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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

UDP-glucuronosyltransferase 1A7 (UGT1A7) is a major UGT contributing to the glucuronidation of xenobiotic phenols in rats. Its expression in rat liver is tightly regulated, with low constitutive and high inducible expression in response to aryl hydrocarbon receptor ligands and oltipraz. Previously, we reported the absence of 3-methylcholanthrene- or oltipraz-responsive elements in the 1.6-kbp region flanking the UGT1A7 promoter. However, potential binding sites were noted for several liver-enriched transcription factors. Here we show that deletion of the hepatic nuclear factor (HNF)3, HNF4, and CCAAT-enhancer binding protein-like binding sites had no effect on the expression of a UGT1A7 reporter plasmid, p(-965/+56)1A7-Luc, in primary rat hepatocytes. The full activity of the promoter was contained in the region between bases -157 and +76. Two sites of binding by rat liver nuclear proteins were detected in this region by DNase footprinting. PR-1 corresponded to the HNF1-like binding site between bases -52 and -38, whereas PR-2 was located between -30 to -6. Gel retardation studies supported the presence of HNF1alpha in the PR-1 DNA-liver nuclear protein complex. Mutation of PR-1 inhibited binding in the gel shift assay, prevented activation by overexpressed HNF1 in human embryonic kidney cells, and reduced by >80% the maximal luciferase activities expressed from basal and 3-methylcholanthrene-responsive UGT1A7 gene reporter constructs in primary rat hepatocytes. These data provide evidence for an important stimulatory role of HNF1 in promoting UGT1A7 gene expression in rat liver.
Mol Pharmacol 2000 Aug
PMID:Involvement of hepatocyte nuclear factor 1 in the regulation of the UDP-glucuronosyltransferase 1A7 (UGT1A7) gene in rat hepatocytes. 1090 99

Recent evidence suggests that certain LEF/TCF family members act as repressors in the absence of Wnt signaling. We show here that repression by LEF1 requires histone deacetylase (HDAC) activity. Further, LEF1 associates in vivo with HDAC1, and transcription of a model LEF1-dependent target gene is modulated by the ratio of HDAC1 to beta-catenin, implying that repression by LEF1 is mediated by promoter-targeted HDAC. Consistent with this hypothesis, under repression conditions the promoter region of a LEF1 target gene is hypoacetylated. By contrast, when the reporter is activated, its promoter becomes hyperacetylated. Coexpression of beta-catenin with LEF1 and HDAC1 results in the formation of a beta-catenin/HDAC1 complex. Surprisingly, the enzymatic activity of HDAC1 associated with beta-catenin is attenuated. Together, these findings imply that activation of LEF1-dependent genes by beta-catenin involves a two-step mechanism. First, HDAC1 is dissociated from LEF1 and its enzymatic activity is attenuated. This first step yields a promoter that is inactive but poised for activation. Second, once HDAC1-dependent repression has been overridden, beta-catenin binds LEF1 and the beta-catenin-LEF1 complex is competent to activate the expression of downstream target genes.
Mol Cell Biol 2000 Sep
PMID:Beta-catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator. 1095 84

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).
J Mol Biol 2000 Sep 29
PMID:Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha. 1099 27

beta-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed beta-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous beta-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5-8 d after transfection) cells overexpressing beta-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous beta-catenin does not induce apoptosis, even though some endogenous beta-catenin moves with the exogenous LEF-1 into the nucleus. TOPFLASH/FOPFLASH reporter assays showed that full-length beta-catenin is able to induce LEF-1-dependent transactivation, whereas Arm beta-catenin totally abolishes the transactivating function. However, Arm beta-catenin, containing deletions of known LEF-1-transactivating domains, has the same apoptotic effects as full-length beta-catenin. Overexpressed beta-catenin also induces apoptosis in cells transfected with nuclear localization signal-deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous beta-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed delta-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to beta-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed beta-catenin, but Bcl-x(L) reduces it. We hypothesize that in vivo apoptosis of cells overexpressing beta-catenin might be a physiological mechanism to eliminate them from the population.
Mol Biol Cell 2000 Oct
PMID:Overexpression of beta-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators. 1102 52

Hepatocyte nuclear factor 4alpha (HNF4alpha) is an orphan receptor of the nuclear receptor superfamily and expressed in vertebrates as a tissue-specific transcription factor in liver, kidney, intestine, stomach, and pancreas. It also plays a crucial role in early embryonic development and has been identified as a maternal component in the Xenopus egg. We now report on an activity present in Xenopus embryos that inhibits the DNA binding of HNF4. This HNF4 inhibitor copurifies with a 25-kDa protein under nondenaturing conditions but can be separated from this protein by sodium dodecyl sulfate treatment. Protease treatment of the inhibitor results in a core fragment of about 5 kDa that retains full inhibitory activity. The activity of the HNF4 inhibitor can also be monitored in the absence of DNA, as it alters the mobility of the HNF4 protein in native polyacrylamide gels and the accessibility of antibodies. Comparing the activity of the HNF4 inhibitor with acyl coenzyme A's, recently proposed to be ligands of HNF4, we observe a more stringent specificity for the HNF4 inhibitor activity. Using deletion constructs of the HNF4 protein, we could show that the potential ligand-binding domain of HNF4 is not required, and thus the HNF4 inhibitor does not represent a classical ligand as defined for the nuclear receptor superfamily. Based on our previous finding that maternal HNF4 is abundantly present in Xenopus embryos but the target gene HNF1alpha is only marginally expressed, we propose that the HNF4 inhibitor functions in the embryo to restrict the activity of the maternal HNF4 proteins.
Mol Cell Biol 2000 Dec
PMID:Inhibitor of the tissue-specific transcription factor HNF4, a potential regulator in early Xenopus development. 1107 69

The Wnt/Wg signaling pathway functions during development to regulate cell fate determination and patterning in various organisms. Two pathways are reported to lie downstream of Wnt signaling in vertebrates. The canonical pathway relies on the activation of target genes through the beta-catenin-Lef/TCF complex, while the noncanonical pathway employs the activation of protein kinase C (PKC) and increases in intracellular calcium to induce target gene expression. cDNA subtractive hybridization between a cell line that overexpresses Wnt-1 (C57MG/Wnt-1) and the parental cell line (C57MG) was performed to identify downstream target genes of Wnt-1 signaling. Among the putative Wnt-1 target genes, we have identified a mouse homolog of the gene encoding human transcription factor basic transcription element binding protein 2 (mBTEB2). The mBTEB2 transcript is found at high levels in mammary tissue taken from a transgenic mouse overexpressing Wnt-1 (both tissue prior to active proliferation and tumor tissue) but is barely detectable in wild-type mouse mammary glands. The regulation of mBTEB2 by Wnt-1 signaling in tissue culture occurs through a beta-catenin-Lef/TCF-independent mechanism, as it is instead partially regulated by PKC. The Wnt-1-induced, PKC-dependent activation of mouse BTEB2 in C57MG cells, as well as the ability of Wnt-1 to stabilize beta-catenin in these cells, is consistent with the hypothesis that both the noncanonical and canonical Wnt pathways are activated concomitantly in the same cell. These results suggest that mBTEB2 is a biologically relevant target of Wnt-1 signaling that is activated through a beta-catenin-independent, PKC-sensitive pathway in response to Wnt-1.
Mol Cell Biol 2001 Jan
PMID:Identification of a mouse homolog of the human BTEB2 transcription factor as a beta-catenin-independent Wnt-1-responsive gene. 1113 43

The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.
J Mol Biol 2001 Mar 09
PMID:Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin. 1123 26

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a nuclear receptor involved in glucose homeostasis and is required for normal beta-cell function. Mutations in the HNF4alpha gene are associated with maturity-onset diabetes of the young type 1. E276Q and R154X mutations were previously shown to impair intrinsic transcriptional activity (without exogenously supplied co-activators) of HNF4alpha. Given that transcriptional partners of HNF4alpha modulate its intrinsic transcriptional activity and play crucial roles in HNF4alpha function, we investigated the effects of these mutations on potentiation of HNF4alpha activity by p300, a key co-activator for HNF4alpha. We show here that loss of HNF4alpha function by both mutations is increased through impaired physical interaction and functional cooperation between HNF4alpha and p300. Impairment of p300-mediated potentiation of HNF4alpha transcriptional activity is of particular importance for the E276Q mutant since its intrinsic transcriptional activity is moderately affected. Together with previous results obtained with chicken ovalbumin upstream promoter-transcription factor II, our results highlight that impairment of recruitment of transcriptional partners represents an important mechanism leading to abnormal HNF4alpha function resulting from the MODY1 E276Q mutation. The impaired potentiations of HNF4alpha activity were observed on the promoter of HNF1alpha, a transcription factor involved in a transcriptional network and required for beta-cell function. Given its involvement in a regulatory signaling cascade, loss of HNF4alpha function may cause reduced beta-cell function secondary to defective HNF1alpha expression. Our results also shed light on a better structure-function relationship of HNF4alpha and on p300 sequences involved in the interaction with HNF4alpha.
Mol Endocrinol 2001 Jul
PMID:Maturity-onset diabetes of the young Type 1 (MODY1)-associated mutations R154X and E276Q in hepatocyte nuclear factor 4alpha (HNF4alpha) gene impair recruitment of p300, a key transcriptional co-activator. 1143 18

Maturity-onset diabetes of the young (MODY), an autosomal dominant, early-onset form of type-2 diabetes, is caused by mutations in five different genes all leading to defect(s) in the pancreatic beta cell. However, some patients with this form of diabetes do not bear a mutation in any of the known (MODY1-MODY5) loci, a notion prompting the search for new MODY genes. Clinical and genetic data point toward a defect in beta cell function in the majority of patients with MODY, and partners of the glucose-sensing device are reasonable functional candidates. The high-capacity glucose transporter GLUT2 has the ideal kinetic features for performing this task. However, complete GLUT2 deficiency in humans leads to hepato-renal glycogenosis (Fanconi-Bickel syndrome), and heterozygous GLUT2 mutations apparently behave in a recessive manner. Furthermore, in the human beta cell GLUT1 mRNA is predominant when compared to GLUT2 and glucose influx appears to be largely mediated by this low-Km transporter. Thus, we looked for the presence of sequence variants by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) within the GLUT1 gene in 90 Italian pedigrees negative at the search for mutations in glucokinase (MODY2) and hepatocyte nuclear factor-1alpha (MODY3), the two genes responsible for about 60% of MODY cases in Italian children. We found three already described silent mutations and a new single base deletion in position -173 of the 5' regulatory region. The -173de1A variant, which was detected in the heterozygous or homozygous state in 30.8% of MODY patients examined and is located in a Nuclear Factor Y binding sequence, is not associated with hyperglycemia in affected relatives of MODY probands. In conclusion, it appears from these results that the glucose transporter gene GLUT1 is unlikely to play a major role in the etiology of MODY diabetes.
J Mol Med (Berl) 2001 Jun
PMID:Single-strand conformation polymorphism analysis of the glucose transporter gene GLUT1 in maturity-onset diabetes of the young. 1148 13

Maturity-onset diabetes of the young (MODY) is a monogenic, autosomal dominant subtype of early-onset diabetes mellitus due to defective insulin secretion by the pancreatic beta-cell in humans. Five different genes have been identified including those encoding the tissue-specific transcription factors expressed in pancreatic beta-cells, i.e. HNF-4alpha (MODY1), HNF-1alpha (MODY3), IPF-1 (also known as PDX-1, MODY4) and HNF-1beta (MODY5). Analyzing the transcription of the HNF-4alpha gene, we now identify an alternative promoter, P2, which is 46 kb 5' to the previously identified P1 promoter of the human gene. Based on RT-PCR this distant upstream P2 promoter represents the major transcription site in pancreatic beta-cells, but is also used in hepatic cells. Transfection assays with various deletions and mutants of the P2 promoter reveal functional binding sites for HNF-1alpha, HNF-1beta and IPF-1, the other transcription factors known to encode MODY genes. We demonstrate the significance of this alternative promoter in a large MODY family where a mutated IPF-1 binding site in the P2 promoter of the HNF-4alpha gene co-segregates with diabetes (LOD score 3.25). These data suggest a regulatory network of the four MODY transcription factors interconnected at the distant upstream P2 promoter of the HNF-4alpha gene.
Hum Mol Genet 2001 Sep 15
PMID:A distant upstream promoter of the HNF-4alpha gene connects the transcription factors involved in maturity-onset diabetes of the young. 1159 Jan 26


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