Tyrosine Aminotransferase Gene Research Articles

Species-specific effects of the hepatocarcinogens 3′-methyl-4-dimethyl-aminoazobenzene and ortho-aminoazotoluene in mouse and rat liver

The effects of rat-specific hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), mouse-specific hepatocarcinogen ortho-aminoazotoluene (OAT), non-species-specific hepatocarcinogen diethylnitrosamine (DENA), and non-carcinogenic 4'-methyl-4-dimethylaminoazobenzene (4'-MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA-binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen-susceptible and -resistant animals. Species-specific hepatocarcinogens 3'-MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non-carcinogenic 4'-MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA-binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species-specific effects of OAT and of 3'-MeDAB on HNF3 DNA-binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32-0.47 g/mL interval of ammonium sulfate concentration. In contrast, non-specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.

Nature of the accessible chromatin at a glucocorticoid-responsive enhancer.

To gain a better understanding of the nature of active chromatin in mammals, we have characterized in living cells the various chromatin modification events triggered by the glucocorticoid receptor (GR) at the rat tyrosine aminotransferase gene. GR promotes a local remodeling at a glucocorticoid-responsive unit (GRU) located 2.5 kb upstream of the transcription start site, creating nuclease hypersensitivity that encompasses 450 bp of DNA. Nucleosomes at the GRU occupy multiple frames that are remodeled without nucleosome repositioning, showing that nucleosome positioning is not the key determinant of chromatin accessibility at this locus. Remodeling affects nucleosomes and adjacent linker sequences, enhancing accessibility at both regions. This is associated with decreased interaction of both the linker histone H1 and the core histone H3 with DNA. Thus, our results indicate that nucleosome and linker histone removal rather than nucleosome repositioning is associated with GR-triggered accessibility. Interestingly, GR induces hyperacetylation of histones H3 and H4, but this is not sufficient either for remodeling or for transcriptional activation. Finally, our data favor the coexistence of several chromatin states within the population, which may account for the previously encountered difficulties in characterizing unambiguously the active chromatin structure in living cells.

Arsenic at very low concentrations alters glucocorticoid receptor (GR)-mediated gene activation but not GR-mediated gene repression: complex dose-response effects are closely correlated with levels of activated GR and require a functional GR DNA binding domain.

Arsenic (As) contamination of drinking water is considered a principal environmental health threat throughout the world. Chronic intake is associated with an increased risk of cancer, diabetes, and cardiovascular disease, and recent studies suggest increased health risks at levels as low as 5-10 ppb. We report here that 0.05-1 microM (6-120 ppb) As showed stimulatory effects on glucocorticoid receptor (GR)-mediated gene activation in rat EDR3 hepatoma cells of both the endogenous tyrosine aminotransferase (TAT) gene and the reporter genes containing TAT glucocorticoid response elements. At slightly higher concentrations (1-3 microM), the effects of As became inhibitory. Thus, over this narrow concentration range, the effects of As changed from a 2- to 4-fold stimulation to a greater than 2-fold suppression in activity. Interestingly, the inhibitory effect of GR on both AP1- and NF-kappa B-mediated gene activation was not affected by As. The magnitude of GR stimulation and inhibition by As was highly dependent on the cellular level of hormone-activated GR. Mutational deletion studies indicated that the central DNA binding domain (DBD) of GR is the minimal region required for the As effect and does not require free sulfhydryls. Point mutations located within the DBD that have known structural consequences significantly altered the GR response to As. In particular, point mutations in the DBD that confer a DNA-bound GR confirmation abolished the low dose As stimulatory effect but enhanced the inhibitory response, further indicating that the DBD is important for mediating these As effects.

Species specific hepatocarcinogen inhibition of the glucocorticoid induction of tyrosine aminotransferase gene in mouse and rat liver.

3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) is a potent hepatocarcinogen in rats and a weak carcinogen in mice, whereas o-aminoazotoluene (OAT) is a potent hepatocarcinogen in mice but weak hepatocarcinogen in rats. They significantly suppress glucocorticoid induction of tyrosine aminotransferase (TAT) in the liver of sensitive animals and have minor effect on the induction of this enzyme in the liver of resistant animals (3'-MeDAB-treated mice and OAT-treated rats). The inhibitory effect of these carcinogens is realized at the level of gene transcription (decreased accumulation of TAT mRNA). This effect is mediated via reduction of DNA-binding activity of transcription factor HNF3 (without decrease of its content) without any involvement of the glucocorticoid receptor. It was shown that carcinogens influence DNA-binding activity of HNF3 via an unknown nuclear factor.

Rat liver tyrosine aminotransferase activity and induction by dexamethasone upon cadmium intoxication

This study was focused on Cd effects on basal and dexamethasone-induced tyrosine aminotransferase (TAT) activity in the rat liver cytosol. Cadmium (Cd), applied in the dose of 2 mg/kg b.w., stimulated both TAT activity and its induction by dexamethasone, inducing the most prominent alterations 24 h after administration. Doses lower than 2 mg Cd/kg b.w. were ineffective while the higher ones (3 and 4 mg Cd/kg b.w) led to the changes similar to those reached by 2 mg Cd/kg. The in vitro application of different Cd concentrations to the liver cytosol rendered the enzyme activity unchanged suggesting that the metal acted at the level of TAT gene transcription.

Methylation and cell memory

It has been known for more than 20 years that DNA methylation at CpG dinucleotides is associated with gene silencing in eukaryotes. The preservation of CpG methylation patterns through cell division suggests that they could be important in generating cell ‘memory’ and hence in embryonic cell determination and differentiation. Several lines of evidence support this hypothesis, but a clear example has been lacking. Thomassin et al. 1xGlucocorticoid-induced DNA methylation and gene memory during development. Thomassin, H. et al. EMBO J. 2001; 20: 1974–1983CrossRef | PubMed | Scopus (176)See all References1 now provide an elegant model system in their study of the liver-specific tyrosine aminotransferase (Tat) gene, and Stancheva et al. 2xLoss of the maintenance methyltransferase, xDnmt1, induces apoptosis in Xenopus embryos. Stancheva, I. et al. EMBO J. 2001; 20: 1963–1973CrossRef | PubMed | Scopus (68)See all References2 investigate the role of DNA methylation in amphibian embryonic development.Tat transcription in the liver is triggered at birth by hypoglycemia. This response is mediated by transcription factors that cooperate with the glucocorticoid receptor, interacting with glucocorticoid responsive units (GRUs) located in the Tat enhancer. Thomassin et al. show that glucocorticoid treatment of cultured rat hepatoma cells induces rapid (<1 hour) remodelling of chromatin and recruitment of transcription factors to the Tat enhancer, but also slower (2–3 day) demethylation of CpGs within one of the GRUs. In contrast to transcriptional activation of Tat, which requires continuous glucocorticoid treatment, this CpG demethylation is stable. Furthermore, a second glucocorticoid stimulus, delivered to the cells even one month later, induces Tat transcription more strongly and rapidly. This suggests that demethylation provides the cells with a memory that sensitizes them to the transcription-activating signal.How do these in vitro observations compare with the situation in the embryo? The authors show that the Tat GRU is demethylated, specifically in the liver, following an increase in embryonic corticosteroids a few days before birth. Furthermore, GRU demethylation is prematurely induced by glucocorticoid treatment of cultured fetal hepatocytes. However, unlike hepatoma cells, glucocorticoid stimulation does not immediately trigger Tat transcription in hepatocytes and the embryo. This is induced at birth, when corticosteroids are low, perhaps by factors interacting with the Tat proximal promoter. Thus, this paper suggests that, at least in the Tat gene, DNA demethylation is not sufficient for tissue-specific expression, but contributes to it by preparing the gene for a rapid response to inducing factors. Glucocorticoid receptor knockout mice do not activate the Tat gene at birth, showing the importance of this gene sensitization.Stancheva et al. have previously shown in Xenopus that antisense depletion of the maintenance methyltransferase, xDnmt1, leads to premature expression of certain embryonic transcripts. They now show that phenotypic abnormalities in xDnmt1-depleted embryos are associated with the appearance of apoptotic cells. Experimental inhibition of apoptosis leads to the growth of abnormal undifferentiated cellular masses. These results confirm the importance of DNA methylation for embryogenesis. Cell differentiation activates an apoptotic pathway to eliminate cells in which appropriate methylation is not maintained. An interesting observation is that the genes prematurely activated by xDnmt1-depletion are involved in the early determinative events of embryonic germ-layer patterning. In future, experiments similar to those of Thomassin et al. might shed light on the specific role of DNA methylation in these events.

In vivo analysis of the model tyrosine aminotransferase gene reveals multiple sequential steps in glucocorticoid receptor action.

We are studying the mechanisms of transcriptional activation by nuclear receptors and we focus our studies on the glucocorticoid regulation of the model tyrosine aminotransferase gene. Rather than using in vitro biochemical approaches, we determine the actual events occurring in the cells. Our experimental approaches include genomic footprinting, chromatin immunoprecipitation, in situ hybridization and transgenic mice. Our results show that the glucocorticoid receptor uses a dynamic multistep mechanism to recruit successively accessory DNA binding proteins that assist in the activation process. Chromatin is first remodelled, DNA is then demethylated, and the synthesis of an accessory factor is induced. Efficient transcription induction is finally achieved upon the formation of a 'stable' multiprotein complex interacting with the regulatory element. We discuss: the relative contribution of histone acetyltransferases and ATP-dependent remodelling machines to the chromatin remodelling event; the nature of the remodelled state; the contribution of regulated DNA demethylation to gene memory during development; the mechanisms of regulated DNA demethylation; the dynamics of protein recruitment at regulatory elements; the control of the frequency of transcription pulses and the control levels of the cell-type specificity of the glucocorticoid response.

Involvement of transcription factor HNF3gamma in the effect of o-aminoazotoluene on glucocorticoid induction of tyrosine aminotransferase in mice sensitive to its hepatocarcinogenic action.

In the rodent liver, hepatocarcinogens inhibit the glucocorticoid induction of several liver-specific genes, including tyrosine aminotransferase (TAT). A distinct positive correlation exists in mice between the extent of inhibition of TAT induction after acute administration of o-aminoazotoluene (OAT) and the frequency of liver tumors after chronic exposure to the carcinogen. To elucidate the mechanism of the carcinogenic action, the effects of OAT on the DNA-binding activity of several transcription factors participating in the glucocorticoid regulation of TAT gene expression were studied. The experimental inbred male mice were sensitive (A/He and SWR/J, tumor induction frequency of 75-100%, TAT induction inhibition of 35-50%) and resistant (CC57BR/Mv and AKR/J, 0-6% and 10-15%, respectively) to OAT. Gel retardation experiments showed that hepatocyte nuclear factor 3 (HNF3)gamma DNA-binding activity was strongly reduced in nuclear extracts from the livers of OAT-treated A/He and SWR/J mice but only slightly reduced in CC57Br/Mv and AKR/J mice. The DNA-binding activities of Ets, AP1 family members, and GME binding proteins were unaffected. HNF3gamma DNA-binding activity was reduced by 1 h after OAT administration and remained low for 1 mo, as did inhibition of TAT induction in the liver. These results suggested that the inhibitory effect of OAT on the glucocorticoid induction of TAT is mediated by reduced HNF3gamma DNA-binding activity.

Glucocorticoid-induced DNA demethylation and gene memory during development.

Glucocorticoid hormones were found to regulate DNA demethylation within a key enhancer of the rat liver-specific tyrosine aminotransferase (Tat) gene. Genomic footprinting analysis shows that the glucocorticoid receptor uses local DNA demethylation as one of several steps to recruit transcription factors in hepatoma cells. Demethylation occurs within 2-3 days following rapid (< 1 h) chromatin remodeling and recruitment of a first transcription factor, HNF-3. Upon demethylation, two additional transcription factors are recruited when chromatin is remodeled. In contrast to chromatin remodeling, the demethylation is stable following hormone withdrawal. As a stronger subsequent glucocorticoid response is observed, demethylation appears to provide memory of the first stimulation. During development, this demethylation occurs before birth, at a stage where the Tat gene is not yet inducible, and it could thus prepare the enhancer for subsequent stimulation by hypoglycemia at birth. In vitro cultures of fetal hepatocytes recapitulate the regulation analyzed in hepatoma cells. There fore, demethylation appears to contribute to the fine-tuning of the enhancer and to the memorization of a regulatory event during development.

Local DNA demethylation in vertebrates: how could it be performed and targeted?

In vertebrates, cytosine methylation is an epigenetic DNA modification that participates in genome stability and gene repression. Methylation patterns are either maintained throughout cell division, or modified by global or local de novo methylation and demethylation. Site-specific demethylation is a rather elusive process that occurs mainly in parallel to gene activation during development. In light of our studies of the glucocorticoid-dependent DNA demethylation of the tyrosine aminotransferase gene, we discuss the potential biochemical mechanisms allowing DNA demethylation and its targeting to specific sequences by transcription factors as well as possible links to DNA replication and chromatin remodelling.

Evidence for a Common Step in Three Different Processes for Modulating the Kinetic Properties of Glucocorticoid Receptor-induced Gene Transcription

The dose-response curve of steroid hormones and the associated EC(50) value are critical parameters both in the development of new pharmacologically active compounds and in the endocrine therapy of various disease states. We have recently described three different variables that can reposition the dose-response curve of agonist-bound glucocorticoid receptors (GRs): a 21-base pair sequence of the rat tyrosine aminotransferase gene called a glucocorticoid modulatory element (GME), GR concentration, and coactivator concentration. At the same time, each of these three components was found to influence the partial agonist activity of antiglucocorticoids. In an effort to determine whether these three processes proceed via independent pathways or a common intermediate, we have examined several mechanistic details. The effects of increasing concentrations of both GR and the coactivator TIF2 are found to be saturable. Furthermore, saturating levels of either GR or TIF2 inhibit the ability of each protein, and the GME, to affect further changes in the dose-response curve or partial agonist activity of antisteroids. This competitive inhibition suggests that all three modulators proceed through a common step involving a titratable factor. Support for this hypothesis comes from the observation that a fragment of the coactivator TIF2 retaining intrinsic transactivation activity is a dominant negative inhibitor of each component (GME, GR, and coactivator). This inhibition was not due to nonspecific effects on the general transcription machinery as the VP16 transactivation domain was inactive. The viral protein E1A also prevented the action of each of the three components in a manner that was independent of E1A's ability to block the histone acetyltransferase activity of CBP. Collectively, these results suggest that three different inputs (GME, GR, and coactivator) for perturbing the dose-response curve, and partial agonist activity, of GR-steroid complexes act by converging at a single step that involves a limiting factor prior to transcription initiation.

The tyrosine aminotransferase from Trypanosoma rangeli: sequence and genomic characterization

The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical p I of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates.

The tyrosine aminotransferase from Trypanosoma rangeli: sequence and genomic characterization.

The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates.

Properties of the glucocorticoid modulatory element binding proteins GMEB-1 and -2: potential new modifiers of glucocorticoid receptor transactivation and members of the family of KDWK proteins.

An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at -3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.

Properties of the Glucocorticoid Modulatory Element Binding Proteins GMEB-1 and -2: Potential New Modifiers of Glucocorticoid Receptor Transactivation and Members of the Family of KDWK Proteins

An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at −3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.

Transducin-like Enhancer of Split Proteins, the Human Homologs of Drosophila Groucho, Interact with Hepatic Nuclear Factor 3β

Members of the hepatic nuclear factor 3 (HNF3) family, including HNF3alpha, HNF3beta, and HNF3gamma, play important roles in embryonic development, the establishment of tissue-specific gene expression, and the regulation of gene expression in differentiated tissues. We found, using the glutathione S-transferase pull-down method, that the transducin-like Enhancer of split (TLE) proteins, which are the human homologs of Drosophila Groucho, directly associate with HNF3beta. Conserved region II of HNF3beta (amino acids 361-388) is responsible for the interaction with TLE1. A mammalian two-hybrid assay was used to confirm that this interaction occurs in vivo. Overexpression of TLE1 in HepG2 and HeLa cells decreases transactivation mediated through the C-terminal domain of HNF3beta, and Grg5, a naturally occurring dominant negative form of Groucho/TLE, also increases the transcriptional activity of this region of HNF3. These results lead us to suggest that TLE proteins could influence the expression of mammalian genes regulated by HNF3.

Ability of the glucocorticoid modulatory element to modify glucocorticoid receptor transactivation indicates parallel pathways for the expression of glucocorticoid modulatory element and glucocorticoid response element activities

The glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is located at −3.6 kb and 1 kb upstream of the glucocorticoid response elements (GREs). The GME has the unique transcriptional properties of modulating both the dose–response curve of agonists bound to the glucocorticoid receptor (GR) and the residual agonist activity of GR-bound antisteroids. The expression of GME activity involves the binding of two novel proteins (GMEB-1 and GMEB-2) that we have recently cloned. However, the mechanistic details are limited. The DNA sequence requirements for GME activity (CGTC) also remain poorly defined, which restricts efforts to identify other GME modulated genes. To help understand the mechanism for the unusual activities of the GME and to identify permissive gene environments for GME activity, we compared the changes in GME activity and GRE action (i.e. the fold induction by GR) caused by modifying several parameters. Phasing between the GME and downstream tandem GREs was unimportant, in contrast to other cis-acting elements like the GRE, while GME activity decreased rapidly when placed at increasingly larger distances 3′ to a tandem GRE. A minimal promoter was less effective in supporting GME than GRE activity. Although CREB binds to the GME, overexpression of CREB reduced GRE, but not GME, activity and a CRE was inactive when substituted for the GME. No effect of the GME was observed on the binding of GRs to a single GRE. However, the GME upstream of a single GRE was also unable to produce a left shift in the Dex dose–response curve under conditions where the GME was active with two GREs. In the absence of any GREs, the GME displayed intrinsic activity by elevating basal level expression. Collectively, these results indicate that an optimal position for a functional GME is within 250 bp upstream of a tandem GRE driving a complex promoter. Furthermore, as the changes in GME activity did not correlate with those for fold induction from the GRE, the mechanisms for expression of GME and GRE activities appear to utilize parallel, as opposed to common pathways.

Molecular mechanisms of extinction: old findings and new ideas

Fusion experiments between somatic cells have been used for a long time as a means to understand the regulation of gene expression. In hybrids between differentiated cells such as hepatocytes or lymphocytes and undifferentiated cells such as fibroblasts a phenomenon called extinction has been described. In such hybrids expression of cell-specific genes derived from the more differentiated parental cell is selectively turned off (extinguished), whereas genes expressed from both cells like housekeeping genes remain active after fusion. Study of the molecular basis of extinction of the liver-specifically expressed tyrosine aminotransferase gene and of the B-cell-specifically expressed immunoglobulin genes has revealed that in hybrids the transcriptional program of the differentiated cells is reset. This is accompanied by a loss of expression or activity of many of the regulatory molecules that were operating in the differentiated cells. In the light of new insights in eukaryotic gene regulation we speculate that molecular mechanisms such as chromatin remodelling, recruitment to heterochromatin or subnuclear localization could underly the extinction process.

Molecular mechanism(s) of action of isoproterenol on the expression of the angiotensinogen gene in opossum kidney proximal tubular cells

beta-adrenoceptors are present in the renal proximal tubules. We have previously reported that isoproterenol stimulates the accumulation of intracellular cAMP and the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells via the beta 1-adrenoceptor. We hypothesized that the molecular mechanism(s) of action of isoproterenol on the expression of the ANG gene is mediated via the interaction of the phosphorylated cAMP-responsive element binding protein (CREB) and the cAMP-responsive element (CRE; that is, ANG N-806/-779) in the 5'-flanking region of the rat ANG gene. The fusion genes containing the putative ANG-CRE of the rat ANG gene inserted upstream of the rat ANG basal promoter (ANG N-53/+18) fused to a human growth hormone (hGH) gene as reporter were stably cotransfected, with or without the plasmid containing the cDNA for 43 kDa CREB, into the OK cells. The effect of various agonists and antagonists of adrenoceptors on the expression of the fusion genes was evaluated by the amount of immunoreactive hGH secreted into the culture medium. The interactions of OK cellular nuclear protein(s) with the ANG N-806/779 were determined by gel mobility shift assays and by Southwestern and Western blot analysis. The addition of isoproterenol, forskolin, or 8-Bromo-cAMP (8-Br-cAMP) stimulated the expression of pOGH (ANG N-806/-779/-53/+18) by 135, 150, and 160%, respectively, but not mutants of the ANG N-806/-779. The stimulatory effect of isoproterenol was blocked in the presence of propranolol, Rp-cAMP, and atenolol, but not by the presence of stauro-sporine, U73122, and ICI 118,551. Transient transfection of the plasmid containing the cDNA for the catalytic subunit of protein kinase A further enhanced the stimulatory effect of 43 kDa CREB on the expression of the fusion gene. The gel mobility shift assays revealed the the nuclear protein(s) of OK cells binds to the radioactive-labeled ANG N-806/-779. The binding of the labeled ANG N-806/-779 to the OK cell nuclear protein(s) was displaced by unlabeled ANG N-806/-779, but not by the CRE of the somatostatin gene, the CRE of the tyrosine amino-transferase gene, or the mutants of the ANG N-806/-779. Southwestern blot analysis revealed that the labeled ANG N-806/-779 binds to two nuclear species of 43 and 35 kDa proteins. Western blot analysis, however, revealed that rabbit polyclonal antibodies against the 43 kDa CREB interacted with only the 43 kDa molecular species but not with the 35 kDa species. These studies demonstrate that the stimulatory effect of isoproterenol on the expression of the ANG gene may be mediated, at least in part, via the interaction of the phosphorylated CREB and the CRE in the 5'-flanking region of the rat ANG gene. The novel 35 kDa nuclear protein that is immunologically different from the 43 kDa CREB may also play a role in the expression of the ANG gene.

Cyclic adenosine-3',5'-monophosphate-mediated activation of a glutamine synthetase composite glucocorticoid response element.

The glutamate synthetase gene (GS) contains a composite glucocorticoid response element (cGRE) comprised of a GRE and an adjacent element with features of both a cAMP-response element (CRE) and a 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE). The CRE/TRE element of the cGRE contributed to two modes of transcriptional activation: 1) enhancement of the response to cortisol and 2) a synergistic response to cortisol and increased cAMP. COS-7 cells transfected with a cGRE-luciferase construct show minimal expression under basal conditions or forskolin treatment. After cortisol treatment, luciferase activity from the cGRE is enhanced 4- to 8-fold greater than the GRE portion of the cGRE or a GRE from the tyrosine aminotransferase gene. Treatment with both forskolin and cortisol produced a 2- to 4-fold synergistic response over cortisol alone. Synergy is also seen with 8-bromo-cAMP, is specific for the cGRE, and occurs in a number of established cell lines. Elimination of the GRE or CRE/TRE reduces the synergy by 70-100%. Altering the CRE/TRE to GRE spacing changed both enhancement and synergy. Moving the elements 3 bp closer or extending 15 bp reduced enhancement. Synergy was markedly reduced when elements were one half of a helical turn out of phase. Western blots verified that CREB (cAMP-responsive binding protein) and ATF-1 (activating transcription factor-1) binds to the cGRE sequence. A specific dominant negative inhibitor of the CREB family, A-CREB, reduced synergy by 50%. These results suggest that the GS cGRE can potentially integrate signaling from both the cAMP and glucocorticoid receptor transduction pathways and that CREB/ATF-1 may play an important role in this process.