Specific Promoter Elements Research Articles

Defective dorsal closure and loss of epidermal decapentaplegic expression in Drosophila fos mutants.

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. Here we show that kayak encodes D-Fos, the Drosophila homologue of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp.

The Transcriptional Silencer Protein NRF: A Repressor of NF-κB Enhancers

NF-κB/rel proteins are present in most cell types. In concert with other transcriptional factors they regulate a variety of genes which contribute to a wide spectrum of physiological activities like inflammation and apoptosis. An excellent example of this combinatorial regulation takes place in the IFN-β promoter. In this promoter the fundamental regulatory elements are assembled within less than 100 base pairs including a NF-κB/rel enhancer and a negative regulatory element, called NRE. NRE is a member of a new class of transcriptional repressor sequences with a silencing capacity targeted to the NF-κB/rel enhancer. NRF is a novel transcriptional factor that binds to NRE. NRF belongs to a major class of transcriptional repressors that interact with specific promoter elements and repress transcription by separable repression domains. Such molecules have been termed active repressors, because they act by inhibitory protein-protein interaction and not simply by steric hindrance.

The adeno-associated virus type 2 p40 promoter requires a proximal Sp1 interaction and a p19 CArG-like element to facilitate Rep transactivation.

We have identified the sequence elements that are required for adeno-associated virus type 2 p40 promoter activity. Mutation of specific promoter elements showed that two Sp1 sites at approximately -50 (Sp1-50) and -70 (GGT-70) bp upstream of the start of the p40 messages were necessary for maximal promoter activity. As expected, the TATA site at -30 was also essential. In vitro DNA binding experiments confirmed that the Sp1-50 and GGT-70 sites were bound by Sp1 or Sp1-like proteins. Two other transcription elements, the ATF-80 and AP1-40 sites, may play a role in p40 activity. Mutation of these elements resulted in a modest decrease in p40 transcription, but DNA binding experiments did not clearly demonstrate binding of transcription factors to these sites. In contrast, a major late transcription factor site at -110 was shown to bind the transcription factor, but mutation of this site had no effect on p40 activity. In a previous report, we have shown that transactivation of the p40 promoter by the viral Rep proteins required an upstream Rep binding element (in the terminal repeat or the p5 promoter), an unidentified p19 promoter element, and a p40 promoter element (D. J. Pereira and N. Muzyczka, J. Virol. 71:1747-1756, 1997). Here we demonstrate that the CArG-140 element in the p19 promoter and the Sp1-50 element in the p40 promoter are the specific p19 and p40 elements required for Rep induction of p40. As in the case of the p19 promoter, Sp1 facilitates interaction of Rep with the p40 promoter by interaction of the two proteins. Furthermore, electron microscopy experiments demonstrated that when Rep is bound to an upstream Rep binding element, it can interact with a proximal Sp1 site by protein contacts and create a loop in the intervening DNA. This finding suggests a common mechanism whereby the Rep binding element in the TR or the p5 promoter induces p19 and p40 activity by interaction with their respective Sp1 sites.

Induction of Sp1 activity by prolactin and interleukin-2 in Nb2 T-cells: differential association of Sp1–DNA complexes with Stats

The induction of the transcription factor Sp1 by prolactin (PRL) and interleukin-2 (IL-2) was investigated in the PRL- and IL-2 responsive rat Nb2 T-cell line. Western analysis showed a rapid increase in Sp1 synthesis in Nb2 cells in response to PRL or IL-2. Elevation of Sp1 protein levels occurred within 15 min following PRL or IL-2 stimulation, reached a maximum by 1 h and was inhibited by cycloheximide, indicating de novo protein synthesis. Interestingly, dilution of confluent, growth-arrested Nb2 cells to low density also caused a rapid elevation in Sp1 suggesting that growth arrest may down-regulate Sp1 synthesis. Electrophoretic mobility shift assays using an Sp1 consensus oligonucleotide as probe showed a rapid but transient formation of a single PRL-inducible complex at 30 min. In contrast, three IL-2-inducible complexes were formed at 30 min and persisted to at least 60 min. Mobility shift interference assays using specific Stat antibodies failed to detect Stat1 α, Stat3 or Stat5 in the 30 min PRL-inducible complex. In contrast, the IL-2 induced complexes contained Stat3 alone at 30 min and both Stat3 and Stat5 at 60 min. The PRL- and IL-2-inducible complexes did not contain the tumor suppressor protein, p53. The time dependent association of the Stat proteins with the IL-2-inducible complexes, but not with the PRL-inducible complex, suggests that the two mitogens may selectively utilize specific promoter elements for transcriptional activation of PRL- and IL-2-responsive genes. Alternatively, the two mitogens may be activating different genes with Sp1-binding promoter elements for their mitogenic action in Nb2 cells.

Comparison between cytomegalovirus promoter and elongation factor-1α promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein

The establishment of stable cell lines expressing the hepatitis C virus (HCV) core protein may be important for studies of HCV pathogenesis. Human and mouse cell lines were generated expressing the HCV core protein using expression vectors driven by either the cytomegalovirus (CMV) or elongation factor-1αa (EF-1α) promoters. Following transient transfection, HCV core protein was expressed in all cell lines. However, stable human hepatocellular carcinoma (HCC) and murine myeloma cell lines expressing the HCV core protein were only established using constructs driven by the EF-1α promoter. In contrast, stable expression of the hepatitis B virus (HBV) middle envelope protein (MHBs) was obtained successfully in these cell lines using an expression vector driven by the CMV promoter. Inhibitory activity of the first 69 amino acids of the HCV core protein on the CMV promoter was found by using chimeric MHBs/HCV core protein constructs. Growth of cloned cell lines expressing the HCV core protein was slower than that of nonexpressing cell lines. However, morphological changes and cell death were not observed in the stable cell lines expressing HCV core protein. These results indicate that the HCV core protein was not directly cytotoxic to HCC and myeloma cell lines but that specific promoter elements are required to establish stable expression of the nucleocapsid structural protein.

Application of Genomic DNA Affinity Chromatography Identifies Multiple Interferon-α-regulated Stat2 Complexes

Interferon-alpha (IFN-alpha)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo-and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA affinity chromatography, for the identification of IFN-alpha-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA affinity chromatography results and identified a Stat2:1 complex that binds with low affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-alpha dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-alpha activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.

Characterization of the core promoter of the Na+/K(+)-ATPase alpha 1 subunit gene. Elements required for transcription by RNA polymerase II and RNA polymerase III in vitro.

We have analyzed the core promoter element of the Na+/K(+)-ATPase alpha 1 subunit gene by means of an in vitro transcription system composed of a HeLa nuclear extract. 5'-deletion and 3'-deletion analyses revealed that this gene is specifically transcribed by RNA polymerase II in a manner that is dependent on the upstream regulatory region of the gene (-102 to -61), and that the 3' boundary of the minimal promoter element does not extend beyond +5. Analysis of linker-substitution mutations and point mutations revealed that the TATA-like sequence (-33 to -26) is required for upstream-sequence-dependent transcription whereas linker-substitution mutations and point mutations near +1 did not abolish transcription. The gene was found to be transcribed by RNA polymerase III when phosphocellulose column fractions were assayed. Deletion analysis mapped the minimal RNA-polymerase-III--specific promoter element from -49 to +17. The phosphocellulose 0.3-M-KCl fraction is absolutely required for transcription by RNA polymerase III, while the 0.85-M-KCl fraction represses aberrant transcription from incorrect initiation sites. Analysis of linker-substitution mutations indicated that the TATA-like sequence is required for RNA-polymerase-III--specific transcription. Although point mutations in the 5' half of the TATA-like sequence did not affect transcription, those in the 3' half shifted the transcription initiation site 3 bp upstream. The results suggest the the Na+/K(+)-ATPase alpha 1 subunit gene promoter contains a TATA-like sequence which can direct transcription by RNA polymerase III in vitro. The mechanism of alternative regulation of RNA polymerase II and RNA polymerase III is discussed.

In vitro analysis of transcription factor binding to nucleosomes and nucleosome disruption/displacement

This chapter discusses the in vitro analysis of transcription factor binding to nucleosomes and nucleosome disruption/displacement. The chapter describes assays to investigate transcription factor interactions with nucleosomes that have been developed with model nucleosome templates. These assays are applicable to examining the roles of diverse factors at specific enhancer and promoter elements contained in nucleosomes. Transcription factor binding to nucleosome cores is analyzed. The use of purified in vitro systems has revealed several criteria that influence the ability of factors to bind nucleosomal DNA. These include (1) the location of the binding site within the nucleosome cores, (2) the inherent nucleosome-binding ability of individual or combinations of factors, (3) acetylation of the core histone amino termini, (4) cooperative binding with additional factors, and (5) the presence of additional activities that participate in nucleosome disruption. The influence of these criteria can be assayed directly in binding assays utilizing reconstituted nucleosome cores as the binding substrate. Nucleosome cores can be formed with probe DNA in purified systems by either reconstitution with purified core histones or histone octamer transfer from purified cellular nucleosome cores.

Role of the duplicated CCAAT box region in gamma-globin gene regulation and hereditary persistence of fetal haemoglobin.

Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means.

Intestinal apolipoprotein AI gene transcription is regulated by multiple distinct DNA elements and is synergistically activated by the orphan nuclear receptor, hepatocyte nuclear factor 4.

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.

A rat gastrin-human gastrin chimeric transgene directs antral G cell-specific expression in transgenic mice

Gastrin gene expression in the gastrointestinal tract is under both developmental and spatial regulation. In the mature animal, gastrin, an important regulator of parietal acid secretion, is expressed primarily in G cells of the antrum. To determine whether specific promoter elements can direct expression to the gastric antrum in vivo, 450 nucleotides of the proximal rat gastrin promoter were cloned and used to construct a rat gastrin-human gastrin reporter chimeric transgene, which was injected into the mouse germ line. Northern blot analysis, in situ hybridization, and double-label immunocytochemistry studies demonstrated expression of the transgene specifically in antral G cells. Low levels of transgene expression were observed in the ileum and colon, where immunohistochemical studies demonstrated colocalization in enteroendocrine cells expressing peptide YY. The same 450-nucleotide rat gastrin promoter, when joined to the human growth hormone gene, did not result in antral expression. Similarly, a human gastrin-human gastrin reporter transgene also did not achieve antral expression, although it did express in the liver. These results suggest that cis-acting elements present in both the basal 450-nucleotide rat gastrin promoter and the intragenic sequences of the human gastrin gene are necessary to direct expression of a transgene specifically to antral G cells.

Neuron-specific expression of the synapsin II gene is directed by a specific core promoter and upstream regulatory elements

Synapsin II is an abundant peripheral membrane protein of synaptic vesicles that is expressed exclusively in neuronal cells. Here we report the isolation and characterization of the 5'-terminal region of the murine synapsin II gene. Primer extension and S1 nuclease protection analysis show that synapsin II gene transcription is initiated from a unique site. The synapsin II gene promoter contains no canonical TATA or CAAT boxes but has putative binding sites for the transcription factors Sp1, AP2, and NGFIA. This promoter is embedded in a large G+C-rich domain with characteristics of a CpG island. Transfection experiments using synapsin II-luciferase fusion genes demonstrate that the 5'-flanking sequence functions as a strong promoter in neuronal but not in nonneuronal cells. Deletion analysis reveals the presence of a neuron-specific core promoter (-79 to 153) and, upstream, two positive and one negative regulatory elements. The 5'-terminal region of the murine synapsin I gene was also cloned and sequenced. Although there is no extensive sequence homology between the 5'-flanking regions of the synapsin I and II genes, comparison analysis has identified two regions of homologous sequences, which may be involved in determining neuron specificity of the core promoters of these two genes.

Nerve growth factor induces transcription of transforming growth factor-beta 1 through a specific promoter element in PC12 cells.

Nerve growth factor (NGF) stimulates the differentiation of PC12 pheochromocytoma cells to those resembling sympathetic neurons. We have investigated whether NGF regulates transforming growth factor (TGF)-beta gene expression and protein secretion in PC12 cells. These cells constitutively express TGF-beta 1 mRNA, whereas TGF-beta 2 and -beta 3 mRNAs are expressed at very low levels. TGF-beta 1 gene expression was stimulated greater than 10-fold when PC12 cells were treated with NGF. Sequences between -119 and -98 in the TGF-beta 1 promoter, homologous to an Egr-1 binding site, were shown to be important for both basal and NGF-induced promoter activity. We also found that a factor(s) present in nuclear extracts from PC12 cells interacted with the sequences between -119 and -98 and that expression of this factor was induced by NGF treatment. Moreover, specific binding to TGF-beta 1 promoter fragments between -119 and -98 was seen using the bacterially expressed transcription factor Egr-1. These results indicate that activation of TGF-beta 1 expression is one of the cellular responses of PC12 cells to NGF and suggest that TGF-beta may play a role in the differentiation of sympathetic neurons.

Inhibition of protein phosphatases activates P4 promoter of the human insulin-like growth factor II gene through the specific promoter element.

To understand the transcriptional regulation of the human insulin-like growth factor II (IGF-II) gene, we examined the effects of okadaic acid, a potent in vitro inhibitor of protein phosphatases, on the activation of human IGF-II gene expression. Treatment of A-549 human lung adenocarcinoma cells with okadaic acid increased expression of the IGF-II mRNAs. Since the 4.8-kb mRNA is transcribed under the control of human IGF-II P4 promoter, we examined the P4 promoter element responsible for the okadaic acid-mediated transcriptional activation. Transfection of IGF-II P4 promoter-chloramphenicol acetyltransferase constructs demonstrated that the effects of okadaic acid on the induction of IGF-II gene expression are mediated through multiple promoter elements, including an Egr-1 consensus element. We have also shown that okadaic acid induced the expression of the transcription factor Egr-1. Moreover, by using a GAL4-Egr-1 fusion protein, we have directly demonstrated that okadaic acid positively regulates Egr-1 transcriptional activity in vivo. These results indicate that protein phosphatases play an important role in the transcriptional regulation of the IGF-II.

Oncogene transfer into the brain.

With the spectacular advances in the characterization of genes associated with brain tumors and other neurological disorders, experimental approaches to stably introduce and express foreign genes in vivo have recently evolved as an important tool to study the specific pathogenetic role of candidate genes. A well-characterized and frequently used model are transgenic mice (Wagner 1990; Hanahan 1989; Pattengale et al. 1989). These animals are generated by introduction of DNA constructs into fertilized eggs or into embryonic stem cells. They carry copies of the transgene in all somatic and germ cells and stably transmit the gene to their offspring in a Mendelian fashion. Transcription of the transgene is critically dependent on regulatory sequences. Using specific promoter elements, it is possible to achieve cell- and tissue-specific expression. Cross-breeding of transgenic lines which carry distinct transgenes allows to study the combined effects of the respective genes in vivo.

1992 ALZA Distinguished Lecture: bioengineering and vascular biology.

The vascular system is naturally dynamic; fluid mechanics and mass transfer are closely integrated with blood and vascular cell function. We are beginning to understand how local wall shear stress and strain modulate endothelial cell metabolism at the gene level. This knowledge may help explain the focal nature of many vascular pathologies, including atherosclerosis. Understanding mechanical control of gene regulation at the level of specific promoter elements and transcription factors involved will lead to development of novel constructs for localized delivery of specific gene products in regions of high or low shear stress or strain in the vascular system. In addition, recent research has shown how local fluid mechanics can alter receptor specificity in cell-to-cell and cell-to-matrix protein adhesion and aggregation. Knowledge of the specific molecular sequences involved in cell-to-cell recognition will allow development of targeted therapeutics, with applications in thrombosis, inflammation, cancer metastasis, and sickle-cell anemia. Bioengineers are uniquely qualified to be leaders in this field, because advances require a synthesis of cell and molecular biology with systems analysis, transport phenomena, and quantitative modeling. Rapid progress in tissue engineering applications will require this new kind of biomedical engineer, which represents both a challenge and an opportunity for our profession.

Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors.

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytes-macrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and Salmonella typhimurium histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-kappa B. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding gamma-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-kappa B pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.

Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors.

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytes-macrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and Salmonella typhimurium histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-kappa B. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding gamma-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-kappa B pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.

Transcriptional regulation and function of yeast metallothioneins

Metallothioneins (MTs) are low molecular weight, cysteine-rich metal binding proteins whose biosynthesis is activated, at the level of transcription, by a wide variety of metal ions and through specific metalresponsive promoter elements [ 11. In the baker’s yeast, Saccharomyces cerevisiae, a single MT gene exists, denoted CUP], which is transcriptionally activated only by the metals copper and silver, through the action of the metalloregulatory transcription factor ACE 1. The ACE 1 protein is a copper-activated sequence-specific DNA binding transcription factor which is constitutively synthesized and located in the S. cerevisiae nucleus poised for immediate activation of CUP1 gene transcription in response to toxic copper levels. In mammals and other higher eukaryotic species, MT protein isoforms are encoded by large gene families which are transcriptionally activated by many distinct metals via promoter metal regulatory elements (MREs) and, as yet unidentified, metal-responsive transcription factors [2]. To begin to understand the mechanisms by whrc;h organisms with MT gene iamriies sense environmental metals and respond by activating transcription of MT gene families, we have begun to study metal-responsive transcription in the yeast Candida glabrata. C. glabrata harbors a single MT-I gene, multiple tandemly amplified MT-Ha genes and a single, unlinked MT-IZb gene. Using a surrogate genetic approach, we have cloned the gene from C. glabrata which encodes a copper metalloregulatory transcription factor, denoted AMTl, and have demonstrated that AMTl binds to multiple sites in the promoter of each MT family member through which it activates MT gene transcription in response to copper. Interestingly, unlike ACEZ in S. cerevisiae, the AMTI gene is subject to rapid positive transcriptional autoregulation in response to copper. This metalloregulatory transcription factor autoregulation plays a critical role in the copper homeostatic machinery, and suggests that organisms harboring MT gene families utilize similar, though distinct mechanisms for metal-responsive transcription.

Identification of promoter elements in the mouse 21-hydroxylase (Cyp21) gene that require a functional cyclic adenosine 3',5'-monophosphate-dependent protein kinase.

The constitutive and cAMP-induced expression of the mouse steroid 21-hydroxylase gene (Cyp21) are impaired in adrenal cell mutants harboring mutations in cAMP-dependent protein kinase (cAMPdPK). These requirements for a functional cAMPdPK have been mapped to the proximal 330 basepairs of the Cyp21 promoter. This study attempts to identify specific promoter elements of Cyp21 that require cAMPdPK for constitutive activity by comparing their abilities to enhance the expression of a reporter gene in Y1 adrenocortical tumor cells and Y1 Kin mutants defective in cAMPdPK activity. As determined in transient transfection assays, Cyp21 promoter elements at -65, -140, -170, -210, and -280 each enhanced the expression of a human GH reporter gene in parent Y1 cells. The relative order of effectiveness of each of these elements was: -170 >> -280 > -140 > -65 > or = -210. The -170 element was 25-fold more effective in enhancing gene expression from the reporter construct in Y1 cells than in Kin mutant cells; the elements at -65, -140, and -210 were 3-fold more effective in Y1 cells than in Kin mutant cells; the -280 element was equally effective in the parent and Kin mutant clones. These studies suggest that the promoter elements at -170, -65, -140, and -210 mediate the requirement for a functional cAMPdPK in the expression of Cyp21. As determined by gel mobility shift assays with these elements, the dependence of the Cyp21 promoter elements on a functional cAMP-dependent protein kinase did not result from decreased expression or binding affinities of their respective DNA-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)