HNF4α Binding Sites Research Articles

Hepatocyte nuclear factor 4α is a critical factor for the production of complement components in the liver.

The complement system plays an important role in biological defense as an effector to eliminate microorganisms that invade an organism and it is composed of more than 50 proteins, most of which are produced in the liver. Of these proteins, the mRNA expression of C3 and Cfb is known to be positively regulated by the nuclear receptor HNF4α. To investigate whether HNF4α regulates the complement system, we analyzed the hepatic expression of genes involved in the complement activation pathway and membrane attack complex (MAC) formation within the complement system using liver-specific Hnf4a-null mice (Hnf4aΔHep mice) and tamoxifen-induced liver-specific Hnf4a-null mice (Hnf4af/f;AlbERT2cre mice). We found that hepatic expression of many complement genes including C8a, C8b, C8g, and C9 that are involved in formation of the MAC was markedly decreased in Hnf4aΔHep mice and Hnf4af/f;AlbERT2cre mice. Furthermore, expression of C8A, C8B, and C8G was also decreased in human hepatoma cell lines in which the expression of HNF4α was suppressed, and expression of C8G and C9 was induced in a human immortalized hepatocyte cell line with forced expression of HNF4α. Transactivation of C8g and C9 was dependent on HNF4α expression of HNF4α binding sites, indicating that C8g and C9 are novel target genes of HNF4α. The results suggest that hepatic HNF4α plays an important role in regulation of the complement system, mainly MAC formation.

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Genetic and Epigenetic Characteristics of Inflammatory Bowel Disease–Associated Colorectal Cancer

Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder associated with an elevated risk of colorectal cancer (CRC). IBD-associated CRC (IBD-CRC) may represent a distinct pathway of tumorigenesis compared to sporadic CRC (sCRC). Our aim was to comprehensively characterize IBD-associated tumorigenesis integrating multiple high-throughput approaches, and to compare the results with in-house data sets from sCRCs. Whole-genome sequencing, single nucleotide polymorphism arrays, RNA sequencing, genome-wide methylation analysis, and immunohistochemistry were performed using fresh-frozen and formalin-fixed tissue samples of tumor and corresponding normal tissues from 31 patients with IBD-CRC. Transcriptome-based tumor subtyping revealed the complete absence of canonical epithelial tumor subtype associated with WNT signaling in IBD-CRCs, dominated instead by mesenchymal stroma-rich subtype. Negative WNT regulators AXIN2 and RNF43 were strongly down-regulated in IBD-CRCs and chromosomal gains at HNF4A, a negative regulator of WNT-induced epithelial-mesenchymal transition (EMT), were less frequent compared to sCRCs. Enrichment of hypomethylation at HNF4α binding sites was detected solely in sCRC genomes. PIGR and OSMR involved in mucosal immunity were dysregulated via epigenetic modifications in IBD-CRCs. Genome-wide analysis showed significant enrichment of noncoding mutations to 5'untranslated region of TP53 in IBD-CRCs. As reported previously, somatic mutations in APC and KRAS were less frequent in IBD-CRCs compared to sCRCs. Distinct mechanisms of WNT pathway dysregulation skew IBD-CRCs toward mesenchymal tumor subtype, which may affect prognosis and treatment options. Increased OSMR signaling may favor the establishment of mesenchymal tumors in patients with IBD.

Selenium-binding protein 1 alters energy metabolism in prostate cancer cells.

ObjectiveThe broad goal of the research described in this study was to investigate the contributions of selenium‐binding protein 1 (SBP1) loss in prostate cancer development and outcome.MethodsSBP1 levels were altered in prostate cancer cell lines and the consequences on oxygen consumption, expression of proteins associated with energy metabolism, and cellular transformation and migration were investigated. The effects of exposing cells to the SBP1 reaction products, H2O2 and H2S were also assessed. In silico analyses identified potential HNF4α binding sites within the SBP1 promoter region and this was investigated using an inhibitor specific for that transcription factor.ResultsUsing in silico analyses, it was determined that the promoter region of SBP1 contains putative binding sites for the HNF4α transcription factor. The potential for HNF4α to regulate SBP1 expression was supported by data indicating that HNF4α inhibition resulted in a dose‐response increase in the levels of SBP1 messenger RNA and protein, identifying HNF4α as a novel negative regulator of SBP1 expression in prostate cancer cells. The consequences of altering the levels of SBP1 were investigated by ectopically expressing SBP1 in PC‐3 prostate cancer cells, where SBP1 expression attenuated anchorage‐independent cellular growth and migration in culture, both properties associated with transformation. SBP1 overexpression reduced oxygen consumption in these cells and increased the activation of AMP‐activated protein kinase (AMPK), a major regulator of energy homeostasis. In addition, the reaction products of SBP1, H2O2, and H2S also activated AMPK.ConclusionsBased on the obtained data, it is hypothesized that SBP1 negatively regulates oxidative phosphorylation (OXPHOS) in the healthy prostate cells by the production of H2O2 and H2S and consequential activation of AMPK. The reduction of SBP1 levels in prostate cancer can occur due to increased binding of HNF4α, acting as a transcriptional inhibitor to the SBP1 promoter. Consequently, there is a reduction in H2O2 and H2S‐mediated signaling, inhibition of AMPK, and stimulation of OXPHOS and building blocks of biomolecules needed for tumor growth and progression. Other effects of SBP1 loss in tumor cells remain to be discovered.

Ser100-Phosphorylated RORα Orchestrates CAR and HNF4α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6.

We have previously shown that the retinoid-related orphan receptor alpha (RORα) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated RORα orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). RORα knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of RORα in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that RORα in the form of phosphorylated (p-) S100 directly bound to a newly identified RORα response element (RORα response element on CYP2B6 promoter, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 RORα was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4α binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORα at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4α formed a complex with Ser 100-phosphorylated RORα, as shown by supershifted complexes with anti-p-Ser100 RORα and anti-HNF4α antibodies. Altogether, the results established that p-Ser100 RORα bridging the PBREM and OARE orchestrates CAR and HNF4α to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT: CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics, and it is prone to induction by xenobiotics, including phenobarbital via constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α). Here, we show that retinoid-related orphan receptor alpha (RORα), through phosphorylated S100 residue, orchestrated CAR-HNF4α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role of RORα in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation sites within the DNA-binding domain of the receptor.

Stearoyl-CoA desaturase (scd1a) is epigenetically regulated by broodstock nutrition in gilthead sea bream (Sparus aurata)

ABSTRACTThe aim of this study was to generate new knowledge on fish epigenetics, assessing the effects of linolenic acid (ALA) conditioning of broodstock in the offspring of the marine fish Sparus aurata. Attention was focused on gene organization, methylation signatures and gene expression patterns of fatty acid desaturase 2 (fads2) and stearoyl-CoA desaturase 1a (scd1a). Blat searches in the genomic IATS-CSIC database (www.nutrigroup-iats.org/seabreamdb) highlighted a conserved exon-intron organization, a conserved PUFA response region, and CG islands at the promoter regions of each gene. The analysed CpG positions in the fads2 promoter were mostly hypomethylated and refractory to broodstock nutrition. The same response was achieved after conditioning of juvenile fish to low water oxygen concentrations, thus methylation susceptibility at individual CpG sites seems to be stringently regulated in fish of different origin and growth trajectories. Conversely, the scd1a promoter was responsive to broodstock nutrition and the offspring of parents fed the ALA-rich diet shared an increased DNA-methylation, mainly in CpG sites neighbouring SP1 and HNF4α binding sites. Cytosine methylation at these sites correlated inversely with the hepatic scd1a expression of the offspring. Co-expression analyses supported that the HNF4α-dependent regulation of scd1a is affected by DNA methylation. The phenotypic output is a regulated liver fat deposition through changes in scd1 expression, which would also allow the preservation of fatty acid unsaturation levels in fish fed reduced levels of n-3 LC-PUFA. Collectively, these findings reveal a reliable mechanism by which parent’s nutrition can shape scd1a gene expression in the fish offspring.

Multiple Plasticity Regulators Reveal Targets Specifying an Induced Predatory Form in Nematodes.

The ability to translate a single genome into multiple phenotypes, or developmental plasticity, defines how phenotype derives from more than just genes. However, to study the evolutionary targets of plasticity and their evolutionary fates, we need to understand how genetic regulators of plasticity control downstream gene expression. Here, we have identified a transcriptional response specific to polyphenism (i.e., discrete plasticity) in the nematode Pristionchus pacificus. This species produces alternative resource-use morphs-microbivorous and predatory forms, differing in the form of their teeth, a morphological novelty-as influenced by resource availability. Transcriptional profiles common to multiple polyphenism-controlling genes in P. pacificus reveal a suite of environmentally sensitive loci, or ultimate target genes, that make up an induced developmental response. Additionally, in vitro assays show that one polyphenism regulator, the nuclear receptor NHR-40, physically binds to promoters with putative HNF4α (the nuclear receptor class including NHR-40) binding sites, suggesting this receptor may directly regulate genes that describe alternative morphs. Among differentially expressed genes were morph-limited genes, highlighting factors with putative "on-off" function in plasticity regulation. Further, predatory morph-biased genes included candidates-namely, all four P. pacificus homologs of Hsp70, which have HNF4α motifs-whose natural variation in expression matches phenotypic differences among P. pacificus wild isolates. In summary, our study links polyphenism regulatory loci to the transcription producing alternative forms of a morphological novelty. Consequently, our findings establish a platform for determining how specific regulators of morph-biased genes may influence selection on plastic phenotypes.

HNF4α and CDX2 Regulate Intestinal YAP1 Promoter Activity.

The Hippo pathway is important for tissue homeostasis, regulation of organ size and growth in most tissues. The co-transcription factor yes-associated protein 1 (YAP1) serves as a main downstream effector of the Hippo pathway and its dysregulation increases cancer development and blocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation of YAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene and transcription factors important for intestinal expression. Bioinformatic analysis of caudal type homeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitated DNA from differentiated Caco-2 cells revealed potential intragenic enhancers in the YAP1 gene. Transfection of luciferase-expressing YAP1 promoter-reporter constructs containing the potential enhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco-2 and T84 cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silico transcription factor binding site analysis and protein-DNA binding was confirmed in vitro using electrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experiments that CDX2 and HNF4α bind to the YAP1 enhancer in Caco-2 cells. These results reveal a previously unknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for high expression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding are important for the YAP1 enhancer activity in intestinal epithelial cells.

Repression of hepatocyte nuclear factor 4 alpha by AP-1 underlies dyslipidemia associated with retinoic acid

All-trans retinoic acid (atRA) is used to treat certain cancers and dermatologic diseases. A common adverse effect of atRA is hypercholesterolemia; cytochrome P450 (CYP) 7A repression is suggested as a driver. However, the underlying molecular mechanisms remain unclear. We investigated CYP7A1 expression in the presence of atRA in human hepatocytes and hepatic cell lines. In HepaRG cells, atRA increased cholesterol levels dose-dependently alongside dramatic decreases in CYP7A1 expression. Lentiviral-mediated CYP7A1 overexpression reversed atRA-induced cholesterol accumulation, suggesting that CYP7A1 repression mediated cholesterol accumulation. In CYP7A1 promoter reporter assays and gene-knockdown studies, altered binding of hepatocyte nuclear factor 4 α (HNF4α) to the proximal promoter was essential for atRA-mediated CYP7A1 repression. Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) and ERK pathways attenuated atRA-mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP-1 (c-Jun/c-Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression. In DNA pull-down and chromatin immunoprecipitation assays, AP-1 exhibited sequence-specific binding to the proximal CYP7A1 promoter region overlapping the HNF4α binding site, and atRA increased AP-1 but decreased HNF4α recruitment to the promoter. Collectively, these results indicate that atRA activates JNK and ERK pathways and the downstream target AP-1 represses HNF4α transactivation of the CYP7A1 promoter, potentially responsible for hypercholesterolemia.

Hepatocyte nuclear factor 4α regulates megalin expression in proximal tubular cells

Hepatocyte nuclear factor 4α (HNF4α) is a member of the nuclear receptor superfamily and upregulates expression of many genes in the liver, pancreas, small intestine, and colon. HNF4α is also highly expressed in proximal tubular epithelial cells (PTECs) in kidney. PTECs reabsorb various substances through transporters, ion channels, and receptors, but the target genes for HNF4α in PTECs have not been investigated in detail. In the present study, we aimed to identify novel HNF4α target genes that are highly expressed in PTECs. Expression of many solute carrier transporter genes was upregulated by HNF4α in human PTEC-derived HK-2 cells. Notably, expression of megalin (LRP2), an endocytic receptor of various molecules involved in development and progression of chronic kidney disease (CKD), was strongly induced by HNF4α, and the transactivation potential of the megalin promoter was dependent on HNF4α expression. Moreover, HNF4α was found to directly bind to an HNF4α binding site near the transcription start site in the megalin gene. These results indicate that HNF4α plays an important role in maintaining reabsorption and metabolism in PTECs by positive regulation of several solute carrier transporter and megalin genes at the transcriptional level.

Derivation and molecular characterization of pancreatic differentiated MODY1-iPSCs

Maturity onset diabetes of the young (MODY) is a hereditary form of diabetes mellitus presenting at childhood or adolescence, which eventually leads to pancreatic β-cells dysfunction. The underlying genetic basis of MODY disorders is haploinsufficiency, where loss-of-function mutations in a single allele cause the diabetic phenotype in heterozygous patients. MODY1 is a type of MODY disorder resulting from a mutation in the transcription factor hepatocyte nuclear factor 4 alpha (HNF4α). In order to establish a human based model to study MODY1, we generated patient-derived induced pluripotent stem cells (iPSCs). Differentiation of these pluripotent cells towards the pancreatic lineage enabled to evaluate the effects of the MODY1 mutation and its impact on endodermal and pancreatic cells. Analyzing the gene expression profiles of differentiated MODY1 cells, revealed the outcome of HNF4α haploinsufficiency on its targets. This molecular analysis suggests that the differential expression of HNF4α target genes in MODY1 is affected by the number of HNF4α binding sites, their distance from the transcription start site, and the number of other transcription factor binding sites. These features may help explain the molecular manifestations of haploinsufficiency in MODY1 disease.

AtRA induces cholesterol accumulation through CYP7A1 repression in the liver

All‐trans retinoic acid (atRA) is one of the most frequently used retinoids to treat certain cancers or dermatological diseases. Common side effects of atRA include hypercholesterolemia, leading to discontinuation of atRA uses. Previous studies have shown that atRA downregulates CYP7A1 (the rate‐liming enzyme converting cholesterol to a bile acid intermediate), potentially responsible for hypercholesterolemia. However, the detailed molecular mechanisms in atRA‐induced hypercholesterolemia is unclear. atRA increased cholesterol levels in a dose dependent manner in HepaRG cells, and this was accompanied by dramatic decrease in CYP7A1 mRNA and protein levels. CYP7A1 overexpression by lentiviral system reversed atRA‐induced cholesterol accumulation in HepaRG cells, suggesting that the cholesterol accumulation is mediated by CYP7A1 repression. CYP7A1 promoter reporter assays in HepG2 cells revealed that the HNF4α binding site in the promoter (−149/−118) is essential for atRA‐mediated CYP7A1 repression. HNF4α depletion by siRNA abrogated CYP7A1 repression by atRA in HepaRG cells. Western blot analysis revealed that atRA activates MAPKs signaling pathways including JNK, ERK, and p38 MAPKs in HepaRG cells. Pharmacological inhibition of JNK and ERK pathways but not p38 pathway by using a specific inhibitor (i.e., SP600125, PD98059, and SB203580 for JNK, ERK, and p38, respectively) attenuated atRA‐mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP‐1 (c‐Jun/c‐Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression in HepG2 cells. In DNA pull‐down assay, AP‐1 exhibited sequence‐specific binding to the HNF4α binding site in CYP7A1 promoter following atRA treatment in HepaRG cells. Chromatin immunoprecipitation assay also showed that AP‐1 binding to the CYP7A1 promoter (−181/−34) was increased following atRA treatment, whereas HNF4α binding was decreased. Collectively, results from this study indicate that atRA‐activated JNK and ERK pathways and downstream target AP‐1 represses HNF4α transactivation of CYP7A1 promoter. These results suggest an important role of CYP7A1 in atRA‐induced cholesterol accumulation and provide a basis to establish future strategy in cholesterol disorder by atRA.Support or Funding InformationThis study was supported by R01 HD089455.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interleukin-35 stimulates hepatitis B virus transcription and replication by targeting transcription factor HNF4α.

Hepatitis B virus (HBV) infection is a major health problem worldwide. Interleukin-35 (IL-35) is a definite immunosuppressive cytokine belonging to the IL-12 family. Nevertheless, the role of IL-35 in HBV replication remains elusive. In this study, we found that the level of HBV DNA replicative intermediates detected by qPCR and Southern blotting analysis was significantly increased by rhIL-35 in a dose-dependent manner. Moreover, HBV 3.5 kb mRNA levels were up-regulated by rhIL-35. The HBV core protein level as well as the HBsAg and HBeAg secretion levels were also increased by rhIL-35. Moreover, a mechanistic study demonstrated that IL-35 promoted HBV replication by enhancing the HBV core promoter activity. Importantly, hepatocyte nuclear factor 4α (HNF4α) was probably the target of IL-35. Mutation of the HNF4α-binding site on HBV core promoter or silencing HNF4α abolished the enhancement of HBV replication induced by IL-35. Finally, rhIL-35 was able to increase HBV replication in HBV transgenic mice. Taken together, our findings demonstrated that IL-35 has a novel role in HBV replication.

Hairy and enhancer of split 6 prevents hepatic lipid accumulation through inhibition of Pparg2 expression.

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a master regulator for white adipocyte differentiation and lipid storage. The increased level of hepatic PPARγ2 isoform reprograms liver for lipid storage and causes abnormal fat accumulation in certain pathophysiologic conditions. The current study aimed to investigate a role of transcriptional repressor hairy and enhancer of split 6 (HES6) in the regulation of Pparg2 expression and hepatic steatosis induced by diet. Liver‐specific overexpression of Hes6 using adenovirus reduced Pparg2 messenger RNA levels by 90% and hepatic triglyceride accumulation by 22% compared to the levels in mice injected with an adenoviral empty vector with Western diet feeding. In sharp contrast, silencing Hes6 gene expression using short hairpin RNA increased hepatic lipid accumulation and Pparg2 messenger RNA levels by 70% and 4‐fold, respectively. To locate hepatocyte nuclear factor 4 alpha (HNF4α) binding site(s), through which repressional activity of HES6 is mediated, a 2.5‐kb Pparg2 promoter‐driven luciferase reporter was constructed for transient transfection assays. Subsequently, chromatin immunoprecipitation and electrophoretic mobility shift assays were performed. An HNF4α binding consensus sequence was identified at 903 base pairs upstream from the transcription start site of Pparg2. Deletion or point mutation of the sequence in a luciferase reporter containing the Pparg2 promoter abolished HNF4α‐mediated activation in HeLa cells. Chromatin immunoprecipitation and electrophoretic mobility shift assays further confirmed direct recruitment and binding of HNF4α to the site. Gene expression analysis with liver samples from subjects with nonalcoholic steatohepatitis suggested that the axis of the Hes6–Hnf4a–Pparg2 transcriptional cascade is also responsible for hepatic fat accumulation in humans. Conclusion: HES6 represses Pparg2 gene expression, thereby preventing hepatic lipid accumulation induced by chronic Western diet feeding or pathophysiologic conditions. (Hepatology Communications 2017;1:1085–1098)

Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3.

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

Abstract 3559: Functional characterization of HNF4α in gastric cancer

Abstract Gastric cancer (GC) is a leading cause of cancer mortality worldwide and particularly common in East Asia. Transcription factors are frequently amplified or overexpressed in GC and might contribute to tumorgenesis, however the underlying mechanisms largely remain unknown. Our previous data indicates that HNF4α is a common target of three amplified transcription factors (GATA4, GATA6 and KLF5) in gastric cancer. HNF4α had also been found to be related to diverse aspects of tumorigenesis. In liver and colon cancers, there are reports indicating that the targets of HNF4α are mainly involved in intermediary metabolism, however, its tissue-specific downstream targets and pathways have not been systemically studied yet. Therefore, a comprehensive functional characterization of HNF4α is required to identify its tissue-specific role in gastric cancer. In this study, we analyzed the expression features of HNF4α in GC, and found the expression level of HNF4α is significantly higher in early stage tumor and in CIN and MSI compared to GS and EBV subtypes of GC according to TCGA classification method. In addition, we performed the HNF4α ChIP in GC cell lines, and the results revealed a number of HNF4α binding sites in GC. Comparison with the published ChIP-seq data of Caco2 and HepG2 cell lines indicated the existence of GC-exclusive targets. Furthermore, functional annotation analysis revealed that the GC-exclusive targets are significantly related to the negative regulation of cellular metabolic process compared to the common targets. These preliminary data suggests that HNF4α might play a tissue specific role in GC, especially in metabolic related processes. Through further target validation and biological function studies, this study hopes to systemically investigate of HNF4α’s role in GC tumorigenesis, especially in metabolism related pathways. Citation Format: Chang Xu, Wen Fong Ooi, Aditi Qamra, Patrick Tan. Functional characterization of HNF4α in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3559. doi:10.1158/1538-7445.AM2017-3559

Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and β-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2.

Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.

Propionate induces the bovine cytosolic phosphoenolpyruvate carboxykinase promoter activity

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a critical enzyme within the metabolic networks for gluconeogenesis, hepatic energy metabolism, and tricarboxylic acid cycle function, and is controlled by several transcription factors including hepatic nuclear factor 4α (HNF4α). The primary objective of the present study was to determine whether propionate regulates bovine PCK1 transcription. The second objective was to determine the action of cyclic AMP (cAMP), glucocorticoids, and insulin, hormonal cues known to modulate glucose metabolism, on bovine PCK1 transcriptional activity. The proximal promoter of the bovine PCK1 gene was ligated to a Firefly luciferase reporter and transfected into H4IIE hepatoma cells. Cells were exposed to treatments for 23 h and luciferase activity was determined in cell lysates. Activity of the PCK1 promoter was linearly induced by propionate, and maximally increased 7-fold with 2.5 mM propionate, which was not muted by 100 nM insulin. Activity of the PCK1 promoter was increased 1-fold by either 1.0 mM cAMP or 5.0µM dexamethasone, and 2.2-fold by their combination. Induction by cAMP and dexamethasone was repressed 50% by 100 nM insulin. Propionate, cAMP, and dexamethasone acted synergistically to induce the PCK1 promoter activity. Propionate-responsive regions, identified by 5′ deletion analysis, were located between −1,238 and −409 bp and between −85 and +221 bp. Deletions of the core sequences of the 2 putative HNF4α sites decreased the responsiveness to propionate by approximately 40%. These data indicate that propionate regulates its own metabolism through transcriptional stimulation of the bovine PCK1 gene. This induction is mediated, in part, by the 2 putative HNF4α binding sites in the bovine PCK1 promoter.

Hepatocyte Nuclear Factor 4α Controls Iron Metabolism and Regulates Transferrin Receptor 2 in Mouse Liver

Iron is an essential element in biological systems, but excess iron promotes the formation of reactive oxygen species, resulting in cellular toxicity. Several iron-related genes are highly expressed in the liver, a tissue in which hepatocyte nuclear factor 4α (HNF4α) plays a critical role in controlling gene expression. Therefore, the role of hepatic HNF4α in iron homeostasis was examined using liver-specific HNF4α-null mice (Hnf4aΔH mice). Hnf4aΔH mice exhibit hypoferremia and a significant change in hepatic gene expression. Notably, the expression of transferrin receptor 2 (Tfr2) mRNA was markedly decreased in Hnf4aΔH mice. Promoter analysis of the Tfr2 gene showed that the basal promoter was located at a GC-rich region upstream of the transcription start site, a region that can be transactivated in an HNF4α-independent manner. HNF4α-dependent expression of Tfr2 was mediated by a proximal promoter containing two HNF4α-binding sites located between the transcription start site and the translation start site. Both the GC-rich region of the basal promoter and the HNF4α-binding sites were required for maximal transactivation. Moreover, siRNA knockdown of HNF4α suppressed TFR2 expression in human HCC cells. These results suggest that Tfr2 is a novel target gene for HNF4α, and hepatic HNF4α plays a critical role in iron homeostasis.

Vanin-1 Is a Key Activator for Hepatic Gluconeogenesis

Vanin-1 (VNN1) is a liver-enriched oxidative stress sensor that has been implicated in the regulation of multiple metabolic pathways. Clinical investigations indicated that the levels of VNN1 were increased in the urine and blood of diabetic patients, but the physiological significance of this phenomenon remains unknown. In this study, we demonstrated that the hepatic expression of VNN1 was induced in fasted mice or mice with insulin resistance. Gain- and loss-of-function studies indicated that VNN1 increased the expression of gluconeogenic genes and hepatic glucose output, which led to hyperglycemia. These effects of VNN1 on gluconeogenesis were mediated by the regulation of the Akt signaling pathway. Mechanistically, vnn1 transcription was activated by the synergistic interaction of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and hepatocyte nuclear factor-4α (HNF-4α). A chromatin immunoprecipitation analysis indicated that PGC-1α was present near the HNF-4α binding site on the proximal vnn1 promoter and activated the chromatin structure. Taken together, our results suggest an important role for VNN1 in regulating hepatic gluconeogenesis. Therefore, VNN1 may serve as a potential therapeutic target for the treatment of metabolic diseases caused by overactivated gluconeogenesis.