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HNF41 Activates Human p21 Promoter Activity Transient transfection assay of HEK293 cells in 12-well plates with the indicated human p21 promoter constructs (0.5 g) driving luciferase (Luc) expression. Shown is fold induction of samples with pMT7.HNF41 (0.5 g) vs. pMT7 empty vector normalized to CMV.-gal (0.2 g). Results are means SD of triplicate samples from one representative experiment out of three independent experiments performed. *, Fold induction is significant at P 0.05. Previously characterized p53 and proximal Sp1 sites and TATA box (10) are shown along with Sp1 sites in the distal region and two HNF4 binding sites predicted by TRANSFAC and HNF4 Motif finder (see supplemental Fig. S3 for the full promoter sequence).
Source publication
The dichotomy between cellular differentiation and proliferation is a fundamental aspect of both normal development and tumor progression; however, the molecular basis of this opposition is not well understood. To address this issue, we investigated the mechanism by which the nuclear receptor hepatocyte nuclear factor 4alpha1 (HNF4alpha1) regulates...
Contexts in source publication
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... determine which portion of the p21 promoter is required for HNF41 activation, transient cotransfec- tion assays with nine luciferase reporter constructs containing different portions of the human p21 pro- moter were performed in HEK293 cells (Fig. 2). The results show that HNF41 activated a 2.4-kb fragment of the promoter (2.4 kb construct) roughly 5-fold (Fig. 2). Smaller fragments of the promoter (1.6 kb, 1.3 kb, 100 bp, and 93 bp) were also activated by HNF41, although to a lesser degree (2-to 3-fold). Similar results were obtained using HepG2 cells (supplemental Fig. S2 ...
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... which portion of the p21 promoter is required for HNF41 activation, transient cotransfec- tion assays with nine luciferase reporter constructs containing different portions of the human p21 pro- moter were performed in HEK293 cells (Fig. 2). The results show that HNF41 activated a 2.4-kb fragment of the promoter (2.4 kb construct) roughly 5-fold (Fig. 2). Smaller fragments of the promoter (1.6 kb, 1.3 kb, 100 bp, and 93 bp) were also activated by HNF41, although to a lesser degree (2-to 3-fold). Similar results were obtained using HepG2 cells (supplemental Fig. S2 ...
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... in HEK293 cells (Fig. 2). The results show that HNF41 activated a 2.4-kb fragment of the promoter (2.4 kb construct) roughly 5-fold (Fig. 2). Smaller fragments of the promoter (1.6 kb, 1.3 kb, 100 bp, and 93 bp) were also activated by HNF41, although to a lesser degree (2-to 3-fold). Similar results were obtained using HepG2 cells (supplemental Fig. S2 ...
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... to the p21 promoter via Sp1, we hypothesized that c-Myc might be able to antagonize HNF41 activation by competing with HNF41 for interaction with Sp1. To test this hypoth- esis, we first overexpressed c-Myc in HepG2 cells where endogenous HNF41 is expressed. We ob- served a significant suppression of the full-length p21 promoter (2.4 kb) activity (Fig. 4Aa, lane 2 (lanes 5-8 vs. lane 3). To determine whether c-Myc could antagonize the ability of HNF41 to activate other promoters, we performed the competition experiment using the promoter of ApoC3 (APOC3), a classical HNF41 target gene (61). Just as with the p21 promoter, c-Myc inhibited the ability of HNF41 to activate the ApoC3 promoter (Fig. ...
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... (Fig. 5A, bottom panel), demonstrated that these two proteins do indeed interact in vivo. We also performed in vitro glutathione-S-transferase (GST) pull-down assays and verified that the interaction between c-Myc and HNF41 is a direct one and showed that c-Myc interacts with multiple regions of HNF41, including the DNA- binding domain (DBD) (Fig. 5B, lane 2-4). Interestingly, c-Myc interacted well with the LBD/F, which contains the ligand-binding domain (LBD) and the 88-amino-acid C- terminal extension termed the F domain (lane 6) as well as the isolated F domain (lane 7). In contrast, it did not interact with the isolated LBD (lane 5). Because the ...
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... next verified that in the cell lines we were using, HCT116 wt and HepG2, ectopically expressed c-Myc was associated with the Sp1-binding regions of the p21 promoter (Fig. 5, C and D, primer sets 1, 2 and 7, 8). Whereas c-Myc has been shown previously to bind the proximal Sp1 sites (17), this is the first report of its binding to the distal sites. Interestingly, in HepG2 cells, which contain endogenous HNF41, we also ob- served c-Myc binding to the region containing the two HNF4 binding sites (Fig. 5D, primer sets 5 and 6). This binding was ...
Citations
... This is interesting when compared to P1-HNF4a, which does not oscillate as dramatically as the P2 isoform throughout the day, remaining at relatively stable levels in both wildtype and CLOCK knockout mice (75). PBMs revealed that P2-HNF4a and P1-HNF4a have essentially identical DNA binding specificity even in the context of liver nuclear extracts; P1-HNF4a also seems to have a preference for GC-rich motifs that bind SP1, consistent with interactions noted previously between these two transcription factors (95,112). ChIPseq analysis also revealed very similar genome-wide binding of the P1 and P2 isoforms, despite a dysregulation of hundreds of genes, although there were some notable differences in chromatin binding in the Apoa1 -Apoc3 -Apoa4 locus that correlated with levels of RNA expression (75) ( Figure 5). ...
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.
... Immunofluorescence revealed an inverse correlation between proliferating cells and differentiated cells in the mutant cultures (HNF4a/FABP1-positive cells are Ki67negative and vice versa), suggesting that proliferation and proper HEP differentiation are mutually exclusive ( Figure 4B-D). HNF4a is a master regulator of liver development, in which it regulates target gene expression in a manner antagonistic to MYC. 26 Our data suggest that the inability of mutant cells to suppress MYC underlies their failure to activate HNF4a and undergo proper HEP differentiation. ...
Background & aims:
Dyskeratosis congenita (DC) is a telomere maintenance disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear.
Methods:
We used isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele to model DC liver pathologies. We differentiated these iPS cells into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships.
Results:
Directed differentiation of iPS cells into hepatocytes and stellate cells, and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, with DC hepatocytes becoming hyperplastic, and also eliciting a pathogenic hyperplastic, pro-inflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant hepatocytes and hepatostellate organoids could be rescued via suppression of AKT activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation.
Conclusions:
Isogenic iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.
... A biochemical study conducted in cancer cell lines suggested that competitive interaction of HNF4α, SP1 and c-myc may take place at gene promoters, depend on c-myc levels and skew cell function [32]. To define the outcome of TF interaction on immune cell activation, we performed in vitro experiments on human PBMC with compounds targeting HNF4α, SP1 and c-myc. ...
Hepatocyte nuclear factor 4 α (HNF4α), a transcription factor (TF) essential for embryonic development, has been recently shown to regulate the expression of inflammatory genes. To characterize HNF4a function in immunity, we measured the effect of HNF4α antagonists on immune cell responses in vitro and in vivo. HNF4α blockade reduced immune activation in vitro and disease severity in the experimental model of multiple sclerosis (MS). Network biology studies of human immune transcriptomes unraveled HNF4α together with SP1 and c-myc as master TF regulating differential expression at all MS stages. TF expression was boosted by immune cell activation, regulated by environmental MS risk factors and higher in MS immune cells compared to controls. Administration of compounds targeting TF expression or function demonstrated non-synergic, interdependent transcriptional control of CNS autoimmunity in vitro and in vivo. Collectively, we identified a coregulatory transcriptional network sustaining neuroinflammation and representing an attractive therapeutic target for MS and other inflammatory disorders.
... Interestingly, the TWEAK ligand was strongly induced in both the HET (↑2.8 fold) and KO (↑3.3 fold) mice, whereas the Fn14 receptor, which is not expressed in normal hepatocytes, was only induced in the KO mice (↑4.0 fold). Additionally, the CDK inhibitor p21, an HNF4α-target gene [46], tended to be downregulated in the HET mice but strongly induced 3.0 fold in the KO mice (Fig. 5A). Taken together, these data clearly demonstrate a critical gene-dosage-dependent role of HNF4α in maintaining hepatocyte differentiation and suppressing hepatocyte proliferation, cholangiocyte transdifferentiation, and liver fibrosis during HFHS intake. ...
Background
Hepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study aimed to elucidate the role of down-regulation of HNF4α and GR in fatty liver and hyperlipidemia.
Methods
Adult mice with liver-specific heterozygote (HET) and knockout (KO) of HNF4α or GR were fed a high-fat-high-sugar diet (HFHS) for 15 days. Alterations in hepatic and circulating lipids were determined with analytical kits, and changes in hepatic mRNA and protein expression in these mice were quantified by real-time PCR and Western blotting. Serum and hepatic levels of bile acids were quantified by LC-MS/MS. The roles of HNF4α and GR in regulating hepatic gene expression were determined using luciferase reporter assays.
Results
Compared to HFHS-fed wildtype mice, HNF4α HET mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4α KO mice had fatty liver but mild hypolipidemia, down-regulation of lipid-efflux genes, and induction of genes for uptake, synthesis, and storage of lipids. Serum levels of chenodeoxycholic acid and deoxycholic acid tended to be decreased in the HNF4α HET mice but dramatically increased in the HNF4α KO mice, which was associated with marked down-regulation of cytochrome P450 7a1, the rate-limiting enzyme for bile acid synthesis. Hepatic mRNA and protein expression of sterol-regulatory-element-binding protein-1 (SREBP-1), a master lipogenic regulator, was induced in HFHS-fed HNF4α HET mice. In reporter assays, HNF4α cooperated with the corepressor small heterodimer partner to potently inhibit the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Hepatic nuclear GR proteins tended to be decreased in the HNF4α KO mice. HFHS-fed mice with liver-specific KO of GR had increased hepatic lipids and induction of SREBP-1C and PPARγ, which was associated with a marked decrease in hepatic levels of HNF4α proteins in these mice. In reporter assays, GR and HNF4α synergistically/additively induced lipid catabolic genes.
Conclusions
induction of lipid catabolic genes and suppression of lipogenic genes by HNF4α and GR may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.
Graphical abstract
... HNF4a may also interact with the co-activator PPARg coactivator 1a (PGC1a) to induce gluconeogenesis during fasting [97,109] or steroid receptor coactivators (SRC-1, -3) to enhance the transcriptional activity of HNF4a, [110,111] whereas interaction with the co-repressor Hes family basic helix-loop-helix transcription factor 6 [112] represses HNF4a transcription activity. HNF4a may also physically interact with FXR, [113,114] p53, [115] sterol regulatory-binding protein element 1 (SREBP1), [116] Smad3/Smad4, [117,118] specificity protein 1 (SP1), [119] cyclin D1, [120] or small heterodimer partner (SHP) [121] to regulate HNF4a activity. ...
Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common chronic liver disease worldwide. It refers to a range of liver conditions affecting people who drink little or no alcohol. NAFLD comprises non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH), the more aggressive form of NAFLD. NASH is featured by steatosis, lobular inflammation, hepatocyte injury, and various degrees of fibrosis. Although much progress has been made over the past decades, the pathogenic mechanism of NAFLD remains to be fully elucidated. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear hormone receptor that is highly expressed in hepatocytes. Hepatic HNF4α expression is markedly reduced in NAFLD patients and mouse models of NASH. HNF4α has been shown to regulate bile acid, lipid, glucose, and drug metabolism. In this review, we summarize the recent advances in the understanding of the pathogenesis of NAFLD with a focus on the regulation of HNF4α and the role of hepatic HNF4α in NAFLD. Several lines of evidence have shown that hepatic HNF4α plays a key role in the initiation and progression of NAFLD. Recent data suggest that hepatic HNF4α may be a promising target for treatment of NAFLD.
... Previous studies have shown that HNF4a levels have reciprocal effects on the expression of the oncoprotein c-Myc. 12,13 Western blot analysis showed induction in c-MYC expression in OGT-KO livers at 28 days after PHX (Figure 11, A). Interestingly, IHC analysis revealed marked cytoplasmic redistribution of HNF4a in OGT-KO mice (Figure 11, B). qPCR analysis of HNF4a target genes indicated the loss of HNF4a function with the decrease of positively regulated genes (Apob, Apoa2, Cyp2c37, Ugt2b1, Dio1, and Ces3) (Figure 11, C) and the increase of negatively regulated genes (Akr1b7 and Ect2) (Figure 11, D). ...
... 22,23 Recent studies from our laboratory have shown that HNF4a, the master regulator of hepatic differentiation, 24,25 is critical for redifferentiation of hepatocytes after proliferation and plays a critical role in termination of liver regeneration. [12][13][14] Previous studies have shown that defective termination of LR will result in significant hepatomegaly and death due to decline in liver function. We hypothesize that in cases where animals do not die due to acute liver failure, defective termination of liver regeneration will lead to hepatic dysplasia that can progress to liver cancer. ...
Background and Aims
The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here we report that O-GlcNAcylation, an intracellular post-translational modification (PTM) regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration (LR) following partial hepatectomy (PHX).
Methods
We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively.
Results
OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-Seq studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and IHC analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes.
Conclusions
These studies show that O-GlcNAcylation plays a critical role in the termination of LR via regulation of HNF4α in hepatocytes.
... HNF4α is a master regulator of liver development where it regulates target gene expression in a manner antagonistic to MYC 27 . Our data suggest that the inability of mutant cells to suppress MYC expression underlies their failure to activate HNF4α and undergo proper hepatocyte differentiation. ...
Patients with dyskeratosis congenita (DC) and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. We model DC liver pathologies using isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele. Differentiation of these iPS cells into hepatocytes or hepatic stellate cells followed by generation of genotype-admixed hepatostellate organoids revealed a dominant phenotype in the parenchyma, with DC hepatocytes eliciting a pathogenic hyperplastic response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes could be rescued via suppression of AKT activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. Thus, isogenic iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.
... A recent study from our group demonstrated that HNF4α expression is decreased in prostate cancer cells, and that ectopic overexpression of HNF4α could significantly inhibit prostate cancer cell proliferation, induce cell-cycle arrest at G2/M phase and trigger the cellular senescence via activation of p21 signal pathway in a p53-independent manner and direct transactivation of cyclin-dependent kinase inhibitor 1, suggesting that HNF4α might have a tumor suppressor role in prostate cancer cells (56). Hwang and Sladek (57) reported that HNF4α competes with the oncoprotein c-Myc for targeting the p21 promoter in order to activate its expression, which could significantly inhibit HCC and colorectal carcinoma cell proliferation. These findings confirmed the critical role of p21 protein in HNF4α-mediated tumor growth inhibition. ...
Hepatocyte nuclear receptor 4 α (HNF4α) is known to be a master transcription regulator of gene expression in multiple biological processes, particularly in liver development and liver function. To date, the function of HNF4α in human cancers has been widely investigated; however, the critical roles of HNF4α in tumorigenesis remain unclear. Numerous controversies exist, even in studies from different research groups but on the same type of cancer. In the present review, the critical roles of HNF4α in tumorigenesis will be summarized and discussed. Furthermore, HNF4α expression profile and alterations will be examined by pan-cancer analysis through bioinformatics, in order to provide a better understanding of the functional roles of this gene in human cancers.
... A similar, albeit less pronounced, preference was noted in the COS-7 extracts ( Figure 4D, bottom left). Consistently, HNF4α1 has been found to interact with SP1 both on and off chromatin, an interaction that involves the N-terminal domain of HNF4α1 (Hwang-Verslues and Sladek, 2008;Kardassis et al., 2002;Takahashi et al., 2002). ...
Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2). 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 expressed at ZT9 and ZT21 in the normal adult liver and orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions. We demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in females. Exon swap mice expressing only P2- HNF4α exhibit subtle differences in circadian gene regulation and disruption of the clock increases expression of P2-HNF4α. Taken together, we propose that the highly conserved two-promoter structure of the Hnfa gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.
... Kinetic analyses revealed that EMT exhibited three consecutive waves of gene expression, and HNF4A was found to particularly regulate the intermediate so-called partial EMT state [163]. Additional mechanisms contributing to HNF4A-related HCC progression include increased cell proliferation [67,164] and reduced apoptosis due to decreased expression of Apoptosis signal-regulated kinase 1 (ASK1/MAP3K5), an apoptosis-promoting tumor suppressor gene which was recently identified as an HNF4A target in hepatocytes [165]. Recent work has revealed that HNF4A-mediated repression of hepatocyte proliferation was based, at least in part, on its inhibition of cyclin D1 [65]. ...
Hepatocyte Nuclear Factor 4 (HNF4) is a transcription factor (TF) belonging to the nuclear receptor family whose expression and activities are restricted to a limited number of organs including the liver and gastrointestinal tract. In this review, we present robust evidence pointing to HNF4 as a master regulator of cellular differentiation during development and a safekeeper of acquired cell identity in adult organs. Importantly, we discuss that transient loss of HNF4 may represent a protective mechanism upon acute organ injury, while prolonged impairment of HNF4 activities could contribute to organ dysfunction. In this context, we describe in detail mechanisms involved in the pathophysiological control of cell identity by HNF4, including how HNF4 works as part of cell-specific TF networks and how its expression/activities are disrupted in injured organs.
