows indicate the conserved region. (F) Human lncRNA XR_007061585.1 with sequence similarity to mouse FincoR annotated in the NCBI genome data viewer. (G) Expression of lncRNA XR_007061585.1 in liver samples from normal individuals or patients with primary biliary cholangitis (PBC) (n=14∼15/group). (H) Expression of lncRNA XR_007061585.1 in liver samples from normal individuals or patients with NAFLD-associated steatosis (n=12∼15/group). (A-D, G-H) Data are presented as mean ± SEM. Statistical significance was determined by the Student’s t test with *p < 0.05, **p < 0.01 and ***p < 0.001.

Hepatic genome browser tracks of FXR, RXRα, LXR, PPARα, and HNF4α binding peaks at the FincoR locus.

PPARα occupancy in the FincoR enhancer region.

C57BL/6 male mice were fasted overnight with or without refeeding for 3 h and then sacrificed. ChIP assays were performed in liver samples to detect PPAR𝛂 occupancy at the FXR binding peak region close to the transcription start site of FincoR.

Sequencing data generated in this study

Public ChIP-Seq dataset

Predicted RNA binding proteins (RBPs) binding to FincoR

Primer sequences used in this study