HNF4A as a potential target of PFOA and PFOS leading to hepatic steatosis: Integrated molecular docking, molecular dynamic and transcriptomic analyses.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are indeed among the most well known and extensively studied Per- and polyfluoroalkyl substances (PFASs), and increasing evidence confirm their effects on human health, especially liver steatosis. Nonetheless, the molecular mechanisms of their initiation of hepatic steatosis is still elusive. Therefore, potential targets of PFOA/PFOS must be explored to ameliorate its adverse consequences. This research aims to investigate the molecular mechanisms of PFOA and PFOS-induced liver steatosis, with emphasis on identifying a potential target that links these PFASs to liver steatosis. The potential target that causes PFOA and PFOS-induced liver steatosis have been explored and determined based on molecular docking, molecular dynamics (MD) simulation, and transcriptomics analysis. In silico results show that PFOA/PFOS can form a stable binding conformation with HNF4A, and PFOA/PFOS may interact with HNF4A to affect the downstream conduction mechanism. Transcriptome data from PFOA/PFOS-induced human stem cell spheres showed that HNF4A was inhibited, suggesting that PFOA/PFOS may constrain its function. PFOS mainly down-regulated genes related to cholesterol synthesis while PFOA mainly up-regulated genes related to fatty acid ß-oxidation. This study explored the toxicological mechanism of liver steatosis caused by PFOA/PFOS. These compounds might inhibit and down-regulate HNF4A, which is the molecular initiation events (MIE) that induces liver steatosis.

Acanthopanax senticosus ameliorates steatohepatitis through HNF4 alpha pathway activation in mice.

Non-alcoholic fatty liver disease is a common liver disease worldwide, and is associated with dysregulation of lipid metabolism, leading to inflammation and fibrosis. Acanthopanax senticosus Harms (ASH) is widely used in traditional medicine as an adaptogen food. We examined the effect of ASH on steatohepatitis using a high-fat diet mouse model. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet with ASH extract (ASHE). After 6 weeks, liver RNA transcriptome sequencing (RNA-Seq) was performed, followed by Ingenuity Pathway Analysis (IPA). Our findings revealed that mice fed a high-fat diet with 5% ASHE exhibited significantly reduced liver steatosis. These mice also demonstrated alleviated inflammation and reduced fibrosis in the liver. IPA of RNA-Seq indicated that hepatocyte nuclear factor 4 alpha (HNF4 alpha), a transcription factor, was the activated upstream regulator (P-value 0.00155, z score = 2.413) in the liver of ASHE-fed mice. Adenosine triphosphate binding cassette transporter 8 and carboxylesterase 2, downstream targets of HNF4 alpha pathway, were upregulated. Finally, ASHE-treated HepG2 cells exposed to palmitate exhibited significantly decreased lipid droplet contents. Our study provides that ASHE can activate HNF4 alpha pathway and promote fat secretion from hepatocytes, thereby serving as a prophylactic treatment for steatohepatitis in mice.

Mechanism and Effect of HNF4α Decrease in a Rat Model of Cirrhosis and Liver Failure.

BACKGROUND & AIMS: HNF4α, a master regulator of liver development and the mature hepatocyte phenotype, is down-regulated in chronic and inflammatory liver disease. We used contemporary transcriptomics and epigenomics to study the cause and effects of this down-regulation and characterized a multicellular etiology. METHODS: Progressive changes in the rat carbon tetrachloride model were studied by deep RNA sequencing and genome-wide chromatin immunoprecipitation sequencing analysis of transcription factor (TF) binding and chromatin modification. Studies compared decompensated cirrhosis with liver failure after 26 weeks of treatment with earlier compensated cirrhosis and with additional rat models of chronic fibrosis. Finally, to resolve cell-specific responses and intercellular signaling, we compared transcriptomes of liver, nonparenchymal, and inflammatory cells. RESULTS: HNF4α was significantly lower in 26-week cirrhosis, part of a general reduction of TFs that regulate metabolism. Nevertheless, increased binding of HNF4α contributed to strong activation of major phenotypic genes, whereas reduced binding to other genes had a moderate phenotypic effect. Decreased Hnf4a expression was the combined effect of STAT3 and nuclear factor kappa B (NFκB) activation, which similarly reduced expression of other metabolic TFs. STAT/NFκB also induced de novo expression of Osmr by hepatocytes to complement induced expression of Osm by nonparenchymal cells. CONCLUSIONS: Liver decompensation by inflammatory STAT3 and NFκB signaling was not a direct consequence of progressive cirrhosis. Despite significant reduction of Hnf4a expression, residual levels of this abundant TF still stimulated strong new gene expression. Reduction of HNF4α was part of a broad hepatocyte transcriptional response to inflammation.

Association between HNF4A rs1800961 polymorphisms and gallstones in a Taiwanese population.

BACKGROUND AND AIM: A large genetic effect of a novel gallstone-associated genetic variant, the hepatocyte nuclear factor 4α (HNF4A) rs1800961 polymorphism, has been identified through recent genome-wide association studies. However, this effect has not been validated in Asian populations. We investigated the association between the rs1800961 variant and gallstones among a Taiwanese population. METHODS: A total of 20 405 participants aged between 30 and 70 years voluntarily enrolled in the Taiwan Biobank. Self-report questionnaires, physical examinations, biochemical tests, and genotyping were used for analysis. The association of the HNF4A rs1800961 variant and other metabolic risks with gallstone disease was analyzed using multiple logistic regression models. RESULTS: The minor T allele of HNF4A rs1800961 was associated with an increased risk of gallstone, and the association remained significant even after adjustment for other risk factors including age, body mass index (BMI), diabetes, hyperlipidemia, hypertension, and cigarette smoking (adjusted odds ratio [OR] = 1.90, 95% confidence interval [CI] = 1.31 to 2.75) in male participants. When further stratified by BMI and age, the lithogenic effect was the most significant in male participants with obesity (adjusted OR = 3.55, 95% CI = 1.92 to 6.56) and who were younger (adjusted OR = 2.45, 95% CI = 1.49 to 4.04). CONCLUSION: The novel gallstone-associated HNF4A rs1800961 variant was associated with the risk of gallstone in the Taiwanese men. Screening for the rs1800961 polymorphism may be particularly useful in assessing the risk of gallstone formation in younger or obese men.

Insulin Determines Transforming Growth Factor ß Effects on Hepatocyte Nuclear Factor 4α Transcription in Hepatocytes.

Loss of hepatocyte nuclear factor 4α (HNF4α) expression is frequently observed in end-stage liver disease and associated with loss of vital liver functions, thus increasing mortality. Loss of HNF4α expression is mediated by inflammatory cytokines, such as transforming growth factor (TGF)-ß. However, details of how HNF4α is suppressed are largely unknown to date. Herein, TGF-ß did not directly inhibit HNF4α but contributed to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CREB-binding protein/p300 to the HNF4α promoter. The recruitment of CREB-binding protein/p300 is indispensable for CCAAT/enhancer-binding protein α (C/EBPα) binding, another essential requirement for constitutive HNF4α expression in hepatocytes. Consistent with the in vitro observation, 67 of 98 patients with hepatic HNF4α expressed both phospho-SMAD2 and C/EBPα, whereas 22 patients without HNF4α expression lacked either phospho-SMAD2 or C/EBPα. In contrast to the observed induction of HNF4α, SMAD2/3 inhibited C/EBPα transcription. Long-term TGF-ß incubation resulted in C/EBPα depletion, which abrogated HNF4α expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBPα promoter was abolished by insulin. Two-thirds of patients without C/EBPα lacked membrane glucose transporter type 2 expression in hepatocytes, indicating insulin resistance. Taken together, these data indicate that hepatic insulin sensitivity is essential for hepatic HNF4α expression in the condition of inflammation.

HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

Hepatocyte nuclear factor 4 a (HNF4α): A perspective in cancer.

HNF4α, a transcription factor, plays a vital role in regulating functional genes and biological processes. Its alternative splicing leads to various transcript variants encoding different isoforms. The spotlight has shifted towards the extensive discussion on tumors interplayed withHNF4α abnormalities. Aberrant HNF4α expression has emerged as sentinel markers of epigenetic shifts, casting reverberations upon downstream target genes and intricate signaling pathways, most notably with cancer. This review provides a comprehensive overview of HNF4α's involvement in tumor progression and metastasis, elucidating its role and underlying mechanisms.

The HNF4A-CHPF pathway promotes proliferation and invasion through interactions with MAD1L1 in glioma.

Chondroitin polymerizing factor (CHPF) is an important glycosyltransferases that participates in the biosynthesis of chondroitin sulfate (CS). Our previous study showed that silencing CHPF expression inhibited glioma cell proliferation in vitro, but the molecular mechanisms by which CHPF contributes to development of glioma have not been characterized. In this study, we found that CHPF was up-regulated in glioma tissues and was positively correlated with malignant clinical pathological characteristics of patients with glioma. Silencing CHPF expression inhibited proliferation, colony formation, migration, and cell cycle of glioma cells. Moreover, silencing CHPF suppressed glioma malignance in vivo. Immunoprecipitation, co-immunoprecipitation, GST pulldown, and liquid chromatography-mass spectrometry (LC-MS/MS) assays were used to verify the interaction between CHPF and Mitotic arrest deficient 1-like 1 (MAD1L1). In addition, Chromatin Immunoprecipitation (ChIP)-PCR analysis showed that HNF4A bound to the CHPF promoter region, which indicated that the transcription factor hepatocyte nuclear factor 4A (HNF4A) could regulate the expression of CHPF in glioma cells.

Inhibiting HNF4A suppresses inflammation whilst promoting trophoblast invasion and migration: A promising target for the treatment of preeclampsia.

Preeclampsia (PE) is a complex disease of pregnancy, and an important cause of this disease is insufficient trophoblast invasion and migration. However, the underlying mechanism of PE remains largely unknown. Here, transcriptome sequencing analysis found the high expression of hepatocyte nuclear factor 4 alpha (HNF4A) in PE placentas. Meanwhile, we found that HNF4A expression was up-regulated in the placentas of PE patients. Thus, we assumed that HNF4A might be involved in PE progression. To validate our hypothesis, l-arginine methyl ester (l-NAME) or lipopolysaccharide (LPS)-treated rats were used to mimic the pathological status of PE in vivo. Consistently, HTR8/SVneo cells were treated with hypoxia/reoxygenation (H/R) or LPS to simulate PE progression in vitro. The results observed an increase in elevated urine protein levels, systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP), which indicated that the PE-like rat model was successfully established. Meanwhile, the expression of pro-inflammatory cytokines interleukin (IL)-6 and IL-1ß was increased in PE placentas. HTR8/SVneo cells were used to further explore the underlying mechanism of PE in vitro. H/R conditions up-regulated the acetylation level of HNF4A. Further analysis showed that HNF4A overexpression inhibited trophoblast invasion and migration, while HNF4A knockdown promoted the progression. Additionally, inhibiting HNF4A was found to reduce the levels of IL-6 and IL-1ß secretion in HTR8/SVneo cells following H/R or LPS exposure. Conclusively, these findings suggest that inhibiting HNF4A suppresses inflammation whilst promoting trophoblast invasion and migration in PE, providing a promising target for the treatment of PE.

Identification and precision therapy for three maturity-onset diabetes of the young (MODY) families caused by mutations in the HNF4A gene.

Background: Heterozygous pathogenic variants in HNF4A gene cause maturity-onset diabetes of the young type 1 (MODY1). The mutation carriers for MODY1 have been reported to be relatively rare, in contrast to the most frequently reported forms of MODY2 and MODY3. Methods: Whole exome sequencing (WES) and Sanger sequencing were performed for genetic analysis of MODY pedigrees. Tertiary structures of the mutated proteins were predicted using PyMOL software. Results: Three heterozygous missense mutations in the HNF4A gene, I159T, W179C, and D260N, were identified in the probands of three unrelated MODY families using WES, one of which (W179C) was novel. Cascade genetic screening revealed that the mutations co-segregated with hyperglycemic phenotypes in their families. The molecular diagnosis of MODY1 has partly transformed its management in clinical practice and improved glycemic control. The proband in family A successfully converted to sulfonylureas and achieved good glycemic control. Proband B responded well to metformin combined with diet therapy because of his higher body mass index (BMI). The proband in family C, with paternal-derived mutations, had markedly defective pancreatic ß-cell function due to the superposition effect of T2DM susceptibility genes from the maternal grandfather, and he is currently treated with insulin. In silico analysis using PyMOL showed that the I159T and D260N mutations altered polar interactions with the surrounding residues, and W179C resulted in a smaller side chain. Discussion: We identified three heterozygous missense mutations of HNF4A from Chinese MODY families. Structural alterations in these mutations may lead to defects in protein function, further contributing to the hyperglycemic phenotype of mutation carriers.

HNF4α isoforms: the fraternal twin master regulators of liver function.

In the more than 30 years since the purification and cloning of Hepatocyte Nuclear Factor 4 (HNF4α), considerable insight into its role in liver function has been gleaned from its target genes and mouse experiments. HNF4α plays a key role in lipid and glucose metabolism and intersects with not just diabetes and circadian rhythms but also with liver cancer, although much remains to be elucidated about those interactions. Similarly, while we are beginning to elucidate the role of the isoforms expressed from its two promoters, we know little about the alternatively spliced variants in other portions of the protein and their impact on the 1000-plus HNF4α target genes. This review will address how HNF4α came to be called the master regulator of liver-specific gene expression with a focus on its role in basic metabolism, the contributions of the various isoforms and the intriguing intersection with the circadian clock.

Spontaneous episodic inflammation in the intestines of mice lacking HNF4A is driven by microbiota and associated with early life microbiota alterations.

The inflammatory bowel diseases (IBD) occur in genetically susceptible individuals who mount inappropriate immune responses to their microbiota leading to chronic intestinal inflammation. Whereas IBD clinical presentation is well described, how interactions between microbiota and host genotype impact early subclinical stages of the disease remains unclear. The transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) has been associated with human IBD, and deletion of Hnf4a in intestinal epithelial cells (IECs) in mice (Hnf4aΔIEC) leads to spontaneous colonic inflammation by 6-12 mo of age. Here, we tested if pathology in Hnf4aΔIEC mice begins earlier in life and if microbiota contribute to that process. Longitudinal analysis revealed that Hnf4aΔIEC mice reared in specific pathogen-free (SPF) conditions develop episodic elevated fecal lipocalin 2 (Lcn2) and loose stools beginning by 4-5 wk of age. Lifetime cumulative Lcn2 levels correlated with histopathological features of colitis at 12 mo. Antibiotic and gnotobiotic tests showed that these phenotypes in Hnf4aΔIEC mice were dependent on microbiota. Fecal 16S rRNA gene sequencing in SPF Hnf4aΔIEC and control mice disclosed that genotype significantly contributed to differences in microbiota composition by 12 mo, and longitudinal analysis of the Hnf4aΔIEC mice with the highest lifetime cumulative Lcn2 revealed that microbial community differences emerged early in life when elevated fecal Lcn2 was first detected. These microbiota differences included enrichment of a novel phylogroup of Akkermansia muciniphila in Hnf4aΔIEC mice. We conclude that HNF4A functions in IEC to shape composition of the gut microbiota and protect against episodic inflammation induced by microbiota throughout the lifespan. IMPORTANCE The inflammatory bowel diseases (IBD), characterized by chronic inflammation of the intestine, affect millions of people around the world. Although significant advances have been made in the clinical management of IBD, the early subclinical stages of IBD are not well defined and are difficult to study in humans. This work explores the subclinical stages of disease in mice lacking the IBD-associated transcription factor HNF4A in the intestinal epithelium. Whereas these mice do not develop overt disease until late in adulthood, we find that they display episodic intestinal inflammation, loose stools, and microbiota changes beginning in very early life stages. Using germ-free and antibiotic-treatment experiments, we reveal that intestinal inflammation in these mice was dependent on the presence of microbiota. These results suggest that interactions between host genotype and microbiota can drive early subclinical pathologies that precede the overt onset of IBD and describe a mouse model to explore those important processes.

Elucidating the Proximal Tubule HNF4A Gene Regulatory Network in Human Kidney Organoids.

SIGNIFICANCE STATEMENT: HNF4 genes promote proximal tubule differentiation in mice, but their function in human nephrogenesis is not fully defined. This study uses human pluripotent stem cell (PSC)-derived kidney organoids as a model to investigate HNF4A and HNF4G functions. The loss of HNF4A , but not HNF4G , impaired reabsorption-related molecule expression and microvilli formation in human proximal tubules. Cleavage under targets and release using nuclease (CUT&RUN) sequencing and CRISPR-mediated transcriptional activation (CRISPRa) further confirm that HNF4A directly regulates its target genes. Human kidney organoids provide a good model for studying transcriptional regulation in human kidney development. BACKGROUND: The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated. METHODS: We generated HNF4A -knock out (KO), HNF4G -KO, and HNF4A/4G -double KO human pluripotent stem cell lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A-binding sites genome-wide by cleavage under targets and release using nuclease sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation. RESULTS: Organoids lacking HNF4A , but not HNF4G , showed reduced expression of transport-related, endocytosis-related, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. Cleavage under targets and release using nuclease revealed that HNF4A primarily bound promoters and enhancers of genes that were downregulated in HNF4A -KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPR-mediated transcriptional activation drove increased expression of selected target genes during kidney organoid differentiation. CONCLUSIONS: This study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.

Structural insights into the HNF4 biology.

Hepatocyte Nuclear Factor 4 (HNF4) is a transcription factor (TF) belonging to the nuclear receptor (NR) family that is expressed in liver, kidney, intestine and pancreas. It is a master regulator of liver-specific gene expression, in particular those genes involved in lipid transport and glucose metabolism and is crucial for the cellular differentiation during development. Dysregulation of HNF4 is linked to human diseases, such as type I diabetes (MODY1) and hemophilia. Here, we review the structures of the isolated HNF4 DNA binding domain (DBD) and ligand binding domain (LBD) and that of the multidomain receptor and compare them with the structures of other NRs. We will further discuss the biology of the HNF4α receptors from a structural perspective, in particular the effect of pathological mutations and of functionally critical post-translational modifications on the structure-function of the receptor.

High-Throughput Sequencing Data Reveal an Antiangiogenic Role of HNF4A-Mediated CACNA1A/VEGFA Axis in Proliferative Diabetic Retinopathy.

Purpose: Proliferative diabetic retinopathy (PDR) is characterized by retinal new vessel formation, pointing to the importance of the antiangiogenic treatment in PDR. Hepatocyte nuclear factor 4A (HNF4A) has been highlighted to inhibit vascular endothelial growth factor (VEGF)-stimulated in vitro angiogenesis. Therefore, this study aims to elucidate the potential antiangiogenic mechanisms of HNF4A in PDR. Methods: PDR-related high-throughput sequencing datasets (GSE94019, GSE102485, and GSE191210) were obtained from the Gene Expression Omnibus (GEO) database, followed by the screening of differentially expressed genes (DEGs). The protein-protein interaction (PPI) network of the candidate DEGs was constructed based on gene set enrichment analysis (GSEA) data and Search Tool for the Retrieval of Interacting Genes (STRING) data. In addition, the key genes and pathways related to angiogenesis were screened by functional enrichment analysis. Furthermore, human retinal microvascular cells were used for further in vitro validation. Results: Four key genes (CACNA1A, CACNA1E, PDE1B, and CHRM3) related to PDR were identified in the grey module. CACNA1A affected angiogenesis in PDR by regulating vascular endothelial growth factor A (VEGFA) expression. Furthermore, HNF4A participated in angiogenesis in PDR by activating CACNA1A. In vitro experiments further identified that inhibition of HNF4A reduced CACNA1A expression and increased VEGFA expression, thus promoting angiogenesis in PDR. Conclusions: In conclusion, the obtained findings suggest that antiangiogenic HNF4A activates the CACNA1A/VEGFA axis in PDR. Our work provides new insights into the angiogenic mechanism of PDR and offers potential targets for translational applications.

Hepatocyte nuclear factor 4α multiple isoforms, their functions, and their interactomes.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a master regulator gene belonging to the nuclear receptor superfamily and is involved in regulating a wide range of critical biological processes in different organs. Structurally, the HNF4A locus is organized into two independent promoters and is subjected to alternative splicing to produce twelve distinct isoforms. However, little is known about the biological impact of each isoform and the mechanisms by which they regulate transcription. Proteomic analyses have led to the identification of proteins that interact with specific HNF4α isoforms. The identification and validation of these interactions and their roles in the co-regulation of targeted gene expression are essential to better understand the role of this transcription factor in different biological processes and pathologies. This review addresses the discoveries of different HNF4α isoforms and the main functions of the P1 and P2 isoform subgroups. It also provides information on the most recent focus areas in research on the nature and function of proteins associated with each of the isoforms in some biological contexts.

Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor ß (RARß) Gene in Hepatocytes.

HNF4α, a member of the nuclear receptor superfamily, regulates the genes involved in lipid and glucose metabolism. The expression of the RARß gene in the liver of HNF4α knock-out mice was higher versus wildtype controls, whereas oppositely, RARß promoter activity was 50% reduced by the overexpression of HNF4α in HepG2 cells, and treatment with retinoic acid (RA), a major metabolite of vitamin A, increased RARß promoter activity 15-fold. The human RARß2 promoter contains two DR5 and one DR8 binding motifs, as RA response elements (RARE) proximal to the transcription start site. While DR5 RARE1 was previously reported to be responsive to RARs but not to other nuclear receptors, we show here that mutation in DR5 RARE2 suppresses the promoter response to HNF4α and RARα/RXRα. Mutational analysis of ligand-binding pocket amino acids shown to be critical for fatty acid (FA) binding indicated that RA may interfere with interactions of FA carboxylic acid headgroups with side chains of S190 and R235, and the aliphatic group with I355. These results could explain the partial suppression of HNF4α transcriptional activation toward gene promoters that lack RARE, including APOC3 and CYP2C9, while conversely, HNF4α may bind to RARE sequences in the promoter of the genes such as CYP26A1 and RARß, activating these genes in the presence of RA. Thus, RA could act as either an antagonist towards HNF4α in genes lacking RAREs, or as an agonist for RARE-containing genes. Overall, RA may interfere with the function of HNF4α and deregulate HNF4α targets genes, including the genes important for lipid and glucose metabolism.