DNA methylation patterns of transcription factor binding regions characterize their functional and evolutionary contexts.

BACKGROUND: DNA methylation is an important epigenetic modification which has numerous roles in modulating genome function. Its levels are spatially correlated across the genome, typically high in repressed regions but low in transcription factor (TF) binding sites and active regulatory regions. However, the mechanisms establishing genome-wide and TF binding site methylation patterns are still unclear. RESULTS: Here we use a comparative approach to investigate the association of DNA methylation to TF binding evolution in mammals. Specifically, we experimentally profile DNA methylation and combine this with published occupancy profiles of five distinct TFs (CTCF, CEBPA, HNF4A, ONECUT1, FOXA1) in the liver of five mammalian species (human, macaque, mouse, rat, dog). TF binding sites are lowly methylated, but they often also have intermediate methylation levels. Furthermore, biding sites are influenced by the methylation status of CpGs in their wider binding regions even when CpGs are absent from the core binding motif. Employing a classification and clustering approach, we extract distinct and species-conserved patterns of DNA methylation levels at TF binding regions. CEBPA, HNF4A, ONECUT1, and FOXA1 share the same methylation patterns, while CTCF's differ. These patterns characterize alternative functions and chromatin landscapes of TF-bound regions. Leveraging our phylogenetic framework, we find DNA methylation gain upon evolutionary loss of TF occupancy, indicating coordinated evolution. Furthermore, each methylation pattern has its own evolutionary trajectory reflecting its genomic contexts. CONCLUSIONS: Our epigenomic analyses indicate a role for DNA methylation in TF binding changes across species including that specific DNA methylation profiles characterize TF binding and are associated with their regulatory activity, chromatin contexts, and evolutionary trajectories.

Abrogating K458 acetylation enhances hepatocyte nuclear factor 4α (HNF4α)-induced differentiation therapy for hepatocellular carcinoma.

OBJECTIVES: In this study we aimed to assess the impact of acetylation of hepatocyte nuclear factor 4α (HNF4α) on lysine 458 on the differentiation therapy of hepatocellular carcinoma (HCC). METHODS: Periodic acid-Schiff (PAS) staining, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and senescence-associated ß-galactosidase (SA-ß-gal) activity analysis were performed to assess the differentiation of HCC cells. HNF4α protein was detected by western blot and immunohistochemistry (IHC). The effects of HNF4α-K458 acetylation on HCC malignancy were evaluated in HCC cell lines, a Huh-7 xenograft mouse model, and an orthotopic model. The differential expression genes in Huh-7 xenograft tumors were screened by RNA-sequencing analysis. RESULTS: K458R significantly enhanced the inhibitory effect of HNF4α on the malignancy of HCC cells, whereas K458Q reduced the inhibitory effects of HNF4α. Moreover, K458R promoted, while K458Q decreased, HNF4α-induced HCC cell differentiation. K458R stabilized HNF4α, while K458Q accelerated the degradation of HNF4α via the ubiquitin proteasome system. K458R also enhanced the ability of HNF4α to inhibit cell growth of HCC in the Huh-7 xenograft mouse model and the orthotopic model. RNA-sequencing analysis revealed that inhibiting K458 acetylation enhanced the transcriptional activity of HNF4α without altering the transcriptome induced by HNF4α in HCC. CONCLUSION: Our data revealed that inhibiting K458 acetylation of HNF4α might provide a more promising candidate for differential therapy of HCC.

Molecular mechanism of HNF-1A-mediated HNF4A gene regulation and promoter-driven HNF4A-MODY diabetes.

Monogenic diabetes is a gateway to precision medicine through molecular mechanistic insight. Hepatocyte nuclear factor 1A (HNF-1A) and HNF-4A are transcription factors that engage in crossregulatory gene transcription networks to maintain glucose-stimulated insulin secretion in pancreatic ß cells. Variants in the HNF1A and HNF4A genes are associated with maturity-onset diabetes of the young (MODY). Here, we explored 4 variants in the P2-HNF4A promoter region: 3 in the HNF-1A binding site and 1 close to the site, which were identified in 63 individuals from 21 families of different MODY disease registries across Europe. Our goal was to study the disease causality for these variants and to investigate diabetes mechanisms on the molecular level. We solved a crystal structure of HNF-1A bound to the P2-HNF4A promoter and established a set of techniques to probe HNF-1A binding and transcriptional activity toward different promoter variants. We used isothermal titration calorimetry, biolayer interferometry, x-ray crystallography, and transactivation assays, which revealed changes in HNF-1A binding or transcriptional activities for all 4 P2-HNF4A variants. Our results suggest distinct disease mechanisms of the promoter variants, which can be correlated with clinical phenotype, such as age of diagnosis of diabetes, and be important tools for clinical utility in precision medicine.

HNF4A and HNF1A exhibit tissue specific target gene regulation in pancreatic beta cells and hepatocytes.

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3, HAAO, HNF1A, MAP3K11) and previously unidentified (ABCD3, CDKN2AIP, USH1C, VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1, GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation.

FGF21 attenuates salt-sensitive hypertension via regulating HNF4α/ACE2 axis in the hypothalamic paraventricular nucleus of mice.

BACKGROUND: Fibroblast growth factor 21 (FGF21) has a protective effect against cardiovascular disease. However, the role of FGF21 in hypertension remains elusive. METHODS: Ten-week-old male C57BL/6 mice were randomly divided into normal-salt (NS) group, NS+FGF21 group, deoxycorticosterone acetate-salt (DOCA) group and DOCA+FGF21 group. The mice in NS group underwent uninephrectomy without receiving DOCA and 1% NaCl and the mice in DOCA group were subjected to uninephrectomy and DOCA-salt (DOCA and 1% NaCl) treatment for 6 weeks. At the same time, the mice were infused with vehicle (artificial cerebrospinal fluid, aCSF) or FGF21 (1 mg/kg) into the bilateral paraventricular nucleus (PVN) of mice. RESULTS: Here, we showed that FGF21 treatment lowered DOCA salt-induced inflammation and oxidative stress in the PVN, which reduced sympathetic nerve activity and hypertension. Mechanistically, FGF21 treatment decreased the expression of HNF4α and inhibited the binding activity of HNF4α to the promoter region of ACE2 in the PVN of DOCA salt-treated mice, which further up-regulated ACE2/Ang (1-7) signals in the PVN. In addition, ACE2 deficiency abolished the protective effect of FGF21 in DOCA salt-treated mice, suggesting that FGF21-mediated antihypertensive effect was dependent on ACE2. CONCLUSIONS: The results demonstrate that FGF21 protects against salt-sensitive hypertension via regulating HNF4α/ACE2/Ang (1-7) axis in the PVN of DOCA salt-treated mice via multi-organ crosstalk between liver, brain and blood vessels.

Molecular subtypes of neuroendocrine carcinomas: A cross-tissue classification framework based on five transcriptional regulators.

Neuroendocrine carcinomas (NECs) are extremely lethal malignancies that can arise at almost any anatomic site. Characterization of NECs is hindered by their rarity and significant inter- and intra-tissue heterogeneity. Herein, through an integrative analysis of over 1,000 NECs originating from 31 various tissues, we reveal their tissue-independent convergence and further unveil molecular divergence driven by distinct transcriptional regulators. Pan-tissue NECs are therefore categorized into five intrinsic subtypes defined by ASCL1, NEUROD1, HNF4A, POU2F3, and YAP1. A comprehensive portrait of these subtypes is depicted, highlighting subtype-specific transcriptional programs, genomic alterations, evolution trajectories, therapeutic vulnerabilities, and clinicopathological presentations. Notably, the newly discovered HNF4A-dominated subtype-H exhibits a gastrointestinal-like signature, wild-type RB1, unique neuroendocrine differentiation, poor chemotherapeutic response, and prevalent large-cell morphology. The proposal of uniform classification paradigm illuminates transcriptional basis of NEC heterogeneity and bridges the gap across different lineages and cytomorphological variants, in which context-dependent prevalence of subtypes underlies their phenotypic disparities.

Tripartite motif-containing protein 21 is involved in IFN-γ-induced suppression of hepatitis B virus by regulating hepatocyte nuclear factors.

The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.

Downregulation of HNF4A enables transcriptomic reprogramming during the hepatic acute-phase response.

The hepatic acute-phase response is characterized by a massive upregulation of serum proteins, such as haptoglobin and serum amyloid A, at the expense of liver homeostatic functions. Although the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) has a well-established role in safeguarding liver function and its cistrome spans around 50% of liver-specific genes, its role in the acute-phase response has received little attention so far. We demonstrate that HNF4A binds to and represses acute-phase genes under basal conditions. The reprogramming of hepatic transcription during inflammation necessitates loss of HNF4A function to allow expression of acute-phase genes while liver homeostatic genes are repressed. In a pre-clinical liver organoid model overexpression of HNF4A maintained liver functionality in spite of inflammation-induced cell damage. Conversely, HNF4A overexpression potently impaired the acute-phase response by retaining chromatin at regulatory regions of acute-phase genes inaccessible to transcription. Taken together, our data extend the understanding of dual HNF4A action as transcriptional activator and repressor, establishing HNF4A as gatekeeper for the hepatic acute-phase response.

Krüppel-like factor 10 protects against metabolic dysfunction-associated steatohepatitis by regulating HNF4α-mediated metabolic pathways.

BACKGROUND: Krüppel-like factor 10 (KLF10), a zinc finger transcription factor, plays a pivotal role in modulating TGF-ß-mediated cellular processes such as growth, apoptosis, and differentiation. Recent studies have implicated KLF10 in regulating lipid metabolism and glucose homeostasis. This study aimed to elucidate the precise role of hepatic KLF10 in developing metabolic dysfunction-associated steatohepatitis (MASH) in diet-induced obese mice. METHODS: We investigated hepatic KLF10 expression under metabolic stress and the effects of overexpression or ablation of hepatic KLF10 on MASH development and lipidemia. We also determined whether hepatocyte nuclear factor 4α (HNF4α) mediated the metabolic effects of KLF10. RESULTS: Hepatic KLF10 was downregulated in MASH patients and genetically or diet-induced obese mice. AAV8-mediated overexpression of KLF10 in hepatocytes prevented Western diet-induced hypercholesterolemia and steatohepatitis, whereas inactivation of hepatocyte KLF10 aggravated Western diet-induced steatohepatitis. Mechanistically, KLF10 reduced hepatic triglyceride and free fatty acid levels by inducing lipolysis and fatty acid oxidation and inhibiting lipogenesis, and reducing hepatic cholesterol levels by promoting bile acid synthesis. KLF10 highly induced HNF4α expression by directly binding to its promoter. The beneficial effect of KLF10 on MASH development was abolished in mice lacking hepatocyte HNF4α. In addition, the inactivation of KLF10 in hepatic stellate cells exacerbated Western diet-induced liver fibrosis by activating the TGF-ß/SMAD2/3 pathway. CONCLUSIONS: Our data collectively suggest that the transcription factor KLF10 plays a hepatoprotective role in MASH development by inducing HNF4α. Targeting hepatic KLF10 may offer a promising strategy for treating MASH.

Role of HNF4α-cMyc Interaction in CDE Diet-Induced Liver Injury and Regeneration.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a nuclear factor essential for liver function that regulates the expression of cMyc and plays an important role during liver regeneration. This study investigated the role of the HNF4α-cMyc interaction in regulating liver injury and regeneration using the choline-deficient and ethionine-supplemented (CDE) diet model. Wild-type (WT), hepatocyte-specific HNF4α-knockout (KO), cMyc-KO, and HNF4α-cMyc double KO (DKO) mice were fed a CDE diet for 1 week to induce subacute liver injury. To study regeneration, normal chow diet was fed for 1 week after CDE diet. WT mice exhibited significant liver injury and decreased HNF4α mRNA and protein expression after CDE diet. HNF4α deletion resulted in significantly higher injury with increased inflammation, fibrosis, proliferation, and hepatic progenitor cell activation compared with WT mice after CDE diet but indicated similar recovery. Deletion of cMyc lowered liver injury with activation of inflammatory genes compared with WT and HNF4α-KO mice after CDE diet. DKO mice had a phenotype comparable to that of the HNF4α-KO mice after CDE diet and a complete recovery. DKO mice exhibited a significant increase in hepatic progenitor cell markers both after injury and recovery phase. Taken together, these data show that HNF4α protects against inflammatory and fibrotic changes after CDE diet-induced injury, which is driven by cMyc.

Stress-induced mucin 13 reductions drive intestinal microbiome shifts and despair behaviors.

Depression is a prevalent psychological condition with limited treatment options. While its etiology is multifactorial, both chronic stress and changes in microbiome composition are associated with disease pathology. Stress is known to induce microbiome dysbiosis, defined here as a change in microbial composition associated with a pathological condition. This state of dysbiosis is known to feedback on depressive symptoms. While studies have demonstrated that targeted restoration of the microbiome can alleviate depressive-like symptoms in mice, translating these findings to human patients has proven challenging due to the complexity of the human microbiome. As such, there is an urgent need to identify factors upstream of microbial dysbiosis. Here we investigate the role of mucin 13 as an upstream mediator of microbiome composition changes in the context of stress. Using a model of chronic stress, we show that the glycocalyx protein, mucin 13, is selectively reduced after psychological stress exposure. We further demonstrate that the reduction of Muc13 is mediated by the Hnf4 transcription factor family. Finally, we determine that deleting Muc13 is sufficient to drive microbiome shifts and despair behaviors. These findings shed light on the mechanisms behind stress-induced microbial changes and reveal a novel regulator of mucin 13 expression.

A MTA2-SATB2 chromatin complex restrains colonic plasticity toward small intestine by retaining HNF4A at colonic chromatin.

Plasticity among cell lineages is a fundamental, but poorly understood, property of regenerative tissues. In the gut tube, the small intestine absorbs nutrients, whereas the colon absorbs electrolytes. In a striking display of inherent plasticity, adult colonic mucosa lacking the chromatin factor SATB2 is converted to small intestine. Using proteomics and CRISPR-Cas9 screening, we identify MTA2 as a crucial component of the molecular machinery that, together with SATB2, restrains colonic plasticity. MTA2 loss in the adult mouse colon activated lipid absorptive genes and functional lipid uptake. Mechanistically, MTA2 co-occupies DNA with HNF4A, an activating pan-intestinal transcription factor (TF), on colonic chromatin. MTA2 loss leads to HNF4A release from colonic chromatin, and accumulation on small intestinal chromatin. SATB2 similarly restrains colonic plasticity through an HNF4A-dependent mechanism. Our study provides a generalizable model of lineage plasticity in which broadly-expressed TFs are retained on tissue-specific enhancers to maintain cell identity and prevent activation of alternative lineages, and their release unleashes plasticity.

Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform.

OBJECTIVES: Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD. METHODS: We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days. RESULTS: Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results. CONCLUSIONS: Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.

Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and ß-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.

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.

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.

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.

Microplastics cause hepatotoxicity in diabetic mice by disrupting glucolipid metabolism via PP2A/AMPK/HNF4A and promoting fibrosis via the Wnt/ß-catenin pathway.

In recent years, microplastics (MPs) have gained significant attention as a persistent environmental pollutant resulting from the decomposition of plastics, leading to their accumulation in the human body. The liver, particularly of individuals with type 2 diabetes mellitus (T2DM), is known to be more susceptible to the adverse effects of environmental pollutants. Therefore, to investigate the potential impact of MPs on the liver of diabetic mice and elucidate the underlying toxicological mechanisms, we exposed db/db mice to 0.5 µm MPs for 3 months. Our results revealed that MPs exposure resulted in several harmful effects, including decreased body weight, disruption of liver structure and function, elevated blood glucose levels, impaired glucose tolerance, and increased glycogen accumulation in the hepatic tissue of the mice. Furthermore, MPs exposure was found to promote hepatic gluconeogenesis by perturbing the PP2A/AMPK/HNF4A signaling pathway. In addition, MPs disrupt redox balance, leading to oxidative damage in the liver. This exposure also disrupted hepatic lipid metabolism, stimulating lipid synthesis while inhibiting catabolism, ultimately resulting in the development of fatty liver. Moreover, MPs were found to induce liver fibrosis by activating the Wnt/ß-catenin signaling pathway. Furthermore, MPs influenced adaptive thermogenesis in brown fat by modulating the expression of uncoupling protein 1 (UCP1) and genes associated with mitochondrial oxidative respiration thermogenesis in brown fat. In conclusion, our study demonstrates that MPs induce oxidative damage in the liver, disturb glucose and lipid metabolism, promote hepatic fibrosis, and influence adaptive thermogenesis in brown fat in diabetic mice. These findings underscore the potential adverse effects of MPs on liver health in individuals with T2DM and highlight the importance of further research in this area.

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.