Transcription Factor HNF1alpha Research Articles

Epistasis of Transcriptomes Reveals Synergism between Transcriptional Activators Hnf1α and Hnf4α

The transcription of individual genes is determined by combinatorial interactions between DNA–binding transcription factors. The current challenge is to understand how such combinatorial interactions regulate broad genetic programs that underlie cellular functions and disease. The transcription factors Hnf1α and Hnf4α control pancreatic islet β-cell function and growth, and mutations in their genes cause closely related forms of diabetes. We have now exploited genetic epistasis to examine how Hnf1α and Hnf4α functionally interact in pancreatic islets. Expression profiling in islets from either Hnf1a+/− or pancreas-specific Hnf4a mutant mice showed that the two transcription factors regulate a strikingly similar set of genes. We integrated expression and genomic binding studies and show that the shared transcriptional phenotype of these two mutant models is linked to common direct targets, rather than to known effects of Hnf1α on Hnf4a gene transcription. Epistasis analysis with transcriptomes of single- and double-mutant islets revealed that Hnf1α and Hnf4α regulate common targets synergistically. Hnf1α binding in Hnf4a-deficient islets was decreased in selected targets, but remained unaltered in others, thus suggesting that the mechanisms for synergistic regulation are gene-specific. These findings provide an in vivo strategy to study combinatorial gene regulation and reveal how Hnf1α and Hnf4α control a common islet-cell regulatory program that is defective in human monogenic diabetes.

Integrated Pathway Analysis of Rat Urine Metabolic Profiles and Kidney Transcriptomic Profiles To Elucidate the Systems Toxicology of Model Nephrotoxicants

In this study, approximately 40 endogenous metabolites were identified and quantified by (1)H NMR in urine samples from male rats dosed with two proximal tubule toxicants, cisplatin and gentamicin. The excreted amount of a majority of those metabolites in urine was found to be dose-dependent and exhibited a strong correlation with histopathology scores of overall proximal tubule damage. MetaCore pathway analysis software (GeneGo Inc.) was employed to identify nephrotoxicant-associated biochemical changes via an integrated quantitative analysis of both urine metabolomic and kidney transcriptomic profiles. Correlation analysis was applied to establish quantitative linkages between pairs of individual metabolite and gene transcript profiles in both cisplatin and gentamicin studies. This analysis revealed that cisplatin and gentamicin treatments were strongly linked to declines in mRNA transcripts for several luminal membrane transporters that handle each of the respective elevated urinary metabolites, such as glucose, amino acids, and monocarboxylic acids. The integrated pathway analysis performed on these studies indicates that cisplatin- or gentamicin-induced renal Fanconi-like syndromes manifested by glucosuria, hyperaminoaciduria, lactic aciduria, and ketonuria might be better explained by the reduction of functional proximal tubule transporters rather than by the perturbation of metabolic pathways inside kidney cells. Furthermore, this analysis suggests that renal transcription factors HNF1alpha, HNF1beta, and HIF-1 might be the central mediators of drug-induced kidney injury and adaptive response pathways.

Hepatic Nuclear Factor 1α Inhibitor Ursodeoxycholic Acid Influences Pharmacokinetics of the Organic Anion Transporting Polypeptide 1B1 Substrate Rosuvastatin and Bilirubin

Expression of the organic anion transporting polypeptide 1B1 (OATP1B1) is regulated by transcription factor hepatic nuclear factor (HNF) 1alpha. The aim of this study was to investigate the effect of ursodeoxycholic acid (UDCA), an inhibitor of transcription factor HNF1alpha, on rosuvastatin and bilirubin kinetics in human healthy volunteers. Both substances are substrates of OATP1B1. Twelve subjects with OATP1B1(*)1b/(*)1b genotype predicting high transport activity were recruited for this randomized, crossover study. Each subject received a single p.o. dose of 20 mg of rosuvastatin after 14 days of p.o. intake of either 500 mg of UDCA or placebo. Plasma concentrations of rosuvastatin were determined on days 15 to 18 of each study period. Subjects were randomly assigned to UDCA or placebo group. Intake of UDCA led to a significant increase in rosuvastatin area under the curve (AUC)(0-72) from 128.5 ng/ml.h to 182.1 ng/ml.h(P = 0.008) compared with the control group. The oral clearance decreased from 155.2 l/h with placebo to 109.8 l/h with UDCA. In addition, the mean values of total bilirubin, conjugated bilirubin, and unconjugated bilirubin significantly increased to 139 +/- 39% (P = 0.003), 127 +/- 29% (P = 0.005), and 151 +/- 52% (P = 0.004), respectively, after UDCA treatment. These results in healthy volunteers confirm the findings from in vitro studies that UDCA inhibits OATP1B1 activity by inhibition of the transcription factor HNF1alpha. They highlight a novel mechanism of OATP1B1-based interaction that is mediated by transcription factor HNF1alpha.

Clinical implications of a molecular genetic classification of monogenic β-cell diabetes

Monogenic diabetes resulting from mutations that primarily reduce beta-cell function accounts for 1-2% of diabetes cases, although it is often misdiagnosed as either type 1 or type 2 diabetes. Knowledge of the genetic etiology of diabetes enables more-appropriate treatment, better prediction of disease progression, screening of family members and genetic counseling. We propose that the old clinical classifications of maturity-onset diabetes of the young and neonatal diabetes are obsolete and that specific genetic etiologies should be sought in four broad clinical situations because of their specific treatment implications. Firstly, diabetes diagnosed before 6 months of age frequently results from mutation of genes that encode Kir6.2 (ATP-sensitive inward rectifier potassium channel) or sulfonylurea receptor 1 subunits of an ATP-sensitive potassium channel, and improved glycemic control can be achieved by treatment with high-dose sulfonylureas rather than insulin. Secondly, patients with stable, mild fasting hyperglycemia detected particularly when they are young could have a glucokinase mutation and might not require specific treatment. Thirdly, individuals with familial, young-onset diabetes that does not fit with either type 1 or type 2 diabetes might have mutations in the transcription factors HNF-1alpha (hepatocyte nuclear factor 1-alpha) or HNF-4alpha, and can be treated with low-dose sulfonylureas. Finally, extrapancreatic features, such as renal disease (caused by mutations in HNF-1beta) or deafness (caused by a mitochondrial m.3243A>G mutation), usually require early treatment with insulin.

Human acyl-CoA:cholesterol acyltransferase 2 gene expression in intestinal Caco-2 cells and in hepatocellular carcinoma.

Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1alpha (hepatocyte nuclear factor 1alpha). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1alpha exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1alpha are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1alpha expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1alpha, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.

The effect of cytostatic drug treatment on intestine-specific transcription factors Cdx2, GATA-4 and HNF-1α in mice

Chemotherapy-induced intestinal damage is a very important dose-limiting side effect for which there is no definitive prophylaxis or treatment. This is in part due to the lack of understanding of its pathophysiology and impact on intestinal differentiation. The objective of this study was to investigate the gene expression of the small intestinal transcription factors HNF-1alpha, Cdx2, GATA-4 in an experimental model of methotrexate (MTX)-induced intestinal damage, and to correlate these alterations with histological damage, epithelial proliferation and differentiation. HNF-1alpha, Cdx2 and GATA-4 are critical transcription factors in epithelial differentiation, and in combination they act as promoting factors of the sucrase-isomaltase (SI) gene, an enterocyte-specific differentiation marker which is distinctly downregulated after MTX treatment. Mice received two doses of MTX i.v. on two consecutive days and were sacrificed 1, 3 and 7 or 9 days after final injection. Segments of the jejunum were taken for morphological, immunohistochemical and quantitative analyses. Intestinal damage was most severe at day 3 and was associated with decreased expression of the transcriptional factors HNF-1alpha, Cdx2 and GATA-4, which correlated well with decreased expression of SI, and seemed inversely correlated with enhanced proliferation of epithelial crypt cells. During severe damage, the epithelium was preferentially concerned with proliferation rather than differentiation, most likely in order to restore the small intestinal barrier function rather than maintaining its absorptive function. Since HNF-1alpha, Cdx2 and GATA-4 are critical for intestine-specific gene expression and therefore crucial in epithelial differentiation, these results may explain, at least in part, why intestinal differentiation is compromised during MTX treatment.

The mouse RNase 4 and RNase 5/ang 1 locus utilizes dual promoters for tissue-specific expression

The ribonuclease A (RNase A) superfamily has been the subject of extensive studies in the areas of protein evolution, structure and biochemistry and are exciting molecules in that they appear to be responding to unique selection pressures, generating proteins capable of multiple and diverse activities. The RNase 4 and RNase 5/ang 1 shared locus breaks a pattern that is otherwise canonical among the members of the RNase A gene superfamily. Conserved among humans, mice and rats, the locus includes two non-coding exons followed by two distinct exons encoding RNase 4 and RNase 5/ang 1. Transcription from this locus is controlled by differential splicing and tissue-specific expression from promoters located 5′ to each of the non-coding exons. Promoter 1, 5′ to exon I, is universally active, while Promoter 2, 5′ to exon II, is active only in hepatic cells in promoter assays in vitro. Transcription from Promoter 2 is dependent on an intact HNF-1 consensus binding site which binds the transcription factor HNF-1α. In summary, RNase 4 and RNase 5/ang 1 are unique among the RNase A ribonuclease genes in that they maintain a complex gene locus that is conserved across species with transcription initiated from tissue-specific dual promoters followed by differential exon splicing.

Differential expression of claudin-2 along the human intestine: Implication of GATA-4 in the maintenance of claudin-2 in differentiating cells.

Claudins, and particularly claudin-2, are important regulatory components of tight junction permeability. A better understanding of the involvement of claudin-2 in intestinal barrier functions requires the characterization of its distribution and regulation in the intestine. Interestingly, the claudin-2 gene promoter harbors a number of similarities to that of sucrase-isomaltase, a marker of enterocyte differentiation. We thus investigated the expression of claudin-2 in relation to the transcription factors CDX2, HNF-1alpha, and GATA-4 in the human intestine. The characterization of claudin-2 and the expression of the above transcription factors were performed by immunofluorescence, Western blot, and RT-PCR in the developing human intestinal epithelium. The functional role of CDX2, HNF-1alpha, and GATA-4 on claudin-2 regulation was also examined by ectopic expression studies in intestinal cell models. Claudin-2 was detected in both crypt and villus cells of the small intestine but restricted to undifferentiated crypt cells in the colon. CDX2 and HNF-1alpha were expressed along the entire intestine whereas GATA-4 was undetectable in the colon. Accordingly, in the colonic Caco-2 cell model, claudin-2 was found to be present only in undifferentiated cells. Like in the colonic epithelium, GATA-4 was found to be also lacking in Caco-2 cells while CDX2 and HNF-1alpha were present at significant levels. Cotransfection experiments showed that the claudin-2 promoter was activated by CDX2, HNF-1alpha, and GATA-4 in a cooperative manner. Furthermore, forced GATA-4 expression in Caco-2 cells enhances maintenance of claudin-2 expression during differentiation. These observations suggest that optimal claudin-2 expression in the gut relies on the presence of GATA-4, suggesting a role for this factor in intestinal regionalization.

The HNF1beta transcription factor has several domains involved in nephrogenesis and partially rescues Pax8/lim1-induced kidney malformations.

The tissue-specific transcription factors HNF1alpha and HNF1beta are closely related homeodomain proteins conserved in vertebrate evolution. Heterozygous mutations in human HNF1beta but not in HNF1alpha genes are associated with kidney malformations. Overexpression of HNF1beta in Xenopus embryos leads to defective pronephros development, while HNF1alpha has no effect. We have defined the regions responsible for this functional difference between HNF1beta and HNF1alpha in transfected HeLa cells as well as in injected Xenopus embryos. Using domain swapping experiments, we located a nuclear localization signal in the POUH domain of HNF1beta, and showed that the POUS and POUH domains of HNF1beta mediate a high transactivation potential in transfected cells. In injected Xenopus embryos three HNF1beta domains are involved in nephrogenesis. These include the dimerization domain, the 26 amino acid segment specific for splice variant A as well as the POUH domain. As HNF1beta together with Pax8 and lim1 constitute the earliest regulators in the pronephric anlage, it is possible that they cooperate during early nephrogenesis. We have shown here that HNF1beta can overcome the enlargement and the induction of an ectopic pronephros mediated by overexpression of Pax8 and lim1. However, the phenotype induced by Pax8 and lim1 overexpression and characterized by cyst-like structures and thickening of the pronephric tubules was not altered by HNF1beta overexpression. Taken together, HNF1beta acts antagonistically to Pax8 and lim1 in only some processes during nephrogenesis, and a simple antagonistic relationship does not completely describe the functions of these genes. We conclude that HNF1beta has some distinct morphogenetic properties during nephrogenesis.

Searching for DNA-protein interactions by lambda phage display.

We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.

Mutations in hepatocyte nuclear factor 4α (HNF4α) gene associated with diabetes result in greater loss of HNF4α function in pancreatic β-cells than in nonpancreatic β-cells and in reduced activation of the apolipoprotein CIII promoter in hepatic cells

Mutations in the HNF4alpha gene have been correlated with maturity-onset diabetes of the young, which is characterized mainly by pancreatic beta-cell dysfunction and is also associated with mild liver abnormalities. HNF4alpha D126Y and D126H mutations were found in a patient with early-onset type 2 diabetes, and the R324H mutation was found in a common type 2 diabetic nephropathic patient. We investigated whether these mutations, which have not yet been functionally characterized, impair HNF4alpha function in three cell models: HEK 293 embryonal kidney cells, HepG2 hepatoma cells, and betaTC3 pancreatic beta-cells. The R324H mutation had no effect on HNF4alpha function with either the HNF1alpha and L-type pyruvate kinase (LPK) promoters, but the D126Y and D126H mutations impaired HNF4alpha transcriptional activities in all tested cell lines. These impairments by D126Y and D126H mutations, which are located in the T box, are not due to a loss of dimerization but to a loss of DNA binding. Interestingly, the strongest functional consequences of these mutations were observed on the HNF1alpha promoter in betaTC3 cells. Given the key role of the transcription factor HNF1alpha in pancreatic beta-cell function, it can be inferred that impairment of HNF4alpha function by these mutations affects metabolic pathways in pancreatic beta-cells and contributes to development of diabetes. Moreover, the HNF4alpha-mediated activation of the apolipoprotein CIII promoter in HepG2 cells was significantly impaired by D126Y and D126H mutations. These results support clinical findings that liver function can also be impaired in diabetic patients having HNF4alpha mutations.

Genetic variation in the hepatocyte nuclear factor (HNF)-3alpha gene does not contribute to maturity-onset diabetes of the young in Japanese.

Heterozygous mutations in the genes encoding transcription factors in the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY), a monogenic form of diabetes mellitus. However, these genes are responsible for only approximately 20% of the cases of MODY in Japanese patients. Searching for a novel MODY gene in this population, we investigated a candidate for encoding the forkhead transcription factor HNF-3alpha, which also belongs to the HNF-transcription cascade. The human HNF-3alpha gene, which was assigned to the segment near microsatellites D14S75 and AFM200ZH4 on chromosome 14 by radiation hybrid mapping, spans approximately 5 kb and consists of two exons. Ninety-five Japanese subjects with MODY/early-onset non-ketotic diabetes were screened for mutations in this gene. Direct sequencing of the exons and flanking regions identified one missense mutation (Ala-83-Thr) in exon 2 and three nucleotide alterations in the non-coding regions. However, their frequencies were not significantly different between MODY and control subjects, indicating that mutations in the HNF-3alpha gene are not a major cause of MODY in Japanese patients.

Hepatocyte Nuclear Factor 4α Regulates the Expression of Pancreatic β-Cell Genes Implicated in Glucose Metabolism and Nutrient-induced Insulin Secretion

Mutations in the HNF4alpha gene are associated with the subtype 1 of maturity-onset diabetes of the young (MODY1), which is characterized by impaired insulin secretory response to glucose in pancreatic beta-cells. Hepatocyte nuclear factor 4alpha (HNF4alpha) is a transcription factor critical for liver development and hepatocyte-specific gene expression. However, the role of HNF4alpha in the regulation of pancreatic beta-cell gene expression and its correlation with metabolism secretion coupling have not been previously investigated. The tetracycline-inducible system was employed to achieve tightly controlled expression of both wild type (WT) and dominant-negative mutant (DN) of HNF4alpha in INS-1 cells. The induction of WT-HNF4alpha resulted in a left shift in glucose-stimulated insulin secretion, whereas DN-HNF4alpha selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4alpha also caused defective mitochondrial function substantiated by reduced [(14)C]pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane hyperpolarization, and blunted nutrient-generated cellular ATP production. Quantitative evaluation of HNF4alpha-regulated pancreatic beta-cell gene expression revealed altered mRNA levels of insulin, glucose transporter-2, L-pyruvate kinase, aldolase B, 2-oxoglutarate dehydrogenase E1 subunit, and mitochondrial uncoupling protein-2. The patterns of HNF4alpha-regulated gene expression are strikingly similar to that of its downstream transcription factor HNF1alpha. Indeed, HNF4alpha changed the HNF1alpha mRNA levels and HNF1alpha promoter luciferase activity through altered HNF4alpha binding. These results demonstrate the importance of HNF4alpha in beta-cell metabolism-secretion coupling.

Loss of HNF1? function in human renal cell carcinoma: Frequent mutations in theVHL gene but not theHNF1? gene

Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue-specific transcription factor hepatocyte nuclear factor (HNF) 1alpha. Detailed analyses of the 10 exons encoding HNF1alpha in 32 human RCCs by single-strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor-associated mutation, whereas with the same probes we frequently found mutations in the von Hippel-Lindau tumor suppressor gene. No mutation leading to loss of HNF1alpha function was detected by analyzing the integrity of the HNF1alpha transcripts in the RNA derived from RCCs by the protein truncation test. Investigating human RCC cell lines by western blotting and gel retardation assays showed a dramatic loss in the expression of the tissue-specific transcription factor HNF1alpha in eight of 10 cell lines. As the HNF1alpha-related transcription factor HNF1beta was expressed in all these tumor cell lines, the loss of HNF1alpha expression was a specific event and was maintained in RCC cell lines. The loss of HNF1alpha expression in RCC cell lines on the RNA level was confirmed by reverse transcription polymerase chain reaction. We propose that tumor-associated mutations in the HNF1alpha gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1alpha gene.

Inactivation of the winged helix transcription factor HNF3alpha affects glucose homeostasis and islet glucagon gene expression in vivo.

Mice homozygous for a null mutation in the winged helix transcription factor HNF3alpha showed severe postnatal growth retardation followed by death between P2 and P12. Homozygous mutant mice were hypoglycemic despite unchanged expression of HNF3 target genes involved in hepatic gluconeogenesis. Whereas insulin and corticosteroid levels were altered as expected, plasma glucagon was reduced markedly in the mutant animals despite the hypoglycemia that should be expected to increase glucagon levels. This correlated with a 70% reduction in pancreatic proglucagon gene expression. We also showed that HNF3alpha could bind to and transactivate the proglucagon gene promoter. These observations invoke a central role for HNF3alpha in the regulatory control of islet genes essential for glucose homeostasis in vivo.

The embryonic expression of the tissue-specific transcription factor HNF1alpha in Xenopus: rapid activation by HNF4 and delayed induction by mesoderm inducers.

The tissue-specific transcription factor HNF1alpha is expressed in kidney, liver, intestine and stomach of Xenopus. We show that the HNF1alpha gene is transcriptionally activated at the onset of zygotic gene transcription and that this transcription is maintained throughout development. Ectodermal explants of blastulae (animal caps) express HNF1alpha mRNA upon stimulation with the mesoderm inducers activin A and BMP4 as well as on overexpression of Smad2 and Smad1, the corresponding members of the intracellular TGF-beta signal transducers, respectively. Beside these factors that mediate their response through serine/threonine kinase receptors, bFGF, which acts via tyrosine kinase receptors, leads to HNF1alpha expression, too. These embryonic inducers result in a delayed appearance of HNF1alpha mRNA, excluding a direct activation of HNF1alpha. In contrast, the maternally expressed nuclear receptors HNF4alpha and HNF4beta activate the initial HNF1alpha transcription, since overexpression of HNF4 leads to a rapid expression of HNF1alpha mRNA in animal caps. Similarly, in entire neurulae HNF4 overexpression results in increased HNF1alpha transcription. Therefore, we assume that the initial activation is dependent on maternal HNF4alpha and HNF4beta transcription factors whereas HNF1alpha induction by growth factors reflects the property of these factors to induce the differentiation of mesodermal and entodermal cell types expressing HNF1alpha.

Chromatin structure and functional analysis of the mouse HNF3alpha gene.

The transcription factor HNF3alpha is a member of the winged-helix family of regulatory proteins. It is expressed in the definitive endoderm, notochord, and neural tube in embryos, but in the adult is expressed primarily in endoderm-derived tissues such as liver, lung, and pancreas. We present here the cloning of the mouse HNF3alpha gene and a characterization of its chromatin structure and regulatory sequences. The HNF3alpha gene is encoded by two exons and its transcription initiates at multiple start sites at a TATA-less promoter that is highly conserved between mouse and rat. We found different patterns of DNaseI hypersensitive sites in HNF3alpha gene chromatin in different adult tissues in which HNF3alpha is expressed, suggesting distinct regulatory mechanisms occurring within different tissue derivatives of the endoderm germ layer. Cell transfection data indicate that sequences spanning certain upstream hypersensitive sites can enhance transcription from the HNF3alpha promoter, but only when stably integrated into chromatin and not when transiently transfected. The results suggest a complex regulatory interplay between distinct genetic regulatory sequences that function specifically in chromatin.

The bifunctional protein DCoH/PCD, a transcription factor with a cytoplasmic enzymatic activity, is a maternal factor in the rat egg and expressed tissue specifically during embryogenesis.

The bifunctional protein DCoH/PCD is both a cytoplasmatic enzyme (PCD) involved in the tetrahydrobiopterin regeneration and a transcription coactivator (DCoH). Originally detected in liver cell nuclei, it forms a 2:2 heterotetrameric complex with the nuclear transcription factors HNF1alpha and the variant form HNF1beta and enhances their transcriptional potential. To address the role of DCoH in tissue specific and developmental gene regulation we analyzed its spatial and temporal expression pattern in the rat. DCoH might have a function in tissue specific gene expression mediated by HNF1 in the adults and in the developing embryo as it is found in the kidney and the liver, organs known to contain HNF1. In addition DCoH is a maternal factor in the rat egg lacking HNF1 transcription factors. The maternal protein enters the cell nuclei at the 8-cell stage suggesting a role in early embryonic gene regulation and excluding a cytoplasmatic enzymatic function. Evidence for a HNF1 independent function of DCoH is also given by the fact that DCoH is present in the eyes (pigmented epithelium) and the brain (ependym cells) of the rat embryos, cell types lacking HNF1 proteins. The tightly regulated expression pattern of DCoH in distinct cell types originating from endo- meso- and ectoderm is conserved between the rat and the frog indicating a fundamental role for DCoH in early gene regulation among the vertebrates.

Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene (CYP7A) in HepG2 cells

A stable HepG2 cell line harboring a human cholesterol 7 alpha-hydroxylase (CYP7A) minigene/luciferase reporter gene construct was selected for studying transcriptional regulation of CYP7A gene promoter. Insulin and phorbol 12-myristate-13-acetate (PMA) strongly repressed the promoter activity as measured with luciferase activity expressed in the cells. The promoter activity of the 5' progressive deletion/luciferase reporter gene constructs was studied in a transient transfection assay in HepG2 cells. PMA represses the promoter activity and the response elements were localized in the -184/-151 and -134/-81 regions. Insulin also represses the promoter activity and response element was mapped in the -298/-81 region. Surprisingly, glucocorticoid receptor (GR) strongly inhibited promoter activity in the presence of dexamethasone, and response elements were localized in the -298/-151 and the -150/+24 regions. Thyroid hormone receptor also repressed promoter activity and response elements were localized in the -150/+24 and upstream regions. Cotransfection of CYP7A chimeric constructs with an expression vector carrying liver-enriched transcription factor HNF3 alpha stimulated the reporter gene activity, but cotransfection with GR plasmid interfered with the HNF3 alpha-stimulated activity possibly through competition for binding to overlapping GR/HNF3 binding sites. Thus, human cholesterol 7 alpha-hydroxylase gene promoter is strongly repressed by insulin, PMA, and steroid/thyroid hormones and results in the low level of cholesterol 7 alpha-hydroxylase expression in the human liver.

Regulation and function of the tissue-specific transcription factor HNF1 alpha (LFB1) during Xenopus development.

We review the data available on the structure, developmental appearance and embryonic regulation of the tissue-specific transcription factor HNF1 alpha (LFB1) in Xenopus. The expression of the HNF1 alpha gene starts early in embryogenesis shortly after mid-blastula transition and the protein accumulates in the region of the embryo where liver, pronephros and gut--tissues that contain HNF1 alpha in the adult--are developing. The cofactor DCoH, known to stabilize dimer formation of HNF1 alpha, is present as a maternal factor in the egg and has a partially distinct tissue distribution compared to HNF1 alpha. This implies that DCoH does not only modulate HNF1 alpha dimerization but may also cooperate with other transcription factors. By injecting HNF1 alpha promoter CAT constructs into fertilized Xenopus eggs we obtained activation of the injected gene restricted to the region of the developing larvae expressing endogenous HNF1 alpha. Deletion analysis allowed to define the OZ-element that is essential for embryonic activation. This element also occurs in other promoters activated at mid-blastula transition in the embryo and interacts with the maternal factor OZ-1. As the HNF1 alpha promoter also contains functional binding sites for HNF4 and HNF1, we postulate that all of these transcription factors contribute to the cascade leading to proper embryonic activation of the HNF1 alpha gene.