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At least 523 records · Page 29Linked to original sources
PubMed1992

Two adjacent AP-1-like binding sites form the electrophile-responsive element of the murine glutathione S-transferase Ya subunit gene.

An electrophile-responsive element (EpRE) in the 5' flanking region of the mouse glutathione S-transferase Ya subunit gene was recently found to be responsible for the induction of gene expression by xenobiotics that contain or acquire by metabolism an electrophilic center. We now find that this EpRE is composed of two adjacent 9-base-pair motifs related in sequence to the AP-1 binding site, a transcriptional enhancer originally identified as the phorbol 12-myristate 13-acetate (PMA) response element and known to be regulated by the binding of protein products of c-jun and c-fos genes. Synthetic oligonucleotides representing each of the AP-1-like binding sites of the EpRE and the AP-1 site consensus sequence were prepared and assayed for their enhancer activity and inducibility by tert-butylhydroquinone, beta-naphthoflavone, and PMA. Single AP-1-like sequences showed a lower enhancer activity than an AP-1 consensus sequence and no inducibility. Two adjacent AP-1-like sites were found to act synergistically and to confer inducibility beyond that observed for a single AP-1 consensus sequence. Examination of the PMA-responsive region of a number of genes shows the presence of adjacent AP-1-like sites and indicates that the structure of the EpRE found in the Ya gene may occur more generally and may be important in regulating the magnitude of the electrophilic response. The present study demonstrates the binding and transactivation of the EpRE by Jun and Fos and indicates that the AP-1 site is part of the EpRE. The induction by PMA or tert-butylhydroquinone appears to be independent of protein kinase C activity since it is not affected by inhibitors of this enzyme.

Animals↗
PubMed1997

Characterization of the response element and DNA binding properties of the nuclear orphan receptor germ cell nuclear factor/retinoid receptor-related testis-associated receptor.

Recently, we have reported the cloning of the germ cell-specific, nuclear orphan receptor germ cell nuclear factor (GCNF)/RTR. In this study, we characterize the RTR response elements by an electrophoretic mobility shift assay/polymerase chain reaction-based, DNA binding site selection strategy. RTR binds with the greatest affinity to response elements containing TCA(AG(G/T)TCA)2 (consensus RTR response element; conRTRE), to which it binds as a homodimer. RTR is also able to bind as a monomer to a single core motif TCAAG(G/T)TCA, albeit with a lower affinity. Mutation analysis supports the specific requirements of the 5'-flanking sequence and the core motif of the RTRE for optimal binding of RTR. An RTR-specific antiserum (RTR-Ab2) was raised that causes supershift of the RTR-conRTRE complex in EMSA. Based on the sequence of the conRTRE, we located a putative RTRE, referred to as P2-RE, in the 5' promoter-flanking region of the mouse protamine 2 gene, which is induced during the same stage of spermatogenesis as RTR. The ability of RTR-Ab2 to cause a supershift of an RTR-RTRE complex with nuclear extracts from different tissues correlated with the tissue- and development-specific expression of RTR. Transfection of RTR in CV-1 cells was unable to cause RTRE-dependent transactivation of a CAT reporter gene; however, an RTR-VP16 fusion protein could induce transactivation through several RTREs, including P2-RE.

Amino Acid Sequence↗
PubMed2001

Cross-talk between the signals hypoxia and glucose at the glucose response element of the L-type pyruvate kinase gene.

The signals oxygen and glucose play an important role in metabolism, angiogenesis, tumorigenesis, and embryonic development. Little is known about an interaction of these two signals. We demonstrate here the cross-talk between oxygen and glucose in the regulation of L-type pyruvate kinase (L-PK) gene expression in the liver. In the liver the periportal to perivenous drop in O(2) tension was proposed to be an endocrine key regulator for the zonated gene expression. In primary rat hepatocyte cultures the expression of the L-PK gene on mRNA and on protein level was induced by venous pO(2), whereas its glucose-dependent induction occurred predominantly under arterial pO(2). It was shown by transient transfection of L-PK promoter luciferase and glucose response element (Glc(PK)RE) SV40 promoter luciferase gene constructs that the modulation by O(2) of the glucose-dependent induction occurred at the Glc(PK)RE in the L-PK gene promoter. The reduction of the glucose-dependent induction of the L-PK gene expression under venous pO(2) appeared to be mediated via an interference between hypoxia inducible factor-1 (HIF-1) and upstream stimulating factor at the Glc(PK)RE. The glucose response element also functioned as an hypoxia response element which was confirmed in cotransfection assays with Glc(PK)RE luciferase gene constructs and HIF-1alpha expression vectors. Furthermore, it was found by gel shift and supershift assay that HIF-1alpha and USF-1 or USF-2 could bind to the Glc(PK)RE. Our findings implicate that the cross-talk between oxygen and glucose might have a fundamental role in the regulation of several physiological and pathophysiological processes.

Animals↗
PubMed2005

Induction of the protective antioxidant response element pathway by 6-hydroxydopamine in vivo and in vitro.

Parkinson's disease, a progressive neurodegenerative disorder, is characterized by loss of midbrain dopaminergic neurons. The etiology of sporadic Parkinson's disease is unknown; however, oxidative stress is thought to play a major role in disease pathogenesis. Little is known regarding the transcriptional changes that occur in Parkinson's disease. The antioxidant response element is a cis-acting enhancer sequence that is upstream of many phase II detoxification and antioxidant genes. Here we show that 6-hydroxydopamine, a mitochondrial inhibitor used to model Parkinson's disease, activates the antioxidant response element both in cultured neurons and in the striatum and brainstem of 6-OHDA-lesioned mice. Pretreatment with antioxidants or NMDA receptor antagonists reduced but did not abolish activation. Further induction of this pathway with tert-butylhydroquinone was able to significantly reduce cell death due to 6-OHDA in vitro. These observations indicate that 6-OHDA activates the antioxidant response element through components of oxidative stress, excitotoxicity, and potential structural factors. Further induction of this endogenous defense mechanism may suggest a novel therapeutic venue in Parkinson's disease.

Animals↗
PubMed2005

Peroxisome proliferator-activated receptor alpha and its response element are required but not sufficient for transcriptional activation of the mouse heart-type fatty acid binding protein gene.

Expression of heart-type fatty acid binding protein is restricted mainly to the skeletal and cardiac muscles and further regulated by peroxisome proliferator-activated receptor alpha. The molecular basis for the muscle-restricted peroxisome proliferator-activated receptor alpha action on the fatty acid binding gene was analyzed using normal and the receptor-null mice and the cultured cells. Two possible peroxisome proliferator-response elements were found in the promoter region of the mouse gene. A gel shift assay showed that both elements were functional. However, neither the tandem repeats of the elements nor the cloned promoter sequence could be activated by peroxisome proliferator-activated receptor alpha and its ligand in the reporter gene assay using cultured cells. The cloned promoter responded to the ligand only in the muscle when the reporter gene was introduced into the mouse muscle. Using a chimeric receptor with the activation domain of herpes virus VP16 protein and the tandem repeats of the elements with or without mutation, the upstream element was finally demonstrated to be potentially involved in the receptor-dependent transcriptional activation. These results suggest that the peroxisome proliferator-response element of the mouse gene is atypical and there is a muscle-specific mechanism to enhance the weak binding of the receptor to the response element to ensure the muscle-specific action of peroxisome proliferator-activated receptor alpha on the heart-type fatty acid binding protein gene promoter.

Animals↗
PubMed2001

The hairpin loop but not the bulged C of the iron responsive element is essential for high affinity binding to iron regulatory protein-1.

Vertebrates control intracellular iron concentration principally through the interaction of iron regulatory proteins with mRNAs that contain an iron responsive element, a small hairpin with a bulged C. The hairpin loop and bulged C have previously been assumed to be critical for binding and have been proposed to make direct contact with the iron regulatory proteins. However, we show here that a U or G can be substituted for the bulged C provided that specific nucleotides are also present within internal loops. The K(d), IC(50) and chemical modifications of the iron responsive element variants are similar to the wild-type. Results are more consistent with a role in which the C-bulge functions to orient the hairpin for optimal protein binding rather than to directly contact the protein. Characterization of these novel iron responsive element variants may facilitate the identification of additional mRNAs whose expression is controlled by iron regulatory proteins, as well as provide insight into the nature of a critical RNA-protein interaction.

Base Sequence↗
PubMed1995

Steroid hormones upregulate rat angiotensin II type 1A receptor gene: role of glucocorticoid responsive elements in rat angiotensin II type 1A promoter.

The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells. There are three putative glucocorticoid responsive elements in the promoter. However, only one glucocorticoid responsive element was found to respond to dexamethasone (1 microM). The region was located at positions, -756 to -770 bp upstream of the transcription initiation site. A glucocorticoid antagonist, RU38486, completely blocked the induction by dexamethasone, suggesting that the glucocorticoid responsive element was functional through a specific glucocorticoid receptor. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs. We concluded that the dexamethasone-induced increase in the transcription of the angiotensin II type 1A receptor gene occurred through the binding to GRE up the glucocorticoid-specific receptor.

Aldosterone↗
PubMed1995

Identification of a putative steroidogenic factor-1 response element in the DAX-1 promoter.

The nuclear hormone receptor, DAX-1, is responsible for X-linked adrenal hypoplasia congenita and hypogonadotrophic hypogonadism. We recently cloned the 5' flanking region of the human DAX-1 gene and in this report we describe the identification of a putative steroidogenic factor 1 (SF-1) response element approximately 110 bases upstream of the TATA box. Both DAX-1 and SF-1 are expressed in similar tissues including the adrenal cortex, gonads, hypothalamus, and the pituitary gland. Like DAX-1, SF-1 expression has been shown to be essential for the development of the adrenal cortex. We demonstrate that SF-1 is able to efficiently bind to the putative SF-1 response element found in the DAX-1 promoter in vitro. This suggests that SF-1 may directly regulate the expression of DAX-1 and that these two transcription factors may be components of a cascade required for development of steroidogenic tissues.

Base Sequence↗
PubMed2002

An iron-responsive element type II in the 5'-untranslated region of the Alzheimer's amyloid precursor protein transcript.

Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5'-untranslated region (5'-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (+51 to +94 from the 5'-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (+101 to +146). APP 5'-UTR conferred translation was selectively down-regulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5'-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5'-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (Delta4 bases:Delta83AGAG86). Iron regulation of APP mRNA through the APP 5'-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce Abeta peptide burden during Alzheimer's disease.

5' Untranslated Regions↗
PubMed1995

Ischemia and reperfusion enhance ATF-2 and c-Jun binding to cAMP response elements and to an AP-1 binding site from the c-jun promoter.

The transcription factors controlling the complex genetic response to ischemia and their modes of regulation are poorly understood. We found that ATF-2 and c-Jun DNA binding activity is markedly enhanced in post-ischemic kidney or in LLC-PK1 renal tubular epithelial cells exposed to reversible ATP depletion. After 40 min of renal ischemia followed by reperfusion for as little as 5 min, binding of ATF-2 and c-Jun, but not ATF-3 or CREB (cAMP response element binding protein), to oligonucleotides containing either an ATF/cAMP response element (ATF/CRE) or the jun2TRE from the c-jun promoter, was significantly increased. Binding to jun2TRE and ATF/CRE oligonucleotides occurred with an identical time course. In contrast, nuclear protein binding to an oligonucleotide containing a canonical AP-1 element was not detected until 40 min of reperfusion, and although c-Jun was present in the complex, ATF-2 was not. Incubating nuclear extracts from reperfused kidney with protein phosphatase 2A markedly reduced binding to both the ATF/CRE and jun2TRE oligonucleotides, compatible with regulation by an ATF-2 kinase. An ATF-2 kinase, which phosphorylated both the transactivation and DNA binding domains of ATF-2, was activated by reversible ATP depletion. This kinase coeluted on Mono Q column chromatography with a c-Jun amino-terminal kinase and with the peak of stress-activated protein kinase, but not p38, immunoreactivity. In conclusion, DNA binding activity of ATF-2 directed at both ATF/CRE and jun2TRE motifs is modulated in response to the extreme cellular stress of ischemia and reperfusion or reversible ATP depletion. Phosphorylation-dependent activation of the DNA binding activity of ATF-2, which appears to be regulated by the stress-activated protein kinases, may play an important role in the earliest stages of the genetic response to ischemia/reperfusion by targeting ATF-2 and c-Jun to specific promoters, including the c-jun promoter and those containing ATF/CREs.

Activating Transcription Factor 2↗
PubMed2000

A hormone response element in the human apolipoprotein CIII (ApoCIII) enhancer is essential for intestinal expression of the ApoA-I and ApoCIII genes and contributes to the hepatic expression of the two linked genes in transgenic mice.

We have generated transgenic mice carrying wild-type promoters of the human apolipoprotein A-I (apoA-I)-apoCIII gene cluster or promoters mutated in their hormone response elements. The wild-type cluster directed high levels of apoA-I gene expression in liver and intestine, moderate expression in kidney, and low to minimal expression in other tissues. It also directed high levels of chloramphenicol acetyltransferase (CAT) expression (used as a reporter for the apoCIII gene) in liver, low levels in intestine and kidney, and no expression in other tissues. Mutations in the apoCIII promoter and enhancer abolished the intestinal and renal expression of the apoA-I gene, reduced hepatic apoA-I expression by 80%, and abolished CAT expression in all tissues. A similar pattern of expression was obtained by mutations in the apoCIII enhancer alone. Mutations in the proximal apoA-I promoter reduced by 85% hepatic and intestinal apoA-I expression and did not affect CAT expression. The findings suggest that a hormone response element within the apoCIII enhancer is essential for intestinal and renal expression of apoA-I and apoCIII genes and also enhances hepatic expression. The hormone response elements of the proximal apoA-I promoter or the apoCIII enhancer can promote independently low levels of hepatic and intestinal expression of the apoA-I gene in vivo.

Animals↗
PubMed2006

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2).

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.

Amino Acid Sequence↗
PubMed1999

A novel factor binding to the glucose response elements of liver pyruvate kinase and fatty acid synthase genes.

Transcription of the liver type pyruvate kinase and lipogenesis enzyme genes is induced by high carbohydrate in liver. We have found a novel protein factor in rat liver nuclei that binds to the glucose response element (CACGTG motifs) of the pyruvate kinase gene (Liu, Z. , Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268, 12787-12795) and the "insulin response element" of fatty acid synthase gene. The amounts of this DNA-binding protein, termed "glucose response element binding protein" (GRBP) in the nuclear extract, were increased in liver by a high carbohydrate diet and decreased by starvation, high fat, and high protein diet. GRBP also occurs in cytosols of liver and is dependent on carbohydrate. Both the nuclear and the cytosolic GRBP showed similar properties, except the former was more resistant to thermal inactivation than the latter. Kinetics of glucose activation of the cytosolic GRBP in a primary culture of hepatocytes indicated that a half-maximum activation was achieved after 6 h, and glucose concentration required for the maximum activation of the GRBP was approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and forskolin inhibited glucose activation of both GRBP and liver pyruvate kinase transcription. These results suggested that GRBP may be a factor that recognizes the glucose response motif site and may be involved in mediating carbohydrate response of the pyruvate kinase gene.

Animals↗
PubMed1992

Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements.

Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.

Base Sequence↗
PubMed2002

A common drug-responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance.

Upregulation of the ATP-binding cassette (ABC) transporter genes CDR1 and CDR2 (Candida drug resistance 1 and 2) is a common mechanism observed in Candida albicans clinical isolates developing resistance to the class of azole antifungals. In this work, the regulatory elements of both genes were delimited using a reporter system in an azole-susceptible strain exposed to oestradiol, which allows transient induction of these genes. We found two regulatory elements in the CDR1 promoter: one responsible for basal expression (basal expression element; BEE) and the other required for oestradiol responsiveness (drug-responsive element I; DREI). In the CDR2 promoter, a single regulatory element responsible for oestradiol responsiveness (DREII) was detected. Both DREs shared a consensus of 21 bp with the sequence 5'-CGGA(A/T)ATCGGATATTTTTTTT-3' having no equivalent to known eukaryotic regulatory sequence. Consistent with this finding, two other C. albicans genes identified by a search for the presence of DRE in the C. albicans genome sequence database were responsive to oestradiol. Finally, the regulatory elements found in CDR1 and CDR2 were also functional in an azole-resistant strain with constitutive high expression of both transporters. These results suggest that, although CDR1 and CDR2 upregulation can be obtained by transient drug-induced and constitutive upregulation, these two processes converge to the same regulatory elements and probably mobilize the same trans-acting factors.

ATP-Binding Cassette Transporters↗
PubMed1990

Mutually dependent response elements in the cis-regulatory region of the neurotensin/neuromedin N gene integrate environmental stimuli in PC12 cells.

The expression of the gene encoding the neuroendocrine peptides neurotensin (NT) and neuromedin N is strictly dependent on simultaneous exposure to multiple inducers in PC12 pheochromocytoma cells. NT peptide and NT/N mRNA levels are synergistically induced by combinations of NGF, dexamethasone, activators of adenylate cyclase, and lithium ion. We have used transient transfection assays to delineate the rat NT/N gene sequences necessary for this complex regulation. Progressive deletions of the 5' flanking region revealed that sequences between -216 and +56 are sufficient to confer the full spectrum of responses exhibited by the endogenous gene to a reporter gene. Detailed mutational analysis of this region indicates that it is composed of an array of inducible cis-regulatory sequences, including AP-1, cAMP response, and glucocorticoid response elements. Specific mutation of either the AP-1 site or each of two cAMP response elements indicates that they are functionally interdependent. This array of response elements serves to integrate multiple environmental stimuli into a unified transcriptional response.

Adrenal Gland Neoplasms↗
PubMed2000

Estrogen receptor-related receptor alpha 1 interacts with coactivator and constitutively activates the estrogen response elements of the human lactoferrin gene.

The human estrogen receptor-related receptor (ERRalpha1, NR3B1a) was shown to bind a steroidogenic factor binding element (SFRE), TCAAGGTCATC, 26 base pairs upstream from the estrogen response element (ERE) of the human lactoferrin gene promoter. A mutation made at SFRE significantly reduced estrogen-dependent transcription from the lactoferrin ERE in human endometrial cells. In this study, we demonstrated that ERRalpha1 binds both SFRE and ERE elements and constitutively transactivates the lactoferrin gene promoter. In DNase I footprinting protection analysis, both SFRE and ERE regions were protected by glutathione S-transferase-ERRalpha1 fusion protein. The receptor formed two protein-DNA complexes with either SFRE or ERE in electrophoresis mobility shift assay. Homodimerization of ERRalpha1 was confirmed with the mammalian two-hybrid system. ERRalpha1 activates reporter constructs containing various types of estrogen response elements in endometrial and non-endometrial cells in transient transfection experiments. Overexpressing the coactivator, SRC1a or GRIP1, further enhances ERRalpha1-induced transcriptional activity. We demonstrated that the AF2 domain of ERRalpha1 is essential for the transactivation function and that deletion or mutation at this region abrogates the activation capability. Protein-protein interaction between the SRC1a and ERRalpha1 C terminus was confirmed with a GST glutathione S-transferase "pull-down" assay. When comparing ERRalpha1 and the estrogen receptor alpha (ERalpha) in many of the experiments, we found that ERalpha can also bind SFRE of the lactoferrin gene and transactivate the promoter activity in a ligand-dependent manner. The present study demonstrated that ERRalpha1 binds similar DNA elements as ERalpha and confers its transactivation function constitutively. Therefore, ERRalpha1 may actively modulate the estrogen response of lactoferrin gene as well as other estrogen-responsive genes.

DNA Footprinting↗
PubMed1997

AP-1 cis-response elements are involved in basal expression and Vmw110 transactivation of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The promoter of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) has two AP-1 cis-response elements, respectively located at positions -62 and -94 relative to the transcription start site (Wymer et al., 1989. J. Virol. 63, 2773-2784). Chloramphenicol acetyl transferase (CAT) analysis with hybrid constructions of the CAT structural gene and the ICP10 promoter or its mutants and gel retardation studies were used to examine the role of the AP-1 cis-response elements in expression from the ICP10 promoter. Basal expression from the wild-type promoter was significantly (75-90%) reduced by mutation of the upstream or downstream AP-1 element. Mutation in the upstream AP-1 element also caused a 60% reduction in c-Jun-mediated activation. Activation was decreased 40% by mutation in the downstream AP-1 element and it was abrogated by mutation of both elements. Similar results were obtained for ACT-deleted mutants and mutants in which CT was mutated to AG. The trans-activation by Vmw110 was also reduced by mutation of the AP-1 elements (10- and 2-fold for the upstream and downstream element, respectively) and it was abrogated by mutation of both AP-1 elements. Mutation of nucleotides adjacent to the AP-1 cis-response elements had no effect on trans-activation. Gel retardation assays with a DNA probe representing the wild-type ICP10 promoter and nuclear extracts from HSV-1-infected cells identified one complex that was not seen with mock-infected cells or with cells infected with a Vmw110-deleted mutant. The complex was not seen when HSV-1-infected cells were reacted with an AP-1-mutant DNA probe, and its formation was competed by an AP-1 but not a mutant AP-1 oligonucleotide. The migration of this complex was retarded by c-Fos antibody, suggesting that both AP-1 and Vmw110 are involved in its formation. A mutant deleted in all sequences upstream of the TATA box was also activated by Vmw110, but this activation was only 2-fold lower than that seen for the wild type and significantly higher (10-fold) than that seen for the double AP-1 mutants. The data suggest that AP-1 elements play a crucial role in ICP10 gene expression/activation.

Animals↗