Transcriptionally controlled transcription factor. Binds to DNA sites required for the transcription of alpha 1-antitrypsin, apolipoprotein CIII, transthyretin genes and HNF1-alpha. May be essential for development of the liver, kidney and intestine.
Interacting selectively and non-covalently with an activating transcription factor, any protein whose activity is required to initiate or upregulate transcription.
J. Biol. Chem. 274, 33050-33056 (1999)[PubMed:10551874]
The transcriptional regulation of the apoCIII gene by hormonal and metabolic signals plays a significant role in determining plasma triglyceride levels. In the current work we demonstrate that the apoCIII gene is regulated by the mitogen-activated protein (MAP) kinase signaling pathway. In HepG2 cells, repression of MAP kinase activity by treatment with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 caused a 5-8-fold increase in apoCIII transcriptional activity. Activation of MAP kinase by phorbol ester treatment caused a 3-5-fold reduction in apoCIII transcription. The region of the apoCIII promoter responsible for this regulation was mapped in transiently transfected HepG2 cells to a 6-base pair element located at -740. The major protein binding to this site was identified as the nuclear hormone receptor HNF4. An increase in HNF4 mRNA and protein levels was observed in HepG2 cells after treatment with PD98059, indicating that the MAP kinase pathway regulates the expression of the HNF4 gene. These findings demonstrate that the apoCIII gene can be regulated by signals acting through the MAP kinase pathway and that this regulation is mediated, at least in part, by changes in the amount of HNF4.
We have used apolipoprotein genes to investigate the signal transduction mechanisms involved in the control of intestinal specific gene expression. The human apoAI, apoCIII, and apoAIV genes are tandemly organized within a 15-kb DNA segment and are expressed predominantly in the liver and intestine. Transient transfection of various human apoAI gene plasmid constructs into human hepatoma (HepG2) and colon carcinoma (Caco-2) cells showed that apoAI gene transcription is under the control of two separate and distinct cell-specific promoters. The region between nucleotides -192 and -41 is essential for expression in HepG2 cells, whereas the region from -595 to -192 is essential for expression in Caco-2 cells. A third 0.6 kb DNA fragment in the apoCIII gene promoter region, approximately 5 kb down-stream from the human apoAI gene, enhances transcription mediated by either of these two tissue-specific apoAI promoters. In Caco-2 cells, expression of the apoAI gene and activation by the distal enhancer required the presence of a nuclear hormone receptor response element (NHRRE) located in the -214 to -192 apoAI promoter region. Overexpression of the orphan receptor hepatocyte nuclear factor 4 (HNF-4), which binds to the NHRRE, dramatically stimulates apoAI gene expression in Caco-2 cells but not in HepG2 cells. Maximal stimulation of transcription by HNF-4 in Caco-2 cells required the presence of both the intestinal specific promoter, the NHRRE, and distal enhancer elements. Transactivation by HNF-4 thus appears to result from functional synergy between the NHRRE binding HNF-4 and distal DNA elements containing intestinal-specific DNA binding activities. The apoAI gene provides a model system to define the mechanism(s) governing intestinal cell specific gene regulation and the role of nuclear hormone receptors in the establishment and regulation of enterocytic gene transcription.
Interacting selectively and non-covalently with fatty acids, aliphatic monocarboxylic acids liberated from naturally occurring fats and oils by hydrolysis.
The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.
Ligand-activated sequence-specific DNA binding RNA polymerase II transcription factor activitydefinition[GO:0004879]‹silver
Combining with a signal and transmitting the signal to the transcriptional machinery by interacting selectively and non-covalently with a specific DNA sequence in order to modulate transcription by RNA polymerase II.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionBHF-UCL
Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Reversible histone methylation and demethylation are highly regulated processes that are crucial for chromatin reorganization and regulation of gene transcription in response to extracellular conditions. However, the mechanisms that regulate histone-modifying enzymes are largely unknown. Here, we characterized a protein kinase A (PKA)-dependent histone lysine demethylase complex, PHF2-ARID5B. PHF2, a jmjC demethylase, is enzymatically inactive by itself, but becomes an active H3K9Me2 demethylase through PKA-mediated phosphorylation. We found that phosphorylated PHF2 then associates with ARID5B, a DNA-binding protein, and induce demethylation of methylated ARID5B. This modification leads to targeting of the PHF2-ARID5B complex to its target promoters, where it removes the repressive H3K9Me2 mark. These findings suggest that the PHF2-ARID5B complex is a signal-sensing modulator of histone methylation and gene transcription, in which phosphorylation of PHF2 enables subsequent formation of a competent and specific histone demethylase complex.
Evidence
3:
Inferred from Physical InteractionUniProtKB
Alpha-synuclein (SNCA) is an abundant neuronal protein involved in synaptic neurotransmission. SNCA expression levels have been strongly implicated in Parkinson's disease pathogenesis. We have previously demonstrated that in the PC12 cell line elements in intron 1 may mediate SNCA transcriptional regulation in response to neurotrophins. We have now identified transcription factor (TF) binding sites in intron 1 and the 5'-promoter of SNCA. A binding site for the TF zinc finger and SCAN domain containing (ZSCAN)21 in the 5'-region of intron 1 is required for intron 1 transcriptional activity. Small interfering RNA against ZSCAN21 inhibits activation in the luciferase assay and diminishes SNCA protein levels in naïve and neurotrophin-treated PC12 cells and in primary cultured cortical neurons, demonstrating that ZSCAN21 is a novel transcriptional regulator of SNCA in neuronal cells. The 5'-promoter of SNCA has a complex architecture, including multiple binding sites for the TF zinc finger protein (ZNF)219, which functions as both an activator and a repressor. Targeting ZSCAN21 or other TFs controlling SNCA transcriptional activity may provide novel therapeutic avenues not only for Parkinson's disease but also for other synucleopathies.
The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.
Interacting selectively and non-covalently with one or more specific sites on a receptor molecule, a macromolecule that undergoes combination with a hormone, neurotransmitter, drug or intracellular messenger to initiate a change in cell function.
The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.
RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity involved in positive regulation of transcriptiondefinition[GO:0001077]
Interacting selectively and non-covalently with a sequence of DNA that is in cis with and relatively close to a core promoter for RNA polymerase II (RNAP II) in order to activate or increase the frequency, rate or extent of transcription from the RNAP II promoter.
ISSOrtholog Curator
RNA polymerase II distal enhancer sequence-specific DNA binding transcription factor activitydefinition[GO:0003705]‹silver
Interacting selectively and non-covalently with a sequence of DNA that is in a distal enhancer region for RNA polymerase II (RNAP II) in order to modulate transcription by RNAP II.
Interacting selectively and non-covalently with DNA of a specific nucleotide composition, e.g. GC-rich DNA binding, or with a specific sequence motif or type of DNA e.g. promotor binding or rDNA binding.
Interacting selectively and non-covalently with a specific DNA sequence in order to modulate transcription. The transcription factor may or may not also interact selectively with a protein or macromolecular complex.
We have used apolipoprotein genes to investigate the signal transduction mechanisms involved in the control of intestinal specific gene expression. The human apoAI, apoCIII, and apoAIV genes are tandemly organized within a 15-kb DNA segment and are expressed predominantly in the liver and intestine. Transient transfection of various human apoAI gene plasmid constructs into human hepatoma (HepG2) and colon carcinoma (Caco-2) cells showed that apoAI gene transcription is under the control of two separate and distinct cell-specific promoters. The region between nucleotides -192 and -41 is essential for expression in HepG2 cells, whereas the region from -595 to -192 is essential for expression in Caco-2 cells. A third 0.6 kb DNA fragment in the apoCIII gene promoter region, approximately 5 kb down-stream from the human apoAI gene, enhances transcription mediated by either of these two tissue-specific apoAI promoters. In Caco-2 cells, expression of the apoAI gene and activation by the distal enhancer required the presence of a nuclear hormone receptor response element (NHRRE) located in the -214 to -192 apoAI promoter region. Overexpression of the orphan receptor hepatocyte nuclear factor 4 (HNF-4), which binds to the NHRRE, dramatically stimulates apoAI gene expression in Caco-2 cells but not in HepG2 cells. Maximal stimulation of transcription by HNF-4 in Caco-2 cells required the presence of both the intestinal specific promoter, the NHRRE, and distal enhancer elements. Transactivation by HNF-4 thus appears to result from functional synergy between the NHRRE binding HNF-4 and distal DNA elements containing intestinal-specific DNA binding activities. The apoAI gene provides a model system to define the mechanism(s) governing intestinal cell specific gene regulation and the role of nuclear hormone receptors in the establishment and regulation of enterocytic gene transcription.
Am. J. Physiol. 276, G1181-94-G1181-94 (1999)[PubMed:10330009]
There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha1-antitrypsin (alpha1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1alpha (HNF-1alpha), HNF-1beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha1-AT gene. Cotransfection studies showed that HNF-1alpha and HNF-4 had a synergistic effect on alpha1-AT gene expression. RNA blot analysis showed that HNF-1alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1alpha binding activity increase coordinately with alpha1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha1-AT gene expression. Together, these results provide evidence that HNF-1alpha and HNF-4 play a role in the mechanism by which the alpha1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.
Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.
J. Thromb. Haemost. 1, 1688-1698 (2003)[PubMed:12911579]
BACKGROUND: Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. OBJECTIVES: The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. METHODS: We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. RESULTS: We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. CONCLUSIONS: The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.
Interacting selectively and non-covalently with a DNA region that regulates the transcription of a region of DNA, which may be a gene, cistron, or operon. Binding may occur as a sequence specific interaction or as an interaction observed only once a factor has been recruited to the DNA by other factors.
Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.
The sequential process in which the multiple coagulation factors of the blood interact, ultimately resulting in the formation of an insoluble fibrin clot; it may be divided into three stages: stage 1, the formation of intrinsic and extrinsic prothrombin converting principle; stage 2, the formation of thrombin; stage 3, the formation of stable fibrin polymers.
J. Thromb. Haemost. 1, 1688-1698 (2003)[PubMed:12911579]
BACKGROUND: Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. OBJECTIVES: The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. METHODS: We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. RESULTS: We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. CONCLUSIONS: The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.
Drug Metab. Pharmacokinet. 22, 287-298 (2007)[PubMed:17827783]
Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.
The chemical reactions and pathways involving lipids, compounds soluble in an organic solvent but not, or sparingly, in an aqueous solvent. Includes fatty acids; neutral fats, other fatty-acid esters, and soaps; long-chain (fatty) alcohols and waxes; sphingoids and other long-chain bases; glycolipids, phospholipids and sphingolipids; and carotenes, polyprenols, sterols, terpenes and other isoprenoids.
Abstract Few genes are known to be involved in renal cell carcinoma (RCC) development and progression. The cell-specific transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha) is down-regulated in RCC and we have shown that HNF4 alpha inhibits cell proliferation in the embryonic kidney cell line HEK293. To clarify the possible tumor suppressor activity of HNF4 alpha we analyzed the whole human expression profile in HEK293 cells upon HNF4 alpha induction. By comparing induced and uninduced cells, we identified 1411 differentially expressed genes. Using RNA interference, we screened 56 HNF4 alpha-regulated genes for their possible role in mediating inhibition of cell proliferation triggered by HNF4 alpha. We demonstrate that 14 of these regulated genes are able to contribute to the inhibitory effect of HNF4 alpha on cell proliferation, including well-known cancer genes, such as CDKN1A (p21), TGFA, MME (NEP) and ADAMTS1. In addition, the genes SEPP1, THEM2, BPHL, DSC2, ANK3, ALDH6A1, EPHX2, NELL2, EFHD1 and PROS1 are also part of the network of HNF4 alpha target genes that regulate proliferation in HEK293 cells. Therefore, we postulate that HNF4 alpha orchestrates, at least, these 14 genes to regulate cell proliferation in HEK293 cells and that down-regulation of HNF4 alpha could contribute to the progression of kidney cancer.
Abstract Few genes are known to be involved in renal cell carcinoma (RCC) development and progression. The cell-specific transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha) is down-regulated in RCC and we have shown that HNF4 alpha inhibits cell proliferation in the embryonic kidney cell line HEK293. To clarify the possible tumor suppressor activity of HNF4 alpha we analyzed the whole human expression profile in HEK293 cells upon HNF4 alpha induction. By comparing induced and uninduced cells, we identified 1411 differentially expressed genes. Using RNA interference, we screened 56 HNF4 alpha-regulated genes for their possible role in mediating inhibition of cell proliferation triggered by HNF4 alpha. We demonstrate that 14 of these regulated genes are able to contribute to the inhibitory effect of HNF4 alpha on cell proliferation, including well-known cancer genes, such as CDKN1A (p21), TGFA, MME (NEP) and ADAMTS1. In addition, the genes SEPP1, THEM2, BPHL, DSC2, ANK3, ALDH6A1, EPHX2, NELL2, EFHD1 and PROS1 are also part of the network of HNF4 alpha target genes that regulate proliferation in HEK293 cells. Therefore, we postulate that HNF4 alpha orchestrates, at least, these 14 genes to regulate cell proliferation in HEK293 cells and that down-regulation of HNF4 alpha could contribute to the progression of kidney cancer.
The chemical reactions and pathways involving ornithine, an amino acid only rarely found in proteins, but which is important in living organisms as an intermediate in the reactions of the urea cycle and in arginine biosynthesis.
Drug Metab. Pharmacokinet. 22, 287-298 (2007)[PubMed:17827783]
Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.
Drug Metab. Pharmacokinet. 22, 287-298 (2007)[PubMed:17827783]
Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.
We have used apolipoprotein genes to investigate the signal transduction mechanisms involved in the control of intestinal specific gene expression. The human apoAI, apoCIII, and apoAIV genes are tandemly organized within a 15-kb DNA segment and are expressed predominantly in the liver and intestine. Transient transfection of various human apoAI gene plasmid constructs into human hepatoma (HepG2) and colon carcinoma (Caco-2) cells showed that apoAI gene transcription is under the control of two separate and distinct cell-specific promoters. The region between nucleotides -192 and -41 is essential for expression in HepG2 cells, whereas the region from -595 to -192 is essential for expression in Caco-2 cells. A third 0.6 kb DNA fragment in the apoCIII gene promoter region, approximately 5 kb down-stream from the human apoAI gene, enhances transcription mediated by either of these two tissue-specific apoAI promoters. In Caco-2 cells, expression of the apoAI gene and activation by the distal enhancer required the presence of a nuclear hormone receptor response element (NHRRE) located in the -214 to -192 apoAI promoter region. Overexpression of the orphan receptor hepatocyte nuclear factor 4 (HNF-4), which binds to the NHRRE, dramatically stimulates apoAI gene expression in Caco-2 cells but not in HepG2 cells. Maximal stimulation of transcription by HNF-4 in Caco-2 cells required the presence of both the intestinal specific promoter, the NHRRE, and distal enhancer elements. Transactivation by HNF-4 thus appears to result from functional synergy between the NHRRE binding HNF-4 and distal DNA elements containing intestinal-specific DNA binding activities. The apoAI gene provides a model system to define the mechanism(s) governing intestinal cell specific gene regulation and the role of nuclear hormone receptors in the establishment and regulation of enterocytic gene transcription.
Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.
Any process that modulates the rate or extent of gastrulation. Gastrulation is the complex and coordinated series of cellular movements that occurs at the end of cleavage during embryonic development of most animals.
Any process that modulates the rate, frequency or extent of the growth hormone receptor signaling pathway. The growth hormone receptor signaling pathway is the series of molecular signals generated as a consequence of growth hormone receptor binding to its physiological ligand.
Human (h) GH plays an essential role in growth and metabolism, and its effectiveness is modulated by the availability of its specific receptor [hGH receptor (hGHR)] on target cells. The hGHR gene has a complex 5'-regulatory region containing multiple first exons. Seven are clustered within two small regions: V2,V3,V9 (module A) and V1,V4,V7,V8 (module B). Module A-derived mRNAs are ubiquitously expressed whereas those from module B are only found in postnatal liver, suggesting developmental- and liver-specific regulation of module B hGHR gene expression. To characterize the elements regulating module B activity, we studied a 1.8-kb promoter of the highest expressing exon in liver, V1. This promoter was repressed in transfection assays; however, either 5'- or 3'-deletions relieved this, suggesting the presence of multiple negative regulatory elements. Six putative hepatic nuclear factor 4 (HNF-4) response elements were identified. We determined that HNF-4alpha is developmentally regulated in the human liver: HNF-4alpha2 and HNF-4alpha8 are expressed in fetal hepatocytes but only HNF-4alpha2 is expressed in postnatal liver. Transient transfection assays demonstrated that HNF-4alpha2 and HNF-4alpha8 have a similar dual effect on V1 transcription: activation via site 1 in the proximal promoter and repression through site 6, approximately 1.7 kb upstream. EMSA/electrophoretic mobility supershift assays and chromatin immunoprecipitation analyses confirmed these two sites are bound by HNF-4alpha. Based on these data, we speculate there are multiple regions working together to repress the expression of V1 hGHR transcripts in tissues other than the normal postnatal liver, and that HNF-4alpha is a good candidate for regulating V1 hGHR expression in the human hepatocyte.
J. Biol. Chem. 274, 33050-33056 (1999)[PubMed:10551874]
The transcriptional regulation of the apoCIII gene by hormonal and metabolic signals plays a significant role in determining plasma triglyceride levels. In the current work we demonstrate that the apoCIII gene is regulated by the mitogen-activated protein (MAP) kinase signaling pathway. In HepG2 cells, repression of MAP kinase activity by treatment with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 caused a 5-8-fold increase in apoCIII transcriptional activity. Activation of MAP kinase by phorbol ester treatment caused a 3-5-fold reduction in apoCIII transcription. The region of the apoCIII promoter responsible for this regulation was mapped in transiently transfected HepG2 cells to a 6-base pair element located at -740. The major protein binding to this site was identified as the nuclear hormone receptor HNF4. An increase in HNF4 mRNA and protein levels was observed in HepG2 cells after treatment with PD98059, indicating that the MAP kinase pathway regulates the expression of the HNF4 gene. These findings demonstrate that the apoCIII gene can be regulated by signals acting through the MAP kinase pathway and that this regulation is mediated, at least in part, by changes in the amount of HNF4.
J. Thromb. Haemost. 1, 1688-1698 (2003)[PubMed:12911579]
BACKGROUND: Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. OBJECTIVES: The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. METHODS: We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. RESULTS: We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. CONCLUSIONS: The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.
Am. J. Physiol. 276, G1181-94-G1181-94 (1999)[PubMed:10330009]
There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha1-antitrypsin (alpha1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1alpha (HNF-1alpha), HNF-1beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha1-AT gene. Cotransfection studies showed that HNF-1alpha and HNF-4 had a synergistic effect on alpha1-AT gene expression. RNA blot analysis showed that HNF-1alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1alpha binding activity increase coordinately with alpha1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha1-AT gene expression. Together, these results provide evidence that HNF-1alpha and HNF-4 play a role in the mechanism by which the alpha1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.
Abstract Few genes are known to be involved in renal cell carcinoma (RCC) development and progression. The cell-specific transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha) is down-regulated in RCC and we have shown that HNF4 alpha inhibits cell proliferation in the embryonic kidney cell line HEK293. To clarify the possible tumor suppressor activity of HNF4 alpha we analyzed the whole human expression profile in HEK293 cells upon HNF4 alpha induction. By comparing induced and uninduced cells, we identified 1411 differentially expressed genes. Using RNA interference, we screened 56 HNF4 alpha-regulated genes for their possible role in mediating inhibition of cell proliferation triggered by HNF4 alpha. We demonstrate that 14 of these regulated genes are able to contribute to the inhibitory effect of HNF4 alpha on cell proliferation, including well-known cancer genes, such as CDKN1A (p21), TGFA, MME (NEP) and ADAMTS1. In addition, the genes SEPP1, THEM2, BPHL, DSC2, ANK3, ALDH6A1, EPHX2, NELL2, EFHD1 and PROS1 are also part of the network of HNF4 alpha target genes that regulate proliferation in HEK293 cells. Therefore, we postulate that HNF4 alpha orchestrates, at least, these 14 genes to regulate cell proliferation in HEK293 cells and that down-regulation of HNF4 alpha could contribute to the progression of kidney cancer.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a glucose stimulus.
Any process that modulates the frequency, rate or extent of gene expression as a consequence of a process in which a signal is released and/or conveyed from one location to another.
The cascade of processes by which a signal interacts with a receptor, causing a change in the activity of a SMAD protein, and ultimately effecting a change in the functioning of the cell.
The chemical reactions and pathways involving a xenobiotic compound, a compound foreign to living organisms. Used of chemical compounds, e.g. a xenobiotic chemical, such as a pesticide.
Drug Metab. Pharmacokinet. 22, 287-298 (2007)[PubMed:17827783]
Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.
Protein involved in the transfer of genetic information from DNA to messenger RNA (mRNA) by DNA-directed RNA polymerase. In the case of some RNA viruses, protein involved in the transfer of genetic information from RNA to messenger RNA (mRNA) by RNA-directed RNA polymerase.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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