6-Gingerol Ameliorates Hepatic Steatosis via HNF4α/miR-467b-3p/GPAT1 Cascade.

BACKGROUND & AIMS: The development of nonalcoholic fatty liver disease (NAFLD) can be modulated by microRNAs (miRNA). Dietary polyphenols modulate the expression of miRNA such as miR-467b-3p in the liver. In addition, 6-gingerol (6-G), the functional polyphenol of ginger, has been reported to ameliorate hepatic steatosis; however, the exact mechanism involved and the role of miRNA remain elusive. In this study, we assessed the role of miR-467b-3p in the pathogenesis of hepatic steatosis and the regulation of miR-467b-3p by 6-G through the hepatocyte nuclear factor 4α (HNF4α). METHODS: miR-467b-3p expression was measured in free fatty acid (FFA)-treated hepatocytes or liver from high-fat diet (HFD)-fed mice. Gain- or loss-of-function of miR-467b-3p was induced using miR-467b-3p-specific miRNA mimic or miRNA inhibitor, respectively. 6-G was exposed to FFA-treated cells and HFD-fed mice. The HNF4α/miR-467b-3p/GPAT1 axis was measured in mouse and human fatty liver tissues. RESULTS: We found that miR-467b-3p was down-regulated in liver tissues from HFD-fed mice and in FFA-treated Hepa1-6 cells. Overexpression of miR-467b-3p decreased intracellular lipid accumulation in FFA-treated hepatocytes and mitigated hepatic steatosis in HFD-fed mice via negative regulation of glycerol-3-phosphate acyltransferase-1 (GPAT1). In addition, miR-467b-3p up-regulation by 6-G was observed. 6-G inhibited FFA-induced lipid accumulation and mitigated hepatic steatosis. Moreover, it increased the transcriptional activity of HNF4α, resulting in the increase of miR-467b-3p and subsequent decrease of GPAT1. HNF4α/miR-467b-3p/GPAT1 signaling also was observed in human samples with hepatic steatosis. CONCLUSIONS: Our findings establish a novel mechanism by which 6-G improves NAFLD. This suggests that targeting of the HNF4α/miR-467b-3p/GPAT1 cascade may be used as a potential therapeutic strategy to control NAFLD.

Chemical Starting Matter for HNF4α Ligand Discovery and Chemogenomics.

Hepatocyte nuclear factor 4α (HNF4α) is a ligand-sensing transcription factor and presents as a potential drug target in metabolic diseases and cancer. In humans, mutations in the HNF4α gene cause maturity-onset diabetes of the young (MODY), and the elevated activity of this protein has been associated with gastrointestinal cancers. Despite the high therapeutic potential, available ligands and structure-activity relationship knowledge for this nuclear receptor are scarce. Here, we disclose a chemically diverse collection of orthogonally validated fragment-like activators as well as inverse agonists, which modulate HNF4α activity in a low micromolar range. These compounds demonstrate the druggability of HNF4α and thus provide a starting point for medicinal chemistry as well as an early tool for chemogenomics.

Dimerization defective MODY mutations of hepatocyte nuclear factor 4α.

HNF4α is a culprit gene product for a monogenic and dominantly-inherited form of diabetes, referred to as MODY1 (Maturity Onset Diabetes of the Young type 1). Reduced HNF4α activities have been linked to impaired insulin secretion and ß-cell function. Numerous mutations have been identified from the patients and they have been instructive as to the individual residue's role in protein structure-function and dysfunction. As a member of the nuclear receptor (NR) superfamily, HNF4α is made of characteristic modular domains and it functions exclusively as a homodimer despite its sequence homology to RXR, a common heterodimer partner of non-steroidal NRs. Transcription factors commonly dimerize to enhance their molecular functions mainly by facilitating the recognition of double helix target DNAs that display an intrinsic pseudo-2-fold symmetry and the recruitment of the remainder of the main transcriptional machinery. HNF4α is no exception and its dimerization is maintained by the ligand binding domain (LBD) mainly through the leucine-zipper-like interactions at the stalk of two interacting helices. Although many MODY1 mutations have been previously characterized, including DNA binding disruptors, ligand binding disruptors, coactivator binding disruptors, and protein stability disruptors, protein dimerization disruptors have not been formally reported. In this report, we present a set of data for the two MODY1 mutations found right at the dimerization interface (L332 P and L328del mutations) which clearly exhibit the disruptive effects of directly affecting dimerization, protein stability, and transcriptional activities. These data reinforced the fact that MODY mutations are loss-of-function mutations and HNF4α dimerization is essential for its optimal function and normal physiology.

Nuclear hormone receptors: Ancient 9aaTAD and evolutionally gained NCoA activation pathways.

In higher metazoans, the nuclear hormone receptors activate transcription trough their specific adaptors, nuclear hormone receptor adaptors NCoA, which are absent in lower metazoans. The Nine amino acid TransActivation Domain, 9aaTAD, was reported for a large number of the transcription activators that recruit general mediators of transcription. In this study, we demonstrated that the 9aaTAD from NHR-49 receptor of nematode C.elegans activates transcription as a small peptide. We showed that the ancient 9aaTAD domains are conserved in the nuclear hormone receptors including human HNF4, RARa, VDR and PPARg. Also their small 9aaTAD peptides effectively activated transcription in absence of the NCoA adaptors. We also showed that adjacent H11 domains in ancient and modern hormone receptors have an inhibitory effect on their 9aaTAD function.

Conjunction of G-quadruplex and stem-loop in the 5' untranslated region of mouse hepatocyte nuclear factor 4-alpha1 mediates strong inhibition of protein expression.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a well-established master regulator of liver development and function. Restoration of HNF4α can treat multiple liver disorders and liver cancers. To date, HNF4α is still "undruggable" due to lack of known activating ligands. Thus, understanding the regulatory mechanism of HNF4α expression may help develop an alternative approach to modulate HNF4α protein levels. G-quadruplexes (G4) are non-canonical stable secondary structures discovered mostly in the promoters of oncogenes. Recent genome-wide studies demonstrate the enrichment of G4s in the 5' untranslated region (UTR). By protoporphyrin IX-binding assay and circular dichroism spectrum, we validated the presence of a chemically highly stable 4-ring G4 within the 5' UTR of mouse Hnf4a1. Our real-time PCR and Western blot data showed that the Hnf4a1 5' UTR caused a remarkable translational suppression regardless of a moderate effect on Hnf4a1 mRNA levels. The subsequent deletion/mutation analysis of Hnf4a1 5' UTR using dual-luciferase reporter assays further demonstrated that although the disruption of the chemically highly stable 4-ring G4 resulted in a marked attenuation of inhibition, the G4 alone only weakly inhibited translation. Likewise, disruption of a long stem-loop adjacent to the 4-ring G4 markedly attenuated translational inhibition, although the stem-loop alone only exerted a weak inhibitory effect. Thus, the tight conjunction of G4s and an adjacent stem-loop within the Hnf4a1 5' UTR was both necessary and sufficient to mediate the very strong translational repression. Our results establish a novel working model that a chemically stable G4 may require co-factors to be bio-stable for exerting biological functions.

Comprehensive screening for monogenic diabetes in 89 Japanese children with insulin-requiring antibody-negative type 1 diabetes.

BACKGROUND: Mutations in causative genes for neonatal diabetes or maturity-onset diabetes of the young have been identified in multiple patients with autoantibody-negative type 1 diabetes (T1D). OBJECTIVES: We aimed to clarify the prevalence and phenotypic characteristics of monogenic abnormalities among 89 children with autoantibody-negative insulin-requiring T1D. METHODS: Mutations in 30 genes were screened using next-generation sequencing, and copy-number alterations of 4 major causative genes were examined using multiplex-ligation-dependent probe amplification. We compared the clinical characteristics between mutation carriers and non-carriers. RESULTS: We identified 11 probable pathogenic substitutions (6 in INS , 2 in HNF1A , 2 in HNF4A , and 1 in HNF1B ) in 11 cases, but no copy-number abnormalities. Only 2 mutation carriers had affected parents. De novo occurrence was confirmed for 3 mutations. The non-carrier group, but not the carrier group, was enriched with susceptible HLA alleles. Mutation carriers exhibited comparable phenotypes to those of non-carriers, except for a relatively normal body mass index (BMI) at diagnosis. CONCLUSIONS: This study demonstrated significant genetic overlap between autoantibody-negative T1D and monogenic diabetes. Mutations in INS and HNF genes, but not those in GCK and other monogenic diabetes genes, likely play critical roles in children with insulin-requiring T1D. This study also suggests the relatively high de novo rates of INS and HNF mutations, and the etiological link between autoimmune abnormalities and T1D in the non-carrier group. Carriers of monogenic mutations show non-specific phenotypes among all T1D cases, although they are more likely to have a normal BMI at diagnosis than non-carriers.

TRIB1 is a positive regulator of hepatocyte nuclear factor 4-alpha.

The TRIB1 locus has been linked to both cardiovascular disease and hepatic steatosis. Recent efforts have revealed TRIB1 to be a major regulator of liver function, largely, but not exclusively, via CEBPA degradation. We recently uncovered a functional interaction between TRIB1 and HNF4A, another key regulator of hepatic function, whose molecular underpinnings remained to be clarified. Here we have extended these findings. In hepatoma models, HNF4A levels were found to depend on TRIB1, independently of its impact on CEBPA. Using a reporter assay model, MTTP reporter activity, which depends on HNF4A, positively correlated with TRIB1 levels. Confocal microscopy demonstrated partial colocalization of TRIB1 and HNF4A. Using overexpressed proteins we demonstrate that TRIB1 and HNF4A can form complexes in vivo. Mapping of the interaction interfaces identified two distinct regions within TRIB1 which associated with the N-terminal region of HNF4A. Lastly, the TRIB1-HNF4A interaction resisted competition with a CEPBA-derived peptide, suggesting different binding modalities. Together these findings establish that TRIB1 is required for HNF4A function. This regulatory axis represents a novel CEBPA-independent aspect of TRIB1 function predicted to play an important role in liver physiology.

RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP.

Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis.

Theoretical modeling of multiprotein complexes by iSPOT: Integration of small-angle X-ray scattering, hydroxyl radical footprinting, and computational docking.

Structural determination of protein-protein complexes such as multidomain nuclear receptors has been challenging for high-resolution structural techniques. Here, we present a combined use of multiple biophysical methods, termed iSPOT, an integration of shape information from small-angle X-ray scattering (SAXS), protection factors probed by hydroxyl radical footprinting, and a large series of computationally docked conformations from rigid-body or molecular dynamics (MD) simulations. Specifically tested on two model systems, the power of iSPOT is demonstrated to accurately predict the structures of a large protein-protein complex (TGFß-FKBP12) and a multidomain nuclear receptor homodimer (HNF-4α), based on the structures of individual components of the complexes. Although neither SAXS nor footprinting alone can yield an unambiguous picture for each complex, the combination of both, seamlessly integrated in iSPOT, narrows down the best-fit structures that are about 3.2Å and 4.2Å in RMSD from their corresponding crystal structures, respectively. Furthermore, this proof-of-principle study based on the data synthetically derived from available crystal structures shows that the iSPOT-using either rigid-body or MD-based flexible docking-is capable of overcoming the shortcomings of standalone computational methods, especially for HNF-4α. By taking advantage of the integration of SAXS-based shape information and footprinting-based protection/accessibility as well as computational docking, this iSPOT platform is set to be a powerful approach towards accurate integrated modeling of many challenging multiprotein complexes.

Identification of HNF4A Mutation p.T130I and HNF1A Mutations p.I27L and p.S487N in a Han Chinese Family with Early-Onset Maternally Inherited Type 2 Diabetes.

Maturity-onset diabetes of the young (MODY) is characterized by the onset of diabetes before the age of 25 years, positive family history, high genetic predisposition, monogenic mutations, and an autosomal dominant mode of inheritance. Here, we aimed to investigate the mutations and to characterize the phenotypes of a Han Chinese family with early-onset maternally inherited type 2 diabetes. Detailed clinical assessments and genetic screening for mutations in the HNF4α, GCK, HNF-1α, IPF-1, HNF1ß, and NEUROD1 genes were carried out in this family. One HNF4A mutation (p.T130I) and two HNF1A polymorphisms (p.I27L and p.S487N) were identified. Mutation p.T130I was associated with both early-onset and late-onset diabetes and caused downregulated HNF4A expression, whereas HNF1A polymorphisms p.I27L and p.S487N were associated with the age of diagnosis of diabetes. We demonstrated that mutation p.T130I in HNF4A was pathogenic as were the predicted polymorphisms p.I27L and p.S487N in HNF1A by genetic and functional analysis. Our results show that mutations in HNF4A and HNF1A genes might account for this early-onset inherited type 2 diabetes.

Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α.

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells.

Cofunctional Subpathways Were Regulated by Transcription Factor with Common Motif, Common Family, or Common Tissue.

Dissecting the characteristics of the transcription factor (TF) regulatory subpathway is helpful for understanding the TF underlying regulatory function in complex biological systems. To gain insight into the influence of TFs on their regulatory subpathways, we constructed a global TF-subpathways network (TSN) to analyze systematically the regulatory effect of common-motif, common-family, or common-tissue TFs on subpathways. We performed cluster analysis to show that the common-motif, common-family, or common-tissue TFs that regulated the same pathway classes tended to cluster together and contribute to the same biological function that led to disease initiation and progression. We analyzed the Jaccard coefficient to show that the functional consistency of subpathways regulated by the TF pairs with common motif, common family, or common tissue was significantly greater than the random TF pairs at the subpathway level, pathway level, and pathway class level. For example, HNF4A (hepatocyte nuclear factor 4, alpha) and NR1I3 (nuclear receptor subfamily 1, group I, member 3) were a pair of TFs with common motif, common family, and common tissue. They were involved in drug metabolism pathways and were liver-specific factors required for physiological transcription. In short, we inferred that the cofunctional subpathways were regulated by common-motif, common-family, or common-tissue TFs.

Is HNF4A a candidate to study zinc finger protein slug?

Protein-Protein Interactions (PPI) play a crucial role in deciphering function besides identifying candidates. While the experimental analysis is often time consuming involving number of experiments like pulldown assays, they are not necessarily limiting the ability to detect novel protein interactors. In this work, we discuss the role and putative interactors of SNAI2, a slug protein which is involved in the development of cancer progression. The protein interactions have been deciphered by domain pair exclusion method which gives confidence to already precluded interaction pairs. Additionally, conservation patterns of the slug protein have also been analysed by estimating site-specific evolutionary rates at structural level. Based upon the computational analysis, we consider HNF4A could be a putative candidate to study zinc finger protein slug. We believe, this candidate study augmented with structural conservation will definitely provide novel insights into the design and discovery of new interactions for zinc finger class of proteins besides providing possible clues for discovery of various cancer types associated with this class of proteins.

TAF4, a subunit of transcription factor II D, directs promoter occupancy of nuclear receptor HNF4A during post-natal hepatocyte differentiation.

The functions of the TAF subunits of mammalian TFIID in physiological processes remain poorly characterised. In this study, we describe a novel function of TAFs in directing genomic occupancy of a transcriptional activator. Using liver-specific inactivation in mice, we show that the TAF4 subunit of TFIID is required for post-natal hepatocyte maturation. TAF4 promotes pre-initiation complex (PIC) formation at post-natal expressed liver function genes and down-regulates a subset of embryonic expressed genes by increased RNA polymerase II pausing. The TAF4-TAF12 heterodimer interacts directly with HNF4A and in vivo TAF4 is necessary to maintain HNF4A-directed embryonic gene expression at post-natal stages and promotes HNF4A occupancy of functional cis-regulatory elements adjacent to the transcription start sites of post-natal expressed genes. Stable HNF4A occupancy of these regulatory elements requires TAF4-dependent PIC formation highlighting that these are mutually dependent events. Local promoter-proximal HNF4A-TFIID interactions therefore act as instructive signals for post-natal hepatocyte differentiation.

Krüppel-like factor 9 promotes hepatic cytochrome P450 2D6 expression during pregnancy in CYP2D6-humanized mice.

Cytochrome P450 2D6 (CYP2D6), a major drug-metabolizing enzyme, is responsible for metabolism of approximately 25% of marketed drugs. Clinical evidence indicates that metabolism of CYP2D6 substrates is increased during pregnancy, but the underlying mechanisms remain unclear. To identify transcription factors potentially responsible for CYP2D6 induction during pregnancy, a panel of genes differentially expressed in the livers of pregnant versus nonpregnant CYP2D6-humanized (tg-CYP2D6) mice was compiled via microarray experiments followed by real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR) verification. As a result, seven transcription factors-activating transcription factor 5 (ATF5), early growth response 1 (EGR1), forkhead box protein A3 (FOXA3), JUNB, Krüppel-like factor 9 (KLF9), KLF10, and REV-ERBα-were found to be up-regulated in liver during pregnancy. Results from transient transfection and promoter reporter gene assays indicate that KLF9 itself is a weak transactivator of CYP2D6 promoter but significantly enhances CYP2D6 promoter transactivation by hepatocyte nuclear factor 4 (HNF4α), a known transcriptional activator of CYP2D6 expression. The results from deletion and mutation analysis of CYP2D6 promoter activity identified a KLF9 putative binding motif at -22/-14 region to be critical in the potentiation of HNF4α-induced transactivation of CYP2D6. Electrophoretic mobility shift assays revealed a direct binding of KLF9 to the putative KLF binding motif. Results from chromatin immunoprecipitation assay showed increased recruitment of KLF9 to CYP2D6 promoter in the livers of tg-CYP2D6 mice during pregnancy. Taken together, our data suggest that increased KLF9 expression is in part responsible for CYP2D6 induction during pregnancy via the potentiation of HNF4α transactivation of CYP2D6.

Adiponectin upregulates SHBG production: molecular mechanisms and potential implications.

Epidemiological studies have shown that plasma SHBG levels correlate with plasma adiponectin levels, both in men and women. There are no reports describing any molecular mechanism by which adiponectin regulates hepatic SHBG production. The aim of the present study is to explore whether adiponectin regulates SHBG production by increasing HNF-4α levels through reducing hepatic lipid content. For this purpose, in vitro studies using human HepG2 cells, as well as human liver biopsies, were performed. Our results show that adiponectin treatment increased SHBG production via AMPK activation in HepG2 cells. Adiponectin treatment decreased the mRNA and protein levels of enzymes related to hepatic lipogenesis (ACC) and increased those related to fatty acid oxidation (ACOX and CPTI). These adiponectin-induced changes in hepatic enzymes resulted in a reduction of total TG and FFA and an increase of HNF-4α. When HNF-4α expression was silenced by using siRNA, adiponectin-induced SHBG overexpression was blocked. Furthermore, adiponectin-induced upregulation of SHBG production via HNF-4α overexpression was abrogated by the inhibition of fatty acid oxidation or by the induction of lipogenesis with a 30mM glucose treatment in HepG2 cells. Finally, adiponectin levels correlated positively and significantly with both HNF-4α and SHBG mRNA levels in human liver biopsies. Our results suggest for the first time that adiponectin increases SHBG production by activating AMPK, which reduces hepatic lipid content and increases HNF-4α levels.

Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α.

Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (~80 Å) to HNF4α, binding with high affinity Kd ~250-300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction altered protein secondary structure. Finally, L-FABP potentiated transactivation of HNF4α in COS7 cells. Taken together, these data suggest that L-FABP provides a signaling path to HNF4α activation in the nucleus.

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

The hepatocyte nuclear factor 4α (HNF-4α; also known as NR2A1) is a member of the nuclear receptor (NR) family of transcription factors, which have conserved DNA-binding domains and ligand-binding domains. HNF-4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF-4α response element. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism. In the pancreas HNF-4α is also a master regulator, controlling an estimated 11% of islet genes. HNF-4α protein mutations are linked to maturity-onset diabetes of the young, type 1 (MODY1) and hyperinsulinaemic hypoglycaemia. Previous structural analyses of NRs, although productive in elucidating the structure of individual domains, have lagged behind in revealing the connectivity patterns of NR domains. Here we describe the 2.9 Å crystal structure of the multidomain human HNF-4α 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 lead to changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the ligand-binding domain and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the interjunctional surfaces of the complex. The overall domain representation of the HNF-4α homodimer is different from that of the PPAR-γ-RXR-α heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and interdomain connections in NRs could be exploited by small-molecule allosteric modulators that affect distal functions in these polypeptides.

MED25 is a mediator component of HNF4α-driven transcription leading to insulin secretion in pancreatic beta-cells.

Unique nuclear receptor Hepatocyte Nuclear Factor 4α (HNF4α) is an essential transcriptional regulator for early development and proper function of pancreatic ß-cells, and its mutations are monogenic causes of a dominant inherited form of diabetes referred to as Maturity Onset Diabetes of the Young 1 (MODY1). As a gene-specific transcription factor, HNF4α exerts its function through various molecular interactions, but its protein recruiting network has not been fully characterized. Here we report the identification of MED25 as one of the HNF4α binding partners in pancreatic ß-cells leading to insulin secretion which is impaired in MODY patients. MED25 is one of the subunits of the Mediator complex that is required for induction of RNA polymerase II transcription by various transcription factors including nuclear receptors. This HNF4α-MED25 interaction was initially identified by a yeast-two-hybrid method, confirmed by in vivo and in vitro analyses, and proven to be mediated through the MED25-LXXLL motif in a ligand-independent manner. Reporter-gene based transcription assays and siRNA/shRNA-based gene silencing approaches revealed that this interaction is crucial for full activation of HNF4α-mediated transcription, especially expression of target genes implicated in glucose-stimulated insulin secretion. Selected MODY mutations at the LXXLL motif binding pocket disrupt these interactions and cause impaired insulin secretion through a 'loss-of-function' mechanism.

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.