Hammerhead-type FXR agonists induce an enhancer RNA Fincor that ameliorates nonalcoholic steatohepatitis in mice

eLife assessment

Using unbiased transcriptional profiling, the study reports a fundamental discovery of FincoR, a novel hepatic lncRNA generated from an enhancer element, which plays a role in FXR biology. The convincing findings have therapeutic implications in the treatment of MASH. The authors use state-of-the-art methodology and use unbiased transcriptomic profiling and epigenetic profiling, including validation in mouse models and human samples.

https://doi.org/10.7554/eLife.91438.3.sa0

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Abstract
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Abstract

The nuclear receptor, farnesoid X receptor (FXR/NR1H4), is increasingly recognized as a promising drug target for metabolic diseases, including nonalcoholic steatohepatitis (NASH). Protein-coding genes regulated by FXR are well known, but whether FXR also acts through regulation of long non-coding RNAs (lncRNAs), which vastly outnumber protein-coding genes, remains unknown. Utilizing RNA-seq and global run-on sequencing (GRO-seq) analyses in mouse liver, we found that FXR activation affects the expression of many RNA transcripts from chromatin regions bearing enhancer features. Among these we discovered a previously unannotated liver-enriched enhancer-derived lncRNA (eRNA), termed FXR-induced non-coding RNA (Fincor). We show that Fincor is specifically induced by the hammerhead-type FXR agonists, including GW4064 and tropifexor. CRISPR/Cas9-mediated liver-specific knockdown of Fincor in dietary NASH mice reduced the beneficial effects of tropifexor, an FXR agonist currently in clinical trials for NASH and primary biliary cholangitis (PBC), indicating that amelioration of liver fibrosis and inflammation in NASH treatment by tropifexor is mediated in part by Fincor. Overall, our findings highlight that pharmacological activation of FXR by hammerhead-type agonists induces a novel eRNA, Fincor, contributing to the amelioration of NASH in mice. Fincor may represent a new drug target for addressing metabolic disorders, including NASH.

eLife digest

Non-alcoholic steatohepatitis, also known as NASH, is a severe condition whereby fat deposits around the liver lead to inflammation, swelling, scarring and lasting damage to the organ. Despite being one of the leading causes of liver-related deaths worldwide, the disease has no approved treatment.

A protein known as Farnesoid X receptor (or FXR) is increasingly being recognized as a promising drug target for non-alcoholic steatohepatitis. Once activated, FXR helps to regulate the activity of DNA regions which are coding for proteins important for liver health. However, less is known about how FXR may act on non-coding regions, the DNA sequences that do not generate proteins but can be transcribed into RNA molecules with important biological roles.

In response, Chen et al. investigated whether FXR activation of non-coding RNAs could be linked to the clinical benefits of hammerhead FXR agonists, a type of synthetic compounds that activates this receptor.

To do so, genetic analyses of mouse livers were performed to identify non-coding RNAs generated when FXR was activated by the agonist. These experiments revealed that agonist-activated FXR induced a range of non-coding RNAs transcribed from DNA sequences known as enhancers, which help to regulate gene expression. In particular, hammerhead FXR agonists led to the production of a liver-specific enhancer RNA called Fincor.

Additional experiments using tropifexor, a hammerhead FXR agonist currently into clinical trials, showed that this investigational new drug had reduced benefits in a mouse model of non-alcoholic steatohepatitis with low Fincor levels. This suggested that this enhancer RNA may play a key role in mediating the clinical benefits of hammerhead FXR agonists, encouraging further research into its role and therapeutic value.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and a leading cause of liver transplants and liver-related death (Friedman et al., 2018). NAFLD begins with simple steatosis but may further progress to a severe form, nonalcoholic steatohepatitis (NASH), and later, fatal cirrhosis and liver cancer (Friedman et al., 2018). Despite its striking global increase and clinical importance, there is no approved drug for NASH. The urgent need for development of therapeutic agents for NASH has greatly increased research interest in the nuclear receptor, farnesoid X receptor (FXR, NR1H4) (Evans and Mangelsdorf, 2014).

FXR is activated by its physiological ligands, bile acids (BAs), and regulates expression of genes involved in BA, lipid, and glucose metabolism and hepatic autophagy, which maintain metabolite levels and metabolic homeostasis (Calkin and Tontonoz, 2012; Kliewer and Mangelsdorf, 2015; Lee et al., 2006; Lee et al., 2014; Seok et al., 2014). Ligand-activated FXR also protects against hepatic inflammation and liver injury (Jung et al., 2020; Wang et al., 2008). The action of FXR, similar to other nuclear receptors, is achieved primarily by its binding to chromatin loci to regulate the transcription of target genes (Calkin and Tontonoz, 2012; Lee et al., 2006; Lee et al., 2012; Thomas et al., 2010). Consistent with its crucial physiological functions, FXR is increasingly recognized as a promising drug target, particularly for liver diseases, such as NASH and primary biliary cholangitis (PBC) (Abenavoli et al., 2018; Ali et al., 2015; Downes et al., 2003; Kremoser, 2021). For example, semi-synthetic or non-steroidal synthetic agonists of FXR, including obeticholic acid (OCA) and hammerhead-type agonists, such as tropifexor and cilofexor, are currently in clinical trials for NASH and PBC patients (Abenavoli et al., 2018; Kremoser, 2021; Sanyal et al., 2023; Tully et al., 2017). However, how pharmacological activation of FXR mediates such beneficial therapeutic effects is poorly understood.

Non-protein-coding RNAs (ncRNAs) are one of the fascinating discoveries of modern biology (Cech and Steitz, 2014). While a significant portion of the genome was initially thought to be ‘junk DNA’, it has been established that many non-coding regions give rise to functional non-coding RNAs (ncRNAs) (Cech and Steitz, 2014). Of these ncRNAs, long non-coding RNAs (lncRNAs) are a group of transcripts longer than 200 nucleotides and play important roles in diverse biological processes (Lam et al., 2014; Li et al., 2016; Mattick et al., 2023; Sartorelli and Lauberth, 2020; Statello et al., 2021). A group of lncRNAs are produced from genomic regions bearing epigenetic features of enhancers (Hon et al., 2017; Mattick et al., 2023). This is consistent with the idea that many transcriptional enhancers actively transcribe ncRNAs that are referred to as enhancer RNAs (eRNAs, >85k in humans and >57k in mice) (Hirabayashi et al., 2019), some of which are functionally important for enhancer functions (Lai and Shiekhattar, 2014; Li et al., 2016; Sartorelli and Lauberth, 2020). A majority of eRNAs have not yet been annotated in current lncRNA databases, such as GENCODE. Exploring eRNA landscapes and functions in diverse biology and disease states will facilitate our understanding of both lncRNAs and enhancers (Li et al., 2016; Mattick et al., 2023). Indeed, the landscape of eRNAs in mouse liver has been minimally explored (Fang et al., 2014), and has not been well studied in response to specific nuclear receptor activation, such as by FXR. Moreover, functional roles of eRNAs in vivo in intact organisms is understudied.

FXR was shown to regulate expression of small non-coding microRNA (miR) genes, e.g., Mir34a and Mir802 (Lee et al., 2010; Seok et al., 2021), but it has not been reported whether FXR achieves its function through regulating lncRNAs, which far outnumber miRs. Because FXR directly regulates expression of its target genes (Lee et al., 2012; Thomas et al., 2010), we examined if FXR regulates eRNAs, and if such transcripts participate in its physiological or pharmacological functions. By utilizing RNA-seq and global run-on sequencing (GRO-seq) analyses of livers from mice treated with FXR ligands, we identified a set of FXR-regulated eRNAs. Among these, we focused on a highly induced and abundantly expressed eRNA that we referred to as FXR-induced non-coding RNA (Fincor) for functional studies. Fincor is highly enriched in mouse liver and is induced specifically by hammerhead-type FXR agonists, including GW4064 and tropifexor. In vivo studies utilizing CRISPR/Cas9-mediated liver-specific knockdown of Fincor in dietary NASH mice indicated that Fincor is critically involved in mediating the beneficial pharmacological effects of tropifexor in reducing liver fibrosis and inflammation.

Results

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

To identify eRNAs potentially regulated by FXR, we first obtained a list of putative enhancers in mouse liver based on ENCODE H3K27ac ChIP-seq data (ENCFF001KMI, see Materials and methods). Then, we performed ribo-depleted total RNA-seq in the livers from mice treated with a specific FXR agonist, GW4064, to identify transcripts produced from these enhancer regions (see Supplementary file 1a). To avoid confounding issues of RNA signals from genes, we specifically focused on intergenic enhancer regions (±3 kb from H3K27ac peak center) that harbor discernible RNA-seq signals (RPKM >1).

This genomic analysis resulted in identification of 190 high-confidence eRNAs in mouse liver. Among these, 14 eRNAs were upregulated and 5 were downregulated by GW4064 treatment (FDR <0.05, log₂FC >1, Figure 1A) (see Supplementary file 1b). FXR-regulated eRNAs were produced adjacent to many genes with important roles in liver metabolism and disease, e.g., Hes1 (Figure 1—figure supplement 1A and B). One of the most robustly induced eRNAs was Fincor, an unannotated novel transcript located on chromosome 19 (Figure 1A, B, and C). Reverse transcription qPCR (RT-qPCR) confirmed that treatment with GW4064 substantially induced expression of Fincor, more than 10-fold in mouse liver, which is similar to induction of Nr0b2, a well-known FXR target gene (Figure 1D; Claudel et al., 2005; Evans and Mangelsdorf, 2014; Goodwin et al., 2000). Induction of Fincor by GW4064 was transient, peaked within 1 hr and then declined gradually, a pattern similar to that of Nr0b2 (Figure 1—figure supplement 1C). Expression of genes adjacent to Fincor, including Gcnt1, Rfk, Pcsk5, and Prune2, did not change after acute 1 hr FXR activation as shown by RNA-seq (Figure 1C), and confirmed for Gcnt1 by time course qPCR (Figure 1—figure supplement 1C).

Figure 1 with 2 supplements see all

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Activation of farnesoid X receptor (FXR) by GW4064 induces FXR-induced non-coding RNA (*Fincor*), a novel enhancer RNA (eRNA), in mouse liver.

(A) Volcano plot from RNA-seq showing significantly induced eRNAs (*Fincor* is highlighted) in the livers of C57BL/6 male mice treated with GW4064 (i.p. injection, 30 mg/kg, 1 hr) 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 upregulated 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) Reverse transcription qPCR (RT-qPCR) data showing GW4064 induction of *Fincor* eRNA and *Nr0b2* 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.

We also found that the short-time GW4064 treatment resulted in 590 upregulated genes and 500 downregulated genes (FDR <0.05) (Figure 1—figure supplement 2A). Gene ontology (GO) enrichment analysis of these differentially expressed genes revealed their roles in the regulation of triglyceride, fatty acid, and cholesterol metabolism (Figure 1—figure supplement 2B), which is consistent with known roles of FXR in these physiological processes (Claudel et al., 2005).

Ligand-activated FXR directly activates transcription of eRNAs, including Fincor

We sought to examine: (1) if these eRNAs were directly activated by FXR, and (2) if the activation takes place transcriptionally using Fxr liver-specific knockout (Fxr-LKO) mice that were treated with GW4064 to determine if FXR was required for induction of eRNAs by GW4064. Fxr-LKO mice were generated from Fxr floxed (Fxr-Flox) mice (Figure 2—figure supplement 1A) and transcription of eRNAs was detected by GRO-seq, a widely used method to detect nascent RNA transcription, including eRNAs (Lam et al., 2014; Li et al., 2016; Sartorelli and Lauberth, 2020). GRO-seq showed that FXR-induced eRNAs were activated transcriptionally by GW4064 in Fxr-flox mice, but such induction was abolished in Fxr-LKO mice (Figure 2A). In particular, Fincor is robustly induced in the GRO-seq analysis and its induction is dependent on hepatic FXR (Figure 2B). RT-qPCR confirmed the FXR-dependent expression of Fincor, similar to that of Nr0b2 (Figure 2—figure supplement 1B).

Figure 2 with 1 supplement see all

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Ligand-activated farnesoid X receptor (FXR) directly activates transcription of enhancer RNAs including FXR-induced non-coding RNA (*Fincor*) in the liver.

(A) Left: experimental outline. *Fxr* floxed (*Fxr-Flox*) and *Fxr* liver-specific knockout (*Fxr-LKO*) male mice were fasted overnight and treated with vehicle or GW4064, and livers were collected 1 hr later with nuclei isolated for global run-on sequencing (GRO-seq) (n=2/group). Right: a boxplot shows the GRO-seq signals for GW4064 upregulated 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 FXR ChIP-seq peak that contains an IR1 motif. (C) ChIP assays were performed in the same liver samples described in Figure 1A to detect FXR, retinoid X receptor alpha (RXRα), and bromodomain-containing protein 4 (BRD4) occupancy at the FXR binding peak region close to the transcription start site of *Fincor* (black arrow in B). (D) HepG2 cells were transfected with luciferase reporter expressing wild-type FXRE or mutant FXRE (see Materials and methods) for 24 hr before treatment with GW4064 for an additional 6 hr. Relative luciferase activities are shown. (**C–D**) Data are presented as mean ± SEM (n=3/group). Statistical significance was determined by the Student’s t test with *p<0.05 and ***p<0.001.

We next examined if FXR binds to the enhancers that produce the identified eRNAs by analyzing published mouse liver ChIP-seq data for FXR (see Data availability) (Lee et al., 2012; Thomas et al., 2010). FXR binding was strongly enriched at the enhancers associated with FXR-induced eRNAs as were the enhancer marks H3K27ac and H3K4me1 (Figure 2—figure supplement 1C). The binding of FXR and the presence of histone marks at the Fincor enhancer region determined by ChIP-seq as compared with nascent transcripts detected by GRO-seq is shown in Figure 2B. These analyses support the conclusion that activation of FXR transcriptionally induces this series of eRNAs via chromatin binding at these enhancers, including Fincor.

We validated FXR binding at the enhancer region that produces Fincor using mouse liver ChIP (Figure 2C). We also examined binding of a well-known DNA binding partner of FXR, retinoid X receptor alpha (RXRα/NR2B1) (Evans and Mangelsdorf, 2014; Zheng et al., 2018), and bromodomain-containing protein 4 (BRD4), an acetylated histone reader protein that often binds at active enhancers (Chen et al., 2016; Li et al., 2016; Rahnamoun et al., 2018; Sartorelli and Lauberth, 2020) and a transcriptional coactivator of FXR (Jung et al., 2020). GW4064 treatment resulted in substantial increases in recruitment of both FXR and RXRα to the enhancer region close to the transcription start site of Fincor (arrow shown below in Figure 2B), whereas binding was not detected at a control region (Figure 2C). We also found that BRD4 occupancy was increased at this enhancer region after GW4064 treatment (Figure 2C). These results indicate that GW4064 activation of FXR leads to increased occupancy of the FXR/RXRα heterodimer and BRD4 to the enhancer region to upregulate Fincor eRNA in the liver.

We identified an inverted repeat1 (IR1) motif that is known to bind FXR (Calkin and Tontonoz, 2012; Lee et al., 2006) within the major FXR binding peak near the start site of Fincor (arrow shown below in Figure 2B), which we also refer to as FXRE (Figure 2B and C). We examined the functionality of this IR1 motif for mediating transcriptional activation by GW4064 using reporter assays (Figure 2D). We cloned the region containing the IR1 motif into the pGL4.23 luciferase reporter and generated a mutated IR1 motif construct as a comparison (Figure 2D). After transfection into human hepatic HepG2 cells, GW4064 treatment significantly elevated the luciferase activity of the reporter with the wild-type IR1 motif, but not with the mutated IR1 motif (Figure 2D). Together, these results suggest that GW4064-activated FXR directly upregulates Fincor expression.

Fincor is a liver-specific nucleus-enriched eRNA

Because enhancers and eRNAs generally act in a tissue-specific manner (Li et al., 2016; Sartorelli and Lauberth, 2020), we examined the tissue-specific expression of Fincor in mice. Strikingly, Fincor is highly expressed in the liver and it is expressed at extremely low levels in most other tissues, except for a detectable, but still fairly low, level in the lung (Figure 3A). GW4064 treatment resulted in induction of Fincor specifically in the liver (Figure 3A). The level of Fincor detected in primary mouse hepatocytes (PMHs) isolated from GW4064-treated mouse liver was similar to that in the liver tissue from the same mouse, suggesting that the majority of Fincor is present in hepatocytes (Figure 3B).

Figure 3

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Farnesoid X receptor-induced non-coding RNA (*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 hr (n=2/group). Data are presented as mean ± SD. BAT, brown adipose tissue; eWAT, epididymal white adipose tissue. (B) C57BL/6 male mice were fasted overnight and injected i.p. with vehicle or GW4064 (30 mg/kg) for 1 hr. 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). Data are presented as mean ± SEM. Statistical significance was determined by the Student’s t-test with ***p<0.001. (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 (n=2/group). Data are presented as mean ± SD.

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By using the 5’ and 3’ rapid amplification of cDNA ends (RACE), we identified one transcript of Fincor that is approximately 3.7 kb in length (Figure 3C). However, based on RNA-seq, the length of Fincor is over 10 kb (Figure 1C), suggesting there are likely additional multiple RNA isoforms that we were not able to identify by RACE. We next analyzed the coding potential of Fincor utilizing a comparative genomic program, PhyloCSF. While adjacent genes Gcnt1 and Prune2 (Figure 3D and E) were correctly predicted to encode proteins, Fincor did not contain a potential protein-coding open reading frame (Figure 3E). Consistent with this bioinformatic prediction, a vector expressing Fincor failed to produce any proteins in an in vitro transcription/translation assay (Figure 3F), confirming that the Fincor transcript is an ncRNA. Fincor transcripts were enriched by binding to oligo-dT beads, suggesting that Fincor is 3’ polyadenylated (Figure 3G). Further, Fincor was detected in the nuclear compartment and GW4064 treatment increased the nuclear abundance of Fincor (Figure 3H), consistent with its potential transcriptional regulatory function. Together, these results from molecular biochemical characterization studies reveal that Fincor is a liver-enriched nuclear polyadenylated eRNA.

Fincor is induced specifically by the hammerhead-type synthetic FXR agonists

To determine whether induction of the FXR-induced Fincor is ligand-specific, we examined the effects of several hammerhead-type synthetic FXR agonists, including GW4064, cilofexor, and tropifexor; a semi-synthetic agonist, OCA; and a non-hammerhead-type gut-specific agonist, fexaramine (Figure 4A; Downes et al., 2003; Fang et al., 2015). Remarkably, treatment with each of the hammerhead-type agonists for 1 hr resulted in a robust induction of Fincor (Figure 4B), whereas Fincor levels were unchanged after treatment with OCA or fexaramine for 1 hr (Figure 4B). Treatment with OCA for 4 hr or even 1 week treatment with OCA failed to induce hepatic Fincor in mice, while expression of Nr0b2 was significantly induced (Figure 4C and D). Acute feeding with a diet supplemented with 0.5% cholic acid (CA), a primary BA, for 6 hr also failed to induce Fincor expression (Figure 4E). Collectively, these results demonstrate that Fincor is induced specifically by hammerhead-type FXR agonists.

Figure 4

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Farnesoid X receptor-induced non-coding RNA (*Fincor*) is induced by the hammerhead class of non-steroidal farnesoid X receptor (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 agonist, 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 obeticholic acid (OCA) (20 mg/kg) for 1 hr (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 hr (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). *Nr0b2* 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 hr (n=3–4/group). In panels B–E, all mice underwent overnight fasting before treatment. (**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-LKD mice

To explore the functional role of hepatic Fincor, we utilized the CRISPR/Cas9 technique to generate Fincor liver-specific knockdown (Fincor-LKD) mice (Figure 5—figure supplement 1A and B). Adenoviral-mediated sgRNA expression in Cas9 mice resulted in downregulation of Fincor specifically in the liver by about 60%, but not in other tissues (Figure 5B). As FXR is a key regulator of BA, cholesterol, lipid, and glucose metabolism and Fincor is specifically regulated by FXR, Fincor may have a role in the metabolic process in physiology and disease. We examined liver triglyceride, cholesterol, BA, glycogen, and serum non-esterified fatty acids (NEFA) but Fincor downregulation did not result in any significant changes under physiological conditions (Figure 5C).

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Generation of CRISPR/Cas9-mediated farnesoid X receptor-induced non-coding RNA (*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 hr 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 non-esterified fatty acids (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 hr 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 downregulated by twofold 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-C**) Data are presented as mean ± SEM. Statistical significance was determined by the Student’s t test with ***p<0.001.

To explore the molecular signatures and pathways affected by Fincor, we examined global gene expression by RNA-seq analysis in mouse liver after Fincor knockdown (Figure 5D). While Fincor was markedly downregulated, the neighboring genes, such as Gcnt1, Rfk, Pcsk5, and Prune2, were largely unchanged (Figure 5D; Figure 5—figure supplement 1C). The RNA-seq analysis revealed 18 upregulated genes and 53 downregulated genes in Fincor-LKD liver (Supplementary file 2; Figure 5E). GO analysis of those downregulated genes indicated that these genes were enriched in pathways involved in fatty acid oxidation, organelle organization, and metabolic process (Figure 5F). Among the downregulated genes, Ppp1r3g, which has a role in controlling glycogen synthesis, and Igfbp2, which functions in insulin resistance, were markedly reduced (Supplementary file 2; Figure 5—figure supplement 1D and E). Among the upregulated genes, expression was substantially increased for Eda2r, which is a member of the tumor necrosis factor receptor superfamily and involved in inflammation, the immune response, and development (Figure 5—figure supplement 1F). Fndc1, which is involved in fibronectin matrix remodeling, was also suppressed by Fincor (and thus upregulated upon its knockdown, Figure 5—figure supplement 1G). These studies suggest that Fincor has a role in modulating metabolic homeostasis by regulating genes involved in metabolism and inflammation.

Amelioration of hepatic steatosis mediated by tropifexor is independent of Fincor in diet-induced NASH mice

Tropifexor, also known as LJN452, is a highly potent hammerhead-type FXR agonist that is currently under clinical trials for NASH and PBC patients (Kremoser, 2021; Sanyal et al., 2023; Tully et al., 2017). Because Fincor is induced specifically by the hammerhead class of FXR agonist (Figure 4) and has a potential role in the regulation of metabolism and inflammation (Figure 5), we hypothesized that Fincor may play a role in tropifexor-mediated beneficial effects on reducing NASH pathologies in mice.

We utilized a mouse model that had been fed the amylin liver NASH-promoting (AMLN) diet (Hernandez et al., 2019; Sun et al., 2022; Zhao et al., 2018), and examined the potential impact of liver-specific downregulation of Fincor on tropifexor’s effects on NASH pathology. Cas9 mice fed the AMLN diet for 12 weeks were injected via tail veins with adenovirus expressing control sgRNA or Fincor sgRNA, respectively, and then treated daily with tropifexor (0.3 mg/kg) for 12 days (Figure 6A). In these mice, as a technical validation of RNA induction and knockdown, Fincor levels were significantly increased by FXR agonist tropifexor, and the increase was blocked by adenovirus expressing sgRNA for Fincor (Figure 6B).

Figure 6

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In diet-induced nonalcoholic steatohepatitis (NASH) mice, tropifexor-mediated beneficial effects on reducing hepatic steatosis are largely independent of farnesoid X receptor-induced non-coding RNA (*Fincor*).

(**A–E**) Male Cas9 mice were fed a NASH diet for 12 weeks. The mice were randomly assigned to three groups and infected with adenovirus expressing sgRNA for *Fincor* or control. Three days later, the mice were daily treated with tropifexor (0.3 mg/kg) or vehicle from day 1 to day 11. On day 12, the mice were given the final treatment of tropifexor or vehicle and fasted for 4 hr 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 ImageJ and the areas of stained field were quantified (n=5/group). (D) Hepatic TG, hepatic cholesterol, gallbladder bile acid (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-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.

We then examined the effect of tropifexor treatment and Fincor downregulation on hepatic steatosis in these mice. Tropifexor treatment markedly reduced neutral lipids determined by Oil Red O staining of liver sections (Figure 6C) and liver TG levels (Figure 6D), and these beneficial effects on reducing fatty liver were not altered by Fincor downregulation. Also, Fincor downregulation had little effect on liver cholesterol and gallbladder BA levels, although gallbladder BA levels were reduced by tropifexor (Figure 6D).

Consistent with the phenotypes, hepatic expression of key genes involved in BA synthesis was dramatically reduced by tropifexor treatment (i.e. Cyp7a1 and Cyp8b1), which is consistent with decreased gallbladder BA levels mediated by this agonist (Figure 6E). Similarly, tropifexor also lowered lipid synthesis genes (Srebp1c, Lpin1, Scd1), consistent with decreased liver TG levels. However, downregulation of Fincor did not result in changes in mRNA levels of these genes (Figure 6E). These results indicate that tropifexor-mediated beneficial effects on reducing hepatic steatosis are independent of Fincor.

Fincor facilitates alleviation of liver inflammation by tropifexor in diet-induced NASH

Tropifexor ameliorated fibrotic NASH pathologies in preclinical studies (Hernandez et al., 2019; Tully et al., 2017) and has recently concluded phase 2 clinical trials for NASH patients (Sanyal et al., 2023). We, therefore, further examined the effects of Fincor downregulation on altering other NASH pathologies, including hepatocellular apoptosis, liver fibrosis, and inflammation.

In the same AMLN diet-fed mice as described above (Figure 6), analyses of liver sections revealed that tropifexor treatment reduced hepatocyte swelling/ballooning (hematoxylin and eosin [H&E] staining), decreased numbers of apoptotic cells (TUNEL staining), alleviated fibrosis (Sirius Red staining), and lowered infiltration of macrophages (F4/80 staining) (Figure 7A). Remarkably, these tropifexor-mediated beneficial effects on NASH pathologies were all markedly diminished by Fincor downregulation (Figure 7A). This was consistently found in various liver lobes and two representative pictures from two different lobes are shown (Figure 7A). In control experiments, Fincor downregulation in vehicle-treated mice did not result in marked changes in NASH pathologies (Figure 7—figure supplement 1). These results demonstrate that Fincor is required for tropifexor-mediated beneficial effects on reducing NASH pathologies, specifically reducing hepatic inflammation, fibrosis, and hepatocyte apoptosis. Consistent with these results, serum ALT and AST levels, indicators of liver damage, were significantly elevated after Fincor downregulation (Figure 7B). Protein levels of key inflammatory markers, IL1β and CCL2, in liver extracts were also elevated after Fincor downregulation (Figure 7C).

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In diet-induced nonalcoholic steatohepatitis (NASH) mice, tropifexor-mediated beneficial effects on reducing liver fibrosis and inflammation are diminished by farnesoid X receptor-induced non-coding RNA (*Fincor*) downregulation.

(A) Representative images from hematoxylin and eosin (H&E), TUNEL, Sirius Red, and F4/80 staining of liver sections from the same cohort of mice described in Figure 6. Scale bar (50 μm). Image analyses were done using ImageJ 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 farnesoid X receptor (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.

Consistent with the phenotypes from histological analyses, expression of hepatic genes involved in the above pathological process was altered by Fincor knockdown. For example, tropifexor treatment reduced mRNA levels of several genes that promote fibrosis (Col1a1, Col1a2, Acta2) and hepatic inflammation (Eda2r, Ifng, Ccl3), whereas these reductions were largely reversed by Fincor downregulation (Figure 7D). We also detected increased expression of inflammatory genes (Ccl2, Ccr2, Lcn2) and an extracellular matrix remodeling gene (Fndc1) in liver with Fincor knockdown (Figure 7D). Tropifexor treatment suppressed hepatic apoptosis by reducing pro-apoptotic genes (Ctsb, Ctss) and upregulating anti-apoptotic genes such as Bcl2 (Warren et al., 2019). Importantly, these effects were significantly reversed by Fincor downregulation (Figure 7D).

Collectively, these results demonstrate that in diet-induced NASH mice, pharmacological activation of FXR by tropifexor reduced fibrosis, apoptosis, and inflammation, which was dependent, at least in part, on the induction of Fincor.

Fincor expression is increased in chronic liver disease with hepatic inflammation and liver injury

To determine whether expression of Fincor is altered in chronic liver disease, we utilized mouse models of NAFLD/NASH and cholestatic liver injury. Hepatic Fincor levels were significantly increased in mice fed with a high-fat diet (HFD) for 12 weeks (Figure 8A) and in mice fed an HFD with high fructose in drinking water for 12 weeks (Figure 8B). Elevated hepatic Fincor levels were also observed in mice treated with α-naphthylisothiocyanate (ANIT), a chemical inducer of liver cholestasis (Jung et al., 2020; Kim et al., 2016; Figure 8C), and in mice with bile duct ligation (BDL), a surgical method to induce cholestatic liver injury (Figure 8D).

Figure 8

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Hepatic expression of farnesoid X receptor-induced non-coding RNA (*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 α-naphthylisothiocyanate (ANIT) (75 mg/kg) for 48 hr and then sacrificed after 5 hr 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 hr 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 long non-coding RNA (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 nonalcoholic fatty liver disease (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.

The sequence of Fincor is moderately conserved between mice and humans as displayed in the UCSC genome browser (Figure 8E). Annotation in the NCBI genome data viewer of the human sequence region with similarity to mouse Fincor revealed an functionally uncharacterized human lncRNA, XR_007061585.1, in this region (Figure 8F). However, whether this sequence (or an as yet to be identified lncRNA) is functional or not has not been determined. To explore the potential changes or role of lncRNA XR_007061585.1 in human liver pathological conditions, we measured hepatic levels of the transcripts in PBC and NAFLD patients. Compared to normal individuals, hepatic lncRNA XR_007061585.1 levels were elevated in patients with PBC or NAFLD, but not in severe NASH-fibrosis patients (Figure 8G and H). These results demonstrate that hepatic levels of a potential human analog of Fincor are elevated in NAFLD and PBC patients, as Fincor is in mouse models of chronic liver disease with hepatic inflammation and liver injury. However, whether human lncRNA XR_007061585.1 is analogous to mouse Fincor in terms of functions and mechanisms, and whether elevated XR_007061585.1 levels may have a role in the disease progression or may be an adaptive response to liver injury remain to be determined.

Discussion

FXR maintains metabolic homeostasis by transcriptional regulation of genes. Direct regulation of protein-coding genes by FXR, including Nr0b2, is well characterized. In this study we show that FXR also mediates its functions by induction of lncRNA genes, which vastly outnumber protein-coding genes. We further show that pharmacological activation of FXR by hammerhead-type agonists induces a liver-specific enhancer-derived lncRNA, which we named Fincor, that contributes to reduction of NASH pathologies in mice.

Fincor is specifically induced by the hammerhead class of FXR agonists, such as GW4064, cilofexor, and tropifexor. GW4064 is the mother compound of these isoxazole-type hammerhead ligands but is not an ideal therapeutic agent because of its poor water solubility and pharmacokinetics (Abel et al., 2010). In contrast, tropifexor and cilofexor have better pharmacokinetics and are generally well tolerated in clinical trials for NASH and PBC patients (Abenavoli et al., 2018; Kremoser, 2021; Sanyal et al., 2023; Tully et al., 2017), but the underlying mechanisms for their beneficial effects are poorly understood. Intriguingly, a recent study showed that the gene signature regulated by tropifexor-activated FXR appears to be broader than that of OCA, partly because the tropifexor backbone allows a more favorable interaction of FXR with coactivators or epigenomic modulators (Hernandez et al., 2019). Further, tropifexor was shown to regulate distinct sets of genes in experimental NASH as compared to other FXR agonists, particularly genes involved in fibrosis, inflammation, and oxidative stress (Hernandez et al., 2019). Utilizing CRISPR/Cas9-mediated liver-specific knockdown of Fincor in diet-induced NASH mice, we demonstrate that beneficial effects on reducing liver fibrosis, inflammation, and apoptosis mediated by tropifexor were largely dependent on Fincor (Model, Figure 7E).

While this study focused on regulation of Fincor by pharmacological activation of FXR, physiological and pathological regulations of Fincor appear to be complex. Although Fincor can be induced by the hammerhead class of FXR agonists, it was not induced by the endogenous FXR ligand, CA. This implies that Fincor may not contribute to the physiological functions of FXR. In an effort to investigate the potential role of Fincor in the pathological conditions, we observed that Fincor levels were elevated in mouse models of cholestasis and NASH as well as in human PBC and NAFLD patients, where BA metabolism is dysregulated. Since different BAs can activate or repress the gene-regulating function of FXR (Wahlström et al., 2016), altered BA composition in these pathological conditions may contribute to induction of Fincor. Further, binding peaks for multiple nuclear receptors, FXR, LXR, PPARα, RXRα, and HNF-4α, were detected in the Fincor locus so that regulation of Fincor likely involves the combinatorial regulation by multiple nuclear receptors (Supplementary file 3a). Interestingly, occupancy of the nuclear receptor PPARα at the enhancer region was increased in fasted mice (Supplementary file 3b). It will be interesting to investigate whether and how Fincor is differently regulated by these nuclear receptors in response to physiological and pathological cues.

The roles of regulatory RNAs in liver function and diseases and their potentials as therapeutic targets are increasingly being appreciated (Brocker et al., 2020; Li et al., 2021; Sallam et al., 2016; Zhao et al., 2018). For example, a critical role for an oxysterol nuclear receptor LXR-induced lncRNA, LeXis, in feedback modulation of cholesterol biosynthesis has been shown (Sallam et al., 2016). Recently, the role of an lncRNA, Pair, in liver phenylalanine metabolism has been demonstrated (Li et al., 2021). Enhancer RNAs are a less-characterized class of lncRNAs and are highly associated with enhancer functions in gene regulation (Li et al., 2016). Numerous studies have revealed transcriptional roles for eRNAs in various cellular processes (Lai and Shiekhattar, 2014; Lam et al., 2014; Li et al., 2016; Sartorelli and Lauberth, 2020) but most previous eRNA studies have used cultured cells (Hsieh et al., 2014; Li et al., 2013), with only a few in vivo studies in mouse models (Mirtschink et al., 2019; Tang et al., 2023). In our current study, through integrative analysis of transcriptome and histone mark ChIP-seq, we identified a group of FXR-regulated eRNAs, including the highly induced Fincor. Our current work characterized the role of Fincor in gene regulation and in mediating beneficial pharmacological effects of tropifexor in NASH, representing important progress in understanding the roles of eRNAs in vivo. Future work is warranted to elucidate the exact mechanisms by which Fincor facilitates action of FXR agonists to alleviate inflammation, fibrosis, and apoptosis.

RNA inside the cells usually associates with different RNA binding proteins (RBPs) (Gerstberger et al., 2014). We identified potential binding proteins of Fincor using the ATtRACT database (Giudice et al., 2016). The top four candidates for Fincor binding are KHDRBS1, RBM38, YBX2, and YBX3 (Supplementary file 4). KHDRBS1 and RBM38 have been reported to have important roles in RNA processing (Bielli et al., 2011; Zou et al., 2021). YBX2 and YBX3 belong to the Y-box (YBX) protein family, which have been linked to diverse forms of RNA metabolism and many other processes, including cell proliferation, DNA repair, stress responses, development, and inflammation (Kleene, 2018). Whether these predicted RBPs interact with Fincor and how they contribute to phenotypes should be investigated in future experimentation to understand the mechanisms involved in Fincor-regulated hepatocyte function.

There is limitation of some of our approaches that cannot fully dissect the underlying mechanisms of Fincor. Currently, the function of eRNA loci can be attributed to a functional eRNA transcript, or binding of transcription factors to this region, or the transcription process itself, or some combination of these effects (Li et al., 2016; Sartorelli and Lauberth, 2020). In our studies to decrease expression of Fincor, the region containing the FXR binding site was deleted (Figure 5—figure supplement 1A, right panel). While the expression of Fincor transcripts was significantly reduced, we cannot rule out whether decreased binding of FXR or decreased transcription contribute to the observed changes in phenotype. To directly investigate the function of Fincor transcripts, downregulation of the transcript with antisense oligonucleotides together with overexpression of Fincor in the liver will be required in future experiments.

FXR is increasingly recognized as an important therapeutic target for enterohepatic diseases, but the development of clinically applicable and more targeted FXR-based therapy is still challenging. In this study, we provide the first characterization of an eRNA, Fincor, induced by pharmacological activation of FXR and show that Fincor has a beneficial role in reducing liver fibrosis and inflammation in dietary NASH mice. Complete understanding of the function and mechanisms of Fincor may provide novel insights for the development of desirable therapy for NASH and other chronic liver diseases.

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Activation of farnesoid X receptor (FXR) by GW4064 induces FXR-induced non-coding RNA (Fincor), a novel enhancer RNA (eRNA), in mouse liver.

Ligand-activated farnesoid X receptor (FXR) directly activates transcription of enhancer RNAs including FXR-induced non-coding RNA (Fincor) in the liver.

Farnesoid X receptor-induced non-coding RNA (Fincor) is a liver-specific nucleus-enriched eRNA.

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Farnesoid X receptor-induced non-coding RNA (Fincor) is induced by the hammerhead class of non-steroidal farnesoid X receptor (FXR) agonists, including GW4064 and tropifexor.

Generation of CRISPR/Cas9-mediated farnesoid X receptor-induced non-coding RNA (Fincor) liver-specific knockdown mice.

In diet-induced nonalcoholic steatohepatitis (NASH) mice, tropifexor-mediated beneficial effects on reducing hepatic steatosis are largely independent of farnesoid X receptor-induced non-coding RNA (Fincor).

In diet-induced nonalcoholic steatohepatitis (NASH) mice, tropifexor-mediated beneficial effects on reducing liver fibrosis and inflammation are diminished by farnesoid X receptor-induced non-coding RNA (Fincor) downregulation.

Hepatic expression of farnesoid X receptor-induced non-coding RNA (Fincor) is elevated in liver disease associated with inflammation and fibrosis.

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Jinjing Chen

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Ruoyu Wang

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Feng Xiong

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Hao Sun

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Byron Kemper

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Wenbo Li

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Jongsook Kemper

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