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Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

High fat diets (HFDs) have been linked to several diseases including obesity, diabetes, fatty liver, inflammatory bowel disease (IBD) and colon cancer. In this study, we examined the impact on intestinal gene expression of three isocaloric HFDs that differed only in their fatty acid composition-coconut oil (saturated fats), conventional soybean oil (polyunsaturated fats) and a genetically modified soybean oil (monounsaturated fats). Four functionally distinct segments of the mouse intestinal tract were analyzed using RNA-seq-duodenum, jejunum, terminal ileum and proximal colon. We found considerable dysregulation of genes in multiple tissues with the different diets, including those encoding nuclear receptors and genes involved in xenobiotic and drug metabolism, epithelial barrier function, IBD and colon cancer as well as genes associated with the microbiome and COVID-19. Network analysis shows that genes involved in metabolism tend to be upregulated by the HFDs while genes related to the immune system are downregulated; neurotransmitter signaling was also dysregulated by the HFDs. Genomic sequencing also revealed a microbiome altered by the HFDs. This study highlights the potential impact of different HFDs on gut health with implications for the organism as a whole and will serve as a reference for gene expression along the length of the intestines., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Deans, Deol, Titova, Radi, Vuong, Evans, Pan, Fahrmann, Yang, Hammock, Fiehn, Fekry, Eckel-Mahan and Sladek.)

Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vemuri, Radi, Sladek and Verzi.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Radi, Vemuri, Martinez-Lomeli and Sladek.)

Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro . These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, and the most common primary liver malignancy to present in the clinic. With the exception of liver transplant, treatment options for advanced HCC are limited, but improved tumor stratification could open the door to new treatment options. Previously, we demonstrated that the circadian regulator Aryl Hydrocarbon-Like Receptor Like 1 (ARNTL, or Bmal1) and the liver-enriched nuclear factor 4 alpha (HNF4α) are robustly co-expressed in healthy liver but incompatible in the context of HCC. Faulty circadian expression of HNF4α- either by isoform switching, or loss of expression- results in an increased risk for HCC, while BMAL1 gain-of-function in HNF4α-positive HCC results in apoptosis and tumor regression. We hypothesize that the transcriptional programs of HNF4α and BMAL1 are antagonistic in liver disease and HCC. Here, we study this antagonism by generating a mouse model with inducible loss of hepatic HNF4α and BMAL1 expression. The results reveal that simultaneous loss of HNF4α and BMAL1 is protective against fatty liver and HCC in carcinogen-induced liver injury and in the "STAM" model of liver disease. Furthermore, our results suggest that targeting Bmal1 expression in the absence of HNF4α inhibits HCC growth and progression. Specifically, pharmacological suppression of Bmal1 in HNF4α-deficient, BMAL1-positive HCC with REV-ERB agonist SR9009 impairs tumor cell proliferation and migration in a REV-ERB-dependent manner, while having no effect on healthy hepatocytes. Collectively, our results suggest that stratification of HCC based on HNF4α and BMAL1 expression may provide a new perspective on HCC properties and potential targeted therapeutics., (© 2022 Federation of American Societies for Experimental Biology.)

Chronic elevated free fatty (FFA) levels are linked to metabolic disorders and tumorigenesis. However, the molecular mechanism by which FFAs induce cancer remains poorly understood. Here, we show that the tumor suppressor PTEN protein levels were decreased in high fat diet (HFD) fed mice. As palmitic acid (PA, C16:0) showed a significant increase in the HFD fed mice, we further investigated its role in PTEN down regulation. Our studies revealed that exposure of cells to high doses of PA induced mTOR/S6K-mediated phosphorylation of PTEN at T366. The phosphorylation subsequently enhanced the interaction of PTEN with the E3 ubiquitin ligase WW domain-containing protein 2 (WWP2), which promoted polyubiquitination of PTEN and protein degradation. Consistent with PTEN degradation, exposure of cells to increased concentrations of PA also promoted PTEN-mediated AKT activation and cell proliferation. Significantly, a higher level of S6K activation, PTEN T366 phosphorylation, and AKT activation were also observed in the livers of the HFD fed mice. These results provide a molecular mechanism by which a HFD and elevated PA regulate cell proliferation through inactivation of tumor suppressor PTEN., (Published by Elsevier B.V.)

Soybean oil consumption has increased greatly in the past half-century and is linked to obesity and diabetes. To test the hypothesis that soybean oil diet alters hypothalamic gene expression in conjunction with metabolic phenotype, we performed RNA sequencing analysis using male mice fed isocaloric, high-fat diets based on conventional soybean oil (high in linoleic acid, LA), a genetically modified, low-LA soybean oil (Plenish), and coconut oil (high in saturated fat, containing no LA). The 2 soybean oil diets had similar but nonidentical effects on the hypothalamic transcriptome, whereas the coconut oil diet had a negligible effect compared to a low-fat control diet. Dysregulated genes were associated with inflammation, neuroendocrine, neurochemical, and insulin signaling. Oxt was the only gene with metabolic, inflammation, and neurological relevance upregulated by both soybean oil diets compared to both control diets. Oxytocin immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus was reduced, whereas plasma oxytocin and hypothalamic Oxt were increased. These central and peripheral effects of soybean oil diets were correlated with glucose intolerance but not body weight. Alterations in hypothalamic Oxt and plasma oxytocin were not observed in the coconut oil diet enriched in stigmasterol, a phytosterol found in soybean oil. We postulate that neither stigmasterol nor LA is responsible for effects of soybean oil diets on oxytocin and that Oxt messenger RNA levels could be associated with the diabetic state. Given the ubiquitous presence of soybean oil in the American diet, its observed effects on hypothalamic gene expression could have important public health ramifications., (© Endocrine Society 2020.)

The incidence of hepatocellular carcinoma (HCC) is on the rise worldwide. Although the incidence of HCC in males is considerably higher than in females, the projected rates of HCC incidence are increasing for both sexes. A recently appreciated risk factor for HCC is the growing problem of nonalcoholic fatty liver disease, which is usually associated with obesity and the metabolic syndrome. In this study, we showed that under conditions of fatty liver, female mice were more likely to develop HCC than expected from previous models. Using an inducible knockout model of the tumor-suppressive isoform of hepatocyte nuclear factor 4 alpha ("P1-HNF4α") in the liver in combination with prolonged high fat (HF) diet, we found that HCC developed equally in male and female mice as early as 38 weeks of age. Similar sex-independent HCC occurred in the "STAM" model of mice, in which severe hyperglycemia and HF feeding results in rapid hepatic lipid deposition, fibrosis, and ultimately HCC. In both sexes, reduced P1-HNF4α activity, which also occurs under chronic HF diet feeding, increased hepatic lipid deposition and produced a greatly augmented circadian rhythm in IL6, a factor previously linked with higher HCC incidence in males. Loss of HNF4α combined with HF feeding induced epithelial-mesenchymal transition in an IL6-dependent manner. Collectively, these data provide a mechanism-based working hypothesis that could explain the rising incidence of aggressive HCC. SIGNIFICANCE: This study provides a mechanism for the growing incidence of hepatocellular carcinoma in both men and women, which is linked to nonalcoholic fatty liver disease., (©2019 American Association for Cancer Research.)

Dimethylsulfoxide (DMSO) is widely used as a solvent and cryopreservative in laboratories and considered to have many beneficial health effects in humans. Oxylipins are a class of biologically active metabolites of polyunsaturated fatty acids (PUFAs) that have been linked to a number of diseases. In this study, we investigated the effect of DMSO on oxylipin levels in mouse liver. Liver tissue from male mice (C57Bl6/N) that were either untreated or injected with 1% DMSO at 18 weeks of age was analyzed for oxylipin levels using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). A decrease in oxylipin diols from linoleic acid (LA, C18:2n6), alpha-linolenic acid (ALA, C18:3n3) and docosahexeanoic acid (DHA, C22:6n3) was observed 2 h after injection with DMSO. In contrast, DMSO had no effect on the epoxide precursors or other oxylipins including those derived from arachidonic acid (C20:4n6) or eicosapentaenoic acid (EPA, C20:5n3). It also did not significantly affect the diol:epoxide ratio, suggesting a pathway distinct from, and potentially complementary to, soluble epoxide hydrolase inhibitors (sEHI). Since oxylipins have been associated with a wide array of pathological conditions, from arthritis pain to obesity, our results suggest one potential mechanism underlying the apparent beneficial health effects of DMSO. They also indicate that caution should be used in the interpretation of results using DMSO as a vehicle in animal experiments.

Nuclear receptors are a superfamily of transcription factors restricted to animals. These transcription factors regulate a wide variety of genes with diverse roles in cellular homeostasis, development, and physiology. The origin and specificity of ligand binding within lineages of nuclear receptors (e.g., subfamilies) continues to be a focus of investigation geared toward understanding how the functions of these proteins were shaped over evolutionary history. Among early-diverging animal lineages, the retinoid X receptor (RXR) is first detected in the placozoan, Trichoplax adhaerens. To gain insight into RXR evolution, we characterized ligand- and DNA-binding activity of the RXR from T. adhaerens (TaRXR). Like bilaterian RXRs, TaRXR specifically bound 9-cis-retinoic acid, which is consistent with a recently published result and supports a conclusion that the ancestral RXR bound ligand. DNA binding site specificity of TaRXR was determined through protein binding microarrays (PBMs) and compared with human RXRɑ. The binding sites for these two RXR proteins were broadly conserved (∼85% shared high-affinity sequences within a targeted array), suggesting evolutionary constraint for the regulation of downstream genes. We searched for predicted binding motifs of the T. adhaerens genome within 1000 bases of annotated genes to identify potential regulatory targets. We identified 648 unique protein coding regions with predicted TaRXR binding sites that had diverse predicted functions, with enriched processes related to intracellular signal transduction and protein transport. Together, our data support hypotheses that the original RXR protein in animals bound a ligand with structural similarity to 9-cis-retinoic acid; the DNA motif recognized by RXR has changed little in more than 1 billion years of evolution; and the suite of processes regulated by this transcription factor diversified early in animal evolution., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of liver-specific gene expression with potent tumor suppressor activity, yet many liver tumors express HNF4α. This study reveals that P1-HNF4α, the predominant isoform expressed in the adult liver, inhibits expression of tumor promoting genes in a circadian manner. In contrast, an additional isoform of HNF4α, driven by an alternative promoter (P2-HNF4α), is induced in HNF4α-positive human hepatocellular carcinoma (HCC). P2-HNF4α represses the circadian clock gene ARNTL (BMAL1), which is robustly expressed in healthy hepatocytes, and causes nuclear to cytoplasmic re-localization of P1-HNF4α. We reveal mechanisms underlying the incompatibility of BMAL1 and P2-HNF4α in HCC, and demonstrate that forced expression of BMAL1 in HNF4α-positive HCC prevents the growth of tumors in vivo. These data suggest that manipulation of the circadian clock in HNF4α-positive HCC could be a tractable strategy to inhibit tumor growth and progression in the liver.

Soybean oil consumption is increasing worldwide and parallels a rise in obesity. Rich in unsaturated fats, especially linoleic acid, soybean oil is assumed to be healthy, and yet it induces obesity, diabetes, insulin resistance, and fatty liver in mice. Here, we show that the genetically modified soybean oil Plenish, which came on the U.S. market in 2014 and is low in linoleic acid, induces less obesity than conventional soybean oil in C57BL/6 male mice. Proteomic analysis of the liver reveals global differences in hepatic proteins when comparing diets rich in the two soybean oils, coconut oil, and a low-fat diet. Metabolomic analysis of the liver and plasma shows a positive correlation between obesity and hepatic C18 oxylipin metabolites of omega-6 (ω6) and omega-3 (ω3) fatty acids (linoleic and α-linolenic acid, respectively) in the cytochrome P450/soluble epoxide hydrolase pathway. While Plenish induced less insulin resistance than conventional soybean oil, it resulted in hepatomegaly and liver dysfunction as did olive oil, which has a similar fatty acid composition. These results implicate a new class of compounds in diet-induced obesity-C18 epoxide and diol oxylipins.

The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb(-/-) mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer.

The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/β-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/β-catenin/TCF4 and AP-1 pathways., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The obesity epidemic in the U.S. has led to extensive research into potential contributing dietary factors, especially fat and fructose. Recently, increased consumption of soybean oil, which is rich in polyunsaturated fatty acids (PUFAs), has been proposed to play a causal role in the epidemic. Here, we designed a series of four isocaloric diets (HFD, SO-HFD, F-HFD, F-SO-HFD) to investigate the effects of saturated versus unsaturated fat, as well as fructose, on obesity and diabetes. C57/BL6 male mice fed a diet moderately high in fat from coconut oil and soybean oil (SO-HFD, 40% kcal total fat) showed statistically significant increases in weight gain, adiposity, diabetes, glucose intolerance and insulin resistance compared to mice on a diet consisting primarily of coconut oil (HFD). They also had fatty livers with hepatocyte ballooning and very large lipid droplets as well as shorter colonic crypt length. While the high fructose diet (F-HFD) did not cause as much obesity or diabetes as SO-HFD, it did cause rectal prolapse and a very fatty liver, but no balloon injury. The coconut oil diet (with or without fructose) increased spleen weight while fructose in the presence of soybean oil increased kidney weight. Metabolomics analysis of the liver showed an increased accumulation of PUFAs and their metabolites as well as γ-tocopherol, but a decrease in cholesterol in SO-HFD. Liver transcriptomics analysis revealed a global dysregulation of cytochrome P450 (Cyp) genes in SO-HFD versus HFD livers, most notably in the Cyp3a and Cyp2c families. Other genes involved in obesity (e.g., Cidec, Cd36), diabetes (Igfbp1), inflammation (Cd63), mitochondrial function (Pdk4) and cancer (H19) were also upregulated by the soybean oil diet. Taken together, our results indicate that in mice a diet high in soybean oil is more detrimental to metabolic health than a diet high in fructose or coconut oil.

Nuclear receptors (NRs) regulate gene expression by binding specific DNA sequences consisting of AG[G/T]TCA or AGAACA half site motifs in a variety of configurations. However, those motifs/configurations alone do not adequately explain the diversity of NR function in vivo. Here, a systematic examination of DNA binding specificity by protein-binding microarrays (PBMs) of three closely related human NRs--HNF4α, retinoid X receptor alpha (RXRα) and COUPTF2--reveals an HNF4-specific binding motif (H4-SBM), xxxxCAAAGTCCA, as well as a previously unrecognized polarity in the classical DR1 motif (AGGTCAxAGGTCA) for HNF4α, RXRα and COUPTF2 homodimers. ChIP-seq data indicate that the H4-SBM is uniquely bound by HNF4α but not 10 other NRs in vivo, while NRs PXR, FXRα, Rev-Erbα appear to bind adjacent to H4-SBMs. HNF4-specific DNA recognition and transactivation are mediated by residues Asp69 and Arg76 in the DNA-binding domain; this combination of amino acids is unique to HNF4 among all human NRs. Expression profiling and ChIP data predict ≈ 100 new human HNF4α target genes with an H4-SBM site, including several Co-enzyme A-related genes and genes with links to disease. These results provide important new insights into NR DNA binding.

Src tyrosine kinase has long been implicated in colon cancer but much remains to be learned about its substrates. The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) has just recently been implicated in colon cancer but its role is poorly defined. Here we show that c-Src phosphorylates human HNF4α on three tyrosines in an interdependent and isoform-specific fashion. The initial phosphorylation site is a Tyr residue (Y14) present in the N-terminal A/B domain of P1- but not P2-driven HNF4α. Phospho-Y14 interacts with the Src SH2 domain, leading to the phosphorylation of two additional tyrosines in the ligand binding domain (LBD) in P1-HNF4α. Phosphomimetic mutants in the LBD decrease P1-HNF4α protein stability, nuclear localization and transactivation function. Immunohistochemical analysis of approximately 450 human colon cancer specimens (Stage III) reveals that P1-HNF4α is either lost or localized in the cytoplasm in approximately 80% of tumors, and that staining for active Src correlates with those events in a subset of samples. Finally, three SNPs in the human HNF4α protein, two of which are in the HNF4α F domain that interacts with the Src SH3 domain, increase phosphorylation by Src and decrease HNF4α protein stability and function, suggesting that individuals with those variants may be more susceptible to Src-mediated effects. This newly identified interaction between Src kinase and HNF4α has important implications for colon and other cancers.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

Background: Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression., Results: Here, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4α, NR2A1), are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs) to show that HNF4α binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP) to demonstrate that HNF4α binds Alu elements in the promoters of target genes (ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR) and luciferase assays to show that at least some of those Alu elements can modulate HNF4α-mediated transactivation in vivo (APOM, PRODH2, TTR, APOA4). HNF4α-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4α binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4α sites., Conclusions: Our findings suggest that HNF4α, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats.

Nuclear receptors (NRs) are a family of highly conserved transcription factors that regulate transcription in response to small lipophilic compounds. They play a role in every aspect of development, physiology and disease in humans. They are also ubiquitous in and unique to the animal kingdom suggesting that they may have played an important role in their evolution. In contrast to the classical endocrine receptors that originally defined the family, recent studies suggest that the first NRs might have been sensors of their environment, binding ligands that were external to the host organism. The purpose of this review is to provide a broad perspective on NR ligands and address the issue of exactly what constitutes a NR ligand from historical, biological and evolutionary perspectives. This discussion will lay the foundation for subsequent reviews in this issue as well as pose new questions for future investigation., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Hepatocyte nuclear factor 4α (HNF4α) is a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors. It is best known as a master regulator of liver-specific gene expression, especially those genes involved in lipid transport and glucose metabolism. However, there is also a growing body of work that indicates the importance of HNF4α in the regulation of genes involved in xenobiotic and drug metabolism. A recent study identifying the essential fatty acid linoleic acid (LA, C18:2) as the endogenous, reversible ligand for HNF4α suggests that HNF4α may also be a potential drug target and that its activity may be regulated by diet. This review will discuss the role of HNF4α in drug metabolism, including the genes it regulates, the factors that regulate its activity, and its potential as a drug target., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Unlabelled: Hepatocyte nuclear factor 4 alpha (HNF4alpha), a member of the nuclear receptor superfamily, is essential for liver function and is linked to several diseases including diabetes, hemophilia, atherosclerosis, and hepatitis. Although many DNA response elements and target genes have been identified for HNF4alpha, the complete repertoire of binding sites and target genes in the human genome is unknown. Here, we adapt protein binding microarrays (PBMs) to examine the DNA-binding characteristics of two HNF4alpha species (rat and human) and isoforms (HNF4alpha2 and HNF4alpha8) in a high-throughput fashion. We identified approximately 1400 new binding sequences and used this dataset to successfully train a Support Vector Machine (SVM) model that predicts an additional approximately 10,000 unique HNF4alpha-binding sequences; we also identify new rules for HNF4alpha DNA binding. We performed expression profiling of an HNF4alpha RNA interference knockdown in HepG2 cells and compared the results to a search of the promoters of all human genes with the PBM and SVM models, as well as published genome-wide location analysis. Using this integrated approach, we identified approximately 240 new direct HNF4alpha human target genes, including new functional categories of genes not typically associated with HNF4alpha, such as cell cycle, immune function, apoptosis, stress response, and other cancer-related genes., Conclusion: We report the first use of PBMs with a full-length liver-enriched transcription factor and greatly expand the repertoire of HNF4alpha-binding sequences and target genes, thereby identifying new functions for HNF4alpha. We also establish a web-based tool, HNF4 Motif Finder, that can be used to identify potential HNF4alpha-binding sites in any sequence.

Mutations in the coding region of hepatocyte nuclear factor 4alpha (HNF4alpha), and its upstream promoter (P2) that drives expression in the pancreas, are known to lead to maturity-onset diabetes of the young 1 (MODY1). HNF4alpha also controls gluconeogenesis and lipid metabolism in the liver, where the proximal promoter (P1) predominates. However, very little is known about the role of hepatic HNF4alpha in diabetes. Here, we examine the expression of hepatic HNF4alpha in two diabetic mouse models, db/db mice (type 2, insulin resistant) and streptozotocin-treated mice (type 1, insulin deficient). We found that the level of HNF4alpha protein and mRNA was decreased in the liver of db/db mice but increased in streptozotocin-treated mice. Because insulin increases the activity of sterol regulatory element-binding proteins (SREBP)-1c and -2, we also examined the effect of SREBPs on hepatic HNF4alpha gene expression and found that, like insulin, ectopic expression of SREBPs decreases the level of hepatic HNF4alpha protein and mRNA both in vitro in primary hepatocytes and in vivo in the liver of C57BL/6 mice. Finally, we use gel shift, chromatin immunoprecipitation, small interfering RNA, and reporter gene analysis to show that SREBP2 binds the human HNF4alpha P1 promoter and negatively regulates its expression. These data indicate that hyperinsulinemia down-regulates HNF4alpha in the liver through the up-regulation of SREBPs, thereby establishing a link between these two critical transcription factor pathways that regulate lipid and glucose metabolism in the liver. These findings also provide new insights into diabetes-associated complications such as fatty liver disease.

Orphan nuclear receptors have been instrumental in identifying novel signaling pathways and therapeutic targets. However, identification of ligands for these receptors has often been based on random compound screens or other biased approaches. As a result, it remains unclear in many cases if the reported ligands are the true endogenous ligands,--i.e., the ligand that is bound to the receptor in an unperturbed in vivo setting. Technical limitations have limited our ability to identify ligands based on this rigorous definition. The orphan receptor hepatocyte nuclear factor 4 alpha (HNF4alpha) is a key regulator of many metabolic pathways and linked to several diseases including diabetes, atherosclerosis, hemophilia and cancer. Here we utilize an affinity isolation/mass-spectrometry (AIMS) approach to demonstrate that HNF4alpha is selectively occupied by linoleic acid (LA, C18:2omega6) in mammalian cells and in the liver of fed mice. Receptor occupancy is dramatically reduced in the fasted state and in a receptor carrying a mutation derived from patients with Maturity Onset Diabetes of the Young 1 (MODY1). Interestingly, however, ligand occupancy does not appear to have a significant effect on HNF4alpha transcriptional activity, as evidenced by genome-wide expression profiling in cells derived from human colon. We also use AIMS to show that LA binding is reversible in intact cells, indicating that HNF4alpha could be a viable drug target. This study establishes a general method to identify true endogenous ligands for nuclear receptors (and other lipid binding proteins), independent of transcriptional function, and to track in vivo receptor occupancy under physiologically relevant conditions.

The type 1, 3, and 5 forms of maturity-onset diabetes of the young (MODY) are caused by mutations of the genes encoding hepatocyte nuclear factor (HNF)-4alpha, HNF-1alpha, and HNF-1beta, respectively [Yamagata, K., Oda, N., Kaisaki, P.J., Menzel, S., Furuta, H., Vaxillaire, M., et al., 1996a. Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3). Nature 384, 455-458; Yamagata, K., Furuta, H., Oda, N., Kaisaki, P.J., Menzel, S., Cox, N.J., et al., 1996b. Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young (MODY1). Nature 384, 458-460; Horikawa, Y., Iwasaki, N., Hara, M., Furuta, H., Hinokio, Y., Cockburn, B.N. et al., 1997. Mutation in hepatocyte nuclear factor-1beta gene (TCF2) associated with MODY. Nat. Genet. 17, 384-385]. Among these transcription factors, the pattern of HNF-4alpha expression during pancreatic differentiation remains largely unknown. We performed an immunohistochemical study to investigate its expression in comparison with the expression of HNF-1alpha and HNF-1beta. We found considerable variation in the level of HNF-4alpha expression by the individual epithelial cells in the pancreatic buds on E9.5. HNF-4alpha and HNF-1beta were initially expressed by Pdx1(+) common progenitor cells and neurogenin3(+) (Ngn3(+)) endocrine precursor cells during the first transition, but expression of HNF-1beta and either HNF-4alpha or HNF-1alpha became complementary around the end of the second transition (E15.5). In the mature pancreas, HNF-4alpha was expressed by glucagon-positive alpha-cells, insulin-positive beta-cells, somatostatin-positive delta-cells, and pancreatic polypeptide-positive PP-cells, as well as by pancreatic exocrine cells and ductal cells. Most of the HNF-4alpha(+) cells were also positive for HNF-1alpha, but HNF-4alpha expression in some non-beta-cells was remarkably high, and this was not paralleled by high HNF-1alpha expression. These results indicate that the expression of MODY proteins in each of the pancreatic cell types is strictly regulated in accordance with the status of differentiation during pancreatic organogenesis.

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 the expression of the human cyclin-dependent kinase inhibitor gene p21/WAF1 (CDKN1A). We found that HNF4alpha1, a transcription factor that plays a central role in differentiation in the liver, pancreas, and intestine, activates the expression of p21 primarily by interacting with promoter-bound Sp1 at both the proximal promoter region and at newly identified sites in a distal region (-2.4 kb). Although HNF4alpha1 also binds two additional regions containing putative HNF4alpha binding sites, HNF4alpha1 mutants deficient in DNA binding activate the p21 promoter to the same extent as wild-type HNF4alpha1, indicating that direct DNA binding by HNF4alpha1 is not necessary for p21 activation. We also observed an in vitro and in vivo interaction between HNF4alpha1 and c-Myc as well as a competition between these two transcription factors for interaction with promoter-bound Sp1 and regulation of p21. Finally, we show that c-Myc competes with HNF4alpha1 for control of apolipoprotein C3 (APOC3), a gene associated with the differentiated hepatic phenotype. These results suggest a general model by which a differentiation factor (HNF4alpha1) and a proliferation factor (c-Myc) may compete for control of genes involved in cell proliferation and differentiation.

Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4alpha (HNF4alpha). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4alpha and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4alpha (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4alpha as well as decreased endogenous HNF4alpha protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor alpha, retinoid X receptor alpha, and thyroid hormone receptor beta) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor alpha and peroxisome proliferator-activated receptor alpha. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

The core promoter of eukaryotic genes is the minimal DNA region that recruits the basal transcription machinery to direct efficient and accurate transcription initiation. The fraction of human and yeast genes that contain specific core promoter elements such as the TATA box and the initiator (INR) remains unclear and core promoter motifs specific for TATA-less genes remain to be identified. Here, we present genome-scale computational analyses indicating that approximately 76% of human core promoters lack TATA-like elements, have a high GC content, and are enriched in Sp1-binding sites. We further identify two motifs - M3 (SCGGAAGY) and M22 (TGCGCANK) - that occur preferentially in human TATA-less core promoters. About 24% of human genes have a TATA-like element and their promoters are generally AT-rich; however, only approximately 10% of these TATA-containing promoters have the canonical TATA box (TATAWAWR). In contrast, approximately 46% of human core promoters contain the consensus INR (YYANWYY) and approximately 30% are INR-containing TATA-less genes. Significantly, approximately 46% of human promoters lack both TATA-like and consensus INR elements. Surprisingly, mammalian-type INR sequences are present - and tend to cluster - in the transcription start site (TSS) region of approximately 40% of yeast core promoters and the frequency of specific core promoter types appears to be conserved in yeast and human genomes. Gene Ontology analyses reveal that TATA-less genes in humans, as in yeast, are frequently involved in basic "housekeeping" processes, while TATA-containing genes are more often highly regulated, such as by biotic or stress stimuli. These results reveal unexpected similarities in the occurrence of specific core promoter types and in their associated biological processes in yeast and humans and point to novel vertebrate-specific DNA motifs that might play a selective role in TATA-independent transcription.

The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumour suppressor protein p53. We have previously shown that p53 inhibits the transactivation function of HNF (hepatocyte nuclear factor) 4alpha1, a nuclear receptor known to be critical for early development and liver differentiation. In the present study we demonstrate that p53 also down-regulates expression of the human HNF4alpha gene via the proximal P1 promoter. Overexpression of wild-type p53 down-regulated endogenous levels of both HNF4alpha protein and mRNA in Hep3B cells. This decrease was also observed when HepG2 cells were exposed to UV irradiation or doxorubicin, both of which increased endogenous p53 protein levels. Ectopically expressed p53, but not a mutant p53 defective in DNA binding (R249S), down-regulated HNF4alpha P1 promoter activity. Chromatin immunoprecipitation also showed that endogenous p53 bound the HNF4alpha P1 promoter in vivo after doxorubicin treatment. The mechanism by which p53 down-regulates the P1 promoter appears to be multifaceted. The down-regulation was partially recovered by inhibition of HDAC activity and appears to involve the positive regulator HNF6alpha. p53 bound HNF6alpha in vivo and in vitro and prevented HNF6alpha from binding DNA in vitro. p53 also repressed stimulation of the P1 promoter by HNF6alpha in vivo. However, since the R249S p53 mutant also bound HNF6alpha, binding HNF6alpha is apparently not sufficient for the repression. Implications of the p53-mediated repression of HNF4alpha expression in response to cellular stress are discussed.

Objective: The regulation of AMP-activated protein kinase (AMPK) is implicated in vascular biology because AMPK can phosphorylate endothelial NO synthase (eNOS). In this study, we investigate the regulation of the AMPK-eNOS pathway in vascular endothelial cells (ECs) by shear stress and the activation of aortic AMPK in a mouse model with a high level of voluntary running (High-Runner)., Methods and Results: By using flow channels with cultured ECs, AMPK Thr172 phosphorylation was increased with changes of flow rate or pulsatility. The activity of LKB1, the upstream kinase of AMPK, and the phosphorylation of eNOS at Ser1179 were concomitant with AMPK activation responding to changes in flow rate or pulsatility. The blockage of AMPK by a dominant-negative mutant of AMPK inhibited shear stress-induced eNOS Ser1179 phosphorylation and NO production. Furthermore, aortic AMPK activity and level of eNOS phosphorylation were significantly elevated in the aortas of High-Runner mice., Conclusions: Our results suggest that shear stress activates AMPK in ECs, which contributes to elevated eNOS activity and subsequent NO production. Hence, AMPK, in addition to serving as an energy sensor, also plays an important role in regulating vascular tone.

Epithelial formation is a central facet of organogenesis that relies on intercellular junction assembly to create functionally distinct apical and basal cell surfaces. How this process is regulated during embryonic development remains obscure. Previous studies using conditional knockout mice have shown that loss of hepatocyte nuclear factor 4alpha (HNF4alpha) blocks the epithelial transformation of the fetal liver, suggesting that HNF4alpha is a central regulator of epithelial morphogenesis. Although HNF4alpha-null hepatocytes do not express E-cadherin (also called CDH1), we show here that E-cadherin is dispensable for liver development, implying that HNF4alpha regulates additional aspects of epithelial formation. Microarray and molecular analyses reveal that HNF4alpha regulates the developmental expression of a myriad of proteins required for cell junction assembly and adhesion. Our findings define a fundamental mechanism through which generation of tissue epithelia during development is coordinated with the onset of organ function.

Background & Aims: Hepatocyte nuclear factor 4 alpha (HNF4alpha) is a transcription factor that has been shown to be required for hepatocyte differentiation and development of the liver. It has also been implicated in regulating expression of genes that act in the epithelium of the lower gastrointestinal tract. This implied that HNF4alpha might be required for development of the gut., Methods: Mouse embryos were generated in which Hnf4a was ablated in the epithelial cells of the fetal colon by using Cre-loxP technology. Embryos were examined by using a combination of histology, immunohistochemistry, DNA microarray, reverse-transcription polymerase chain reaction, electrophoretic mobility shift assays, and chromatin immunoprecipitation analyses to define the consequences of loss of HNF4alpha on colon development., Results: Embryos were recovered at E18.5 that lacked HNF4alpha in their colons. Although early stages of colonic development occurred, HNF4alpha-null colons failed to form normal crypts. In addition, goblet-cell maturation was perturbed and expression of an array of genes that encode proteins with diverse roles in colon function was disrupted. Several genes whose expression in the colon was dependent on HNF4alpha contained HNF4alpha-binding sites within putative transcriptional regulatory regions and a subset of these sites were occupied by HNF4alpha in vivo., Conclusions: HNF4alpha is a transcription factor that is essential for development of the mammalian colon, regulates goblet-cell maturation, and is required for expression of genes that control normal colon function and epithelial cell differentiation.