Human HNF4α Research Articles

Immunohistochemical detection of hepatocyte nuclear factor-4α in vertebrates.

Hepatocyte nuclear factor-4α (HNF4α) presents in multiple isoforms generated using alternative promoter (P1 and P2) and splicing. Neither conservation of tissue distribution of HNF4α isoforms, nor presence of alternative promoter usage is known. In this study, to detect the expression of HNF4α in some species of animals, we have applied monoclonal antibodies against P1 (K9218) and P2 (H6939) promoter-driven and P1/P2 promoter-driven H1415 HNF4α for immunohistochemistry and western blot analysis. Antibody K9218 was observed in the hepatocytes, proximal tubules of the kidney, and epithelial cells in the mucosa of the small intestine and colon of rats, chicken, and tortoise, whereas antibody H6939 signal were detected in the stomach, pancreas, bile duct, and pancreatic duct of human and rats. The signal for antibody K9218 was recognized in tissues of a wide range of mammals, bird, reptile, amphibian, and fish as well. Antibody H1415 displayed a positive reaction in hepatocytes and intestinal epithelial cells in chicken and tortoise, whereas the bile duct, mucosal epithelial cells in the stomach, or pancreas in these animals were negative. Western blotting showed the binding of the antibody with HNF4α protein from each animal. The sequence of human HNF4α was 100% identical to murine and rat HNF4α, 88.9% to chicken, 77.8% to Xenopus HNF4α, and 81.5% to medaka. However, the specific part of human and invertebrate Drosophila HNF4 shares only 14.8% sequence identity. This antibody is useful for detecting HNF4α isoforms in a wide range of vertebrates, and suggests many insights into animal evolution.

Hepatocyte Nuclear Factor 4α Prevents the Steatosis-to-NASH Progression by Regulating p53 and Bile Acid Signaling (in mice).

Hepatocyte nuclear factor 4α (HNF4α) is highly enriched in the liver, but its role in the progression of nonalcoholic liver steatosis (NAFL) to NASH has not been elucidated. In this study, we investigated the effect of gain or loss of HNF4α function on the development and progression of NAFLD in mice. Overexpression of human HNF4α protected against high-fat/cholesterol/fructose (HFCF) diet-induced steatohepatitis, whereas loss of Hnf4α had opposite effects. HNF4α prevented hepatic triglyceride accumulation by promoting hepatic triglyceride lipolysis, fatty acid oxidation, and VLDL secretion. Furthermore, HNF4α suppressed the progression of NAFL to NASH. Overexpression of human HNF4α inhibited HFCF diet-induced steatohepatitis in control mice but not in hepatocyte-specific p53-/- mice. In HFCF diet-fed mice lacking hepatic Hnf4α, recapitulation of hepatic expression of HNF4α targets cholesterol 7α-hydroxylase and sterol 12α-hydroxylase and normalized hepatic triglyceride levels and attenuated steatohepatitis. The current study indicates that HNF4α protects against diet-induced development and progression of NAFLD by coordinating the regulation of lipolytic, p53, and bile acid signaling pathways. Targeting hepatic HNF4α may be useful for treatment of NASH.

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells.

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293Toverexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.

Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter.

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

HNF4α Combinatorial Isoform Heterodimers Activate Distinct Gene Targets that Differ from Their Corresponding Homodimers

Hepatocyte nuclear factor 4α (HNF4α) is a dimeric transcription factor that controls as much as 60% of all liver genes. However, how it achieves such broad functional diversity is unknown. Here, we show that inflammation and immune pathway genes are differentially regulated in an isoform-dependent manner, confirming that each isoform homodimer preferentially regulates a subset of HNF4α targets. With all 12 human HNF4α isoform clones, we tested combinatorial pairings to determine whether isoform heterodimers are functional. Indeed, synergistic and potent pairing combinations of isoform heterodimers were noted for HNF4α3-8, HNF4α6-12, and HNF4α5-8 that activated CYP7A1, IL6, and IL17A genes, respectively. Surprisingly, these genes are not at all activated by their corresponding isoform homodimers, suggesting that a particular heterodimer pair can regulate its own subset of target genes. Given the combinatorial possibility of 66 isoform heterodimers, our data provide the basis for a more detailed understanding of the diverse influence of HNF4α.

Association of sex hormone-binding globulin with nonalcoholic fatty liver disease in Chinese adults

BackgroundSex hormone-binding globulin (SHBG), a glycoprotein synthesized by hepatocytes, has been linked to insulin resistance and hepatic lipid metabolism and is suggested to be associated with nonalcoholic fatty liver disease (NAFLD). This study aimed to investigate the association of SHBG with NAFLD in Chinese adults.MethodsWe conducted a community-based, cross-sectional study in China involving 2912 participants aged 40–75 years old. All participants underwent detection for hepatic fat infiltration by ultrasound in addition to providing complete medical history and undergoing physical and blood biochemical examinations. The association of serum SHBG with the presence of NAFLD was reported by adjusted odds ratio after applying logistic regression models. To further explore the relationship between SHBG and NAFLD, mRNA expression of SHBG and hepatocyte nuclear factor 4-α (HNF4α), as well as intrahepatic triglycerides, were determined from the liver tissues of 32 subjects with different degrees of steatosis.ResultsSerum SHBG levels in patients with NAFLD (median, 43.8 nmol/L; interquartile range, 33.4–56.8 nmol/L) were significantly lower than those in non-NAFLD subjects (median, 63.4 nmol/L; interquartile range, 47.6–83.1 nmol/L). Serum SHBG levels were inversely correlated with WHR, trunk fat percentage, glucose, HOMA-IR, TG, UA and DHEAS, and were positively correlated with HDL-C levels (all p < 0.001). Logistic regression analysis indicated that serum SHBG levels were negatively associated with the presence of NAFLD in all subjects, as well as the subgroups stratified by sex, BMI and HOMA-IR (all p < 0.05). In human liver tissues, SHBG and HNF4α mRNA expression decreased along with the elevated grade of hepatic steatosis. Both SHBG and HNF4α mRNA expression levels were negatively correlated with intrahepatic triglycerides.ConclusionsThese results demonstrate that SHBG levels were negatively associated with the presence of NAFLD in middle-aged and elderly Chinese adults.

Multidomain integration in the structure of the HNF-4α nuclear receptor complex

The hepatocyte nuclear factor 4 alpha (HNF4α, NR2A1) is a member of the nuclear receptor (NR) family of transcription factors that use conserved DNA binding domains (DBDs) and ligand binding domains (LBDs)1,2. HNF4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF4α response element 1,3,4. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism3,5,6. In the pancreas too, HNF4α is a master regulator controlling an estimated 11% of islet genes7. HNF4α protein mutations are linked to Maturity Onset of Diabetes in Young 1 (MODY1) and hyperinsulinemic hypoglycemia (HH)8–11. Prior structural analyses of NRs, while productive with individual domains, have lagged in revealing the connectivity patterns of NR domains. Here, we describe the 2.9 Å crystal structure of the multi-domain HNF4α homodimer bound to its DNA response element and coactivator-derived peptides. A convergence zone connects multiple receptor domains in an asymmetric fashion joining distinct elements from each monomer. An arginine target of PRMT1 methylation protrudes directly into this convergence zone and sustains its integrity. A serine target of protein kinase C is also responsible for maintaining domain-domain interactions. These post-translational modifications manifest into changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the LBD and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the inter-junctional surfaces of the complex. The overall domain representation of the HNF4α homodimer is different from that of the PPARγ-RXRα heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and inter-domain connections in NRs could be exploited by small-molecule allosteric modulators that impact distal functions in these polypeptides.

Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors

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 phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer

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.

Construction and characterization of hepatocyte nuclear factor HNF4alpha1 over-expressing cell line derived from human hepatoma HepG2 cells

Cancer cell lines derived from hepatocytes have an altered phenotype and they lack hepatocyte-specific functions. It is at least partly due to the under-expression of transcription factors such as hepatocyte nuclear factor 4α (HNF4α), steroid receptor co-activator 1 (SRC1) etc. Recently, a strategy of transient transfection of human hepatic cells with HNF4α revealed improved hepatospecific functions, including the expression of drug-metabolizing enzymes. In the current study we established a human cell line derived from HepG2 cells stably transfected with human HNF4α, and we examined this line for hepatospecific markers. Of the 9 clones analyzed, we found an increased secretion of fibrinogen (9 clones), albumin (5 clones) and plasminogen (3 clones), while secretion of alpha1-antitrypsin was not changed. The expression of pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins but not mRNAs was slightly increased. TCDD-dependent induction of CYP1A1 mRNA and protein was augmented in 50% of clones, but there was no correlation between the CYP1A1 inducibility and expression levels of AhR and HNF4α. Induction of CYP3A4 mRNA by rifampicin was about 1.5-2.5 fold (clones 2, 4, 6, 7) and it was not significantly different from CYP3A4 mRNA induction in parent HepG2. The basal expression of CYP3A4 protein was increased in all clones, but rifampicin-induced expression of CYP3A4 protein was in all clones lower than in parent HepG2. Overall, the stable over-expression of HNF4α in HepG2 cells restores some of the hepatospecific functions, but it has a minor effect on the expression of xenobiotic-metabolizing enzymes and their regulators.

Proteomic Analysis of Native Hepatocyte Nuclear Factor-4α (HNF4α) Isoforms, Phosphorylation Status, and Interactive Cofactors

Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor that has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady-state remains to be elucidated. Here we report the native HNF4α isoform, phosphorylation status, and complexes in the steady-state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4α, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semiquantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4α and hepatocyte nuclear factor-4γ was found. A biochemical and genomewide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4α and its cofactor complexes described here are important for an accurate understanding of transcriptional regulation.

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

Functional Targets of the Monogenic Diabetes Transcription Factors HNF-1α and HNF-4α Are Highly Conserved Between Mice and Humans

OBJECTIVEThe evolutionary conservation of transcriptional mechanisms has been widely exploited to understand human biology and disease. Recent findings, however, unexpectedly showed that the transcriptional regulators hepatocyte nuclear factor (HNF)-1α and -4α rarely bind to the same genes in mice and humans, leading to the proposal that tissue-specific transcriptional regulation has undergone extensive divergence in the two species. Such observations have major implications for the use of mouse models to understand HNF-1α– and HNF-4α–deficient diabetes. However, the significance of studies that assess binding without considering regulatory function is poorly understood.RESEARCH DESIGN AND METHODSWe compared previously reported mouse and human HNF-1α and HNF-4α binding studies with independent binding experiments. We also integrated binding studies with mouse and human loss-of-function gene expression datasets.RESULTSFirst, we confirmed the existence of species-specific HNF-1α and -4α binding, yet observed incomplete detection of binding in the different datasets, causing an underestimation of binding conservation. Second, only a minor fraction of HNF-1α– and HNF-4α–bound genes were downregulated in the absence of these regulators. This subset of functional targets did not show evidence for evolutionary divergence of binding or binding sequence motifs. Finally, we observed differences between conserved and species-specific binding properties. For example, conserved binding was more frequently located near transcriptional start sites and was more likely to involve multiple binding events in the same gene.CONCLUSIONSDespite evolutionary changes in binding, essential direct transcriptional functions of HNF-1α and -4α are largely conserved between mice and humans.

Hepatocyte nuclear factor 4α regulation of bile acid and drug metabolism

The hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched nuclear receptor that plays a critical role in early morphogenesis, fetal liver development, liver differentiation and metabolism. Human HNF4α gene mutations cause maturity on-set diabetes of the young type 1, an autosomal dominant non-insulin-dependent diabetes mellitus. HNF4α is an orphan nuclear receptor because of which the endogenous ligand has not been firmly identified. The trans-activating activity of HNF4α is enhanced by interacting with co-activators and inhibited by corepressors. Recent studies have revealed that HNF4α plays a central role in regulation of bile acid metabolism in the liver. Bile acids are required for biliary excretion of cholesterol and metabolites, and intestinal absorption of fat, nutrients, drug and xenobiotics for transport and distribution to liver and other tissues. Bile acids are signaling molecules that activate nuclear receptors to control lipids and drug metabolism in the liver and intestine. Therefore, HNF4α plays a central role in coordinated regulation of bile acid and xenobiotics metabolism. Drugs that specifically activate HNF4α could be developed for treating metabolic diseases such as diabetes, dyslipidemia and cholestasis, as well as drug metabolism and detoxification.

Nuclear Receptor CAR Requires Early Growth Response 1 to Activate the Human Cytochrome P450 2B6 Gene

The nuclear receptor CAR (constitutive active/androstane receptor) is a drug-sensing transcription factor, regulating the hepatic genes that encode various drug-metabolizing enzymes. We have now characterized the novel regulatory mechanism by which the signal molecule EGR1 (early growth response 1) determines CAR-mediated activation of the human CYP2B6 (cytochrome P450 2B6) gene. The CYP2B6 enzyme metabolizes commonly used therapeutics and also activates pro-drugs. The CAR directly binds to the distal enhancer element of the CYP2B6 promoter, which is essential in converging to its drug-sensing function onto promoter activity. However, this binding alone is not sufficient to activate the CYP2B6 promoter; the promoter requires EGR1 to enable CAR to activate the CYP2B6 promoter. Upon stimulation by protein kinase C, EGR1 directly binds to the proximal promoter and coordinates the nearby HNF4alpha (hepatocyte-enriched nuclear factor 4alpha) with CAR at the distal enhancer element to activate the promoter. Thus, synergy of drug activation and the stimulation of cellular signal are necessary for CAR to activate the CYP2B6 gene.

Tumour suppressor p53 down-regulates the expression of the human hepatocyte nuclear factor 4α (HNF4α) gene

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.

PI-32The effects of folate supplementation on human CYP2C9 transactivation

BACKGROUND/AIMS Folic acid (FA) supplementation can increase the metabolism of phenytoin and warfarin, both of which are CYP2C9 substrates, but the mechanisms underlying this are unknown. This study examined the hypothesis that FA affects transactivation of a defined core promoter in human CYP2C9 gene, in which CAR, PXR, HNF1α, and HNF4α binding sites exist. METHODS CYP2C9-luciferase reporter constructs (-3kb) were transfected into HepG2 cells, and human CAR, PXR, HNF1α, and HNF4α were co-transfected either alone or in combination. The transfected cells were treated with or without FA (100μM), and the cells co-transfected with PXR were simultaneously treated with rifampin (Rif, 10μM), followed by dual luciferase assays. Data were expressed as fold activation versus vector control. RESULTS Compared with each of the vehicle controls, FA treatment did not significantly affect luciferase activities (FA vs DMSO: 1.9 ± 0.6 vs 1.8 ± 0.6 for CAR; 12.9 ± 4.2 vs 12.5 ± 3.8 for CAR+ HNF1α; 14.9 ± 7.8 vs 15.4 ± 9.3 for CAR+ HNF4α; 1.3 ± 0.3 vs 1.3 ± 0.4 for PXR/Rif; 9.4 ± 2.8 vs 8.9 ± 2.6 for PXR/Rif+ HNF1α; 3.1 ± 1.5 vs 3.1 ± 1.8 for PXR/Rif+ HNF4α; all P> 0.05, n = 6 each). CONCLUSIONS In transfected HepG2 cells, acute administration of folic acid has no effect on CYP2C9 transcriptional activity, regardless of co-transfection with CAR, PXR, HNF1α, and HNF4α, alone or in combination. The effect of folic acid on the metabolism of CYP2C9 substrates is not mediated by alteration of transcription by key nuclear receptors. Clinical Pharmacology & Therapeutics (2005) 79, P15–P15; doi: 10.1016/j.clpt.2005.12.053

Comparative genomic analysis of the HNF-4α transcription factor gene

Hepatocyte nuclear factor-4α (HNF-4α), the gene for the maturity-onset diabetes of the young type 1 (MODY1) form of type 2 diabetes mellitus (T2DM), is within the T2DM-linked region on chromosome 20q12-q13.1 and consequently, is a positional candidate gene for T2DM. Mutations in the coding region of HNF-4α are rare in diabetes affected subjects. Altered regulation of HNF-4α gene expression, controlled by distant enhancer sequences, may contribute to the development of type 2 diabetes. Comparative sequence analysis was performed between 13 kb of genomic DNA 5 ′ to the P1 promoter sequences of the human, mouse, and rat HNF-4α coding sequences. Three regions, located at −10.5 kb (295 bp in length), −6.25 kb (421 bp in length), and −5.36 kb (263 bp in length), have significant sequence identity between the species. These three regions were functionally characterized using the chloramphenicol acetyltransferase (CAT) reporter assay, in which the conserved 5 ′ regions of mouse HNF-4α were cloned in front of the herpes simplex virus thymidine kinase promoter driving transcription of the CAT gene. A fragment containing the 421 bp conserved region significantly increased CAT activity in differentiated rat hepatoma cells (13.7- ± 1.9-fold control), while only a modest increase in CAT activity was observed in pancreatic cells (2.5- ± 0.9-fold control; 1.6- ± 0.1-fold control) and dedifferentiated hepatoma cells (1.7- ± 0.4-fold control). The remaining two conserved regions increased CAT activity minimally in pancreatic (1.1- ± 0.1-fold control to 1.9- ± 0.1-fold control) and hepatic (1.6- ± 0.5-fold control to 2.3- ± 0.4-fold control) cell lines. Denaturing high-performance liquid chromatography (DHPLC) was used to search for sequence variants in DNA from 259 T2DM individuals. Two single nucleotide polymorphisms (SNPs) were identified, both of which increased CAT activity in the insulinoma cell lines in the CAT reporter assay (1.4-fold increase over wild-type; 1.7-fold increase over wild-type). These results suggest that comparative sequence analysis can efficiently identify regulatory elements and that sequence variants in regulatory elements of HNF-4α can contribute to altered HNF-4α gene expression.

Hepatocyte nuclear factor 1α: A key mediator of the effect of bile acids on gene expression

Bile acids regulate the expression of genes involved in cholesterol homeostasis. They are ligands of the farnesoid X receptor, which induces small heterodimer partner (SHP)-1, a transcriptional repressor of bile acid synthetic enzymes. In cholestatic liver disease, hepatic bile acid concentrations are elevated and expression of the major Na+−independent bile acid uptake system, organic anion transporting polypeptide (OATP)-C (solute carrier gene family SLC21A6), is markedly decreased. Because the OATP-C gene is transcriptionally dependent on the hepatocyte nuclear factor (HNF) 1α, we hypothesized that bile acids decrease OATP-C expression through direct repression of HNF1α. To test this hypothesis, we studied the regulation of the human HNF1α gene by bile acids. HNF1α expression in cultured hepatoma cells was decreased ∼50% after 12 hours' exposure to 100 μmol/L chenodeoxycholic acid (CDCA). Characterization of the human HNF1α gene promoter identified a consensus bile acid response element that binds and is activated by HNF4α. Mutagenesis of the HNF4α site abolished baseline HNF1α promoter activity. The central mechanism by which bile acids repress HNF1α is decreased activation by HNF4α. SHP directly inhibits HNF4α-mediated transactivation of the HNF1α promoter in cotransfection assays. In addition, HNF4α nuclear binding activity is decreased by CDCA and the human HNF4α gene promoter is repressed by CDCA through an SHP-independent mechanism. In conclusion, we show that repression of HNF1α is an important new mechanism by which bile acids regulate the expression of HNF1α-dependent genes in man. This explains the suppressive effect of bile acids on the OATP-C gene promoter, leading to decreased expression in cholestatic liver disease. (H EPATOLOGY 2003;37:622-631.)

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BackgroundHepatocyte nuclear factor-4α (HNF4α; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4α causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4α in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4α protein, due, in part, to the limited availability of human HNF4α-specific antibodies.ResultsMonoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4α fusion proteins as the immunizing agent. The mouse anti-human HNF4α monoclonal antibody (K9218) generated against human HNF4α1/α2/α3 amino acids 3–49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4α proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4α protein, but HEK293 showed no expression of HNF4α protein. Nuclear-specific localization of the HNF4α protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4α protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4α protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4α in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4α mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis.ConclusionThese results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4α and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4α isoforms in humans and in several other mammalian species.