Analysis of c-Fos and c-Jun redox-dependent DNA binding activity.
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Amino Acid Sequence↗
Biomedical subjects
S Xanthoudakis
Publications and source records attributed to S Xanthoudakis.
Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia.
Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells are restored to a normally oxygenated environment. Nuclear run-on analysis demonstrates that induction of transcription is the mechanism of ref-1 mRNA elevation. Electrophoretic mobility shift assays and immunodepletion assays were used to further define the interaction of Ref-1 with specific AP-1-binding proteins under hypoxic conditions. These data demonstrate that the induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.
Carbon-Oxygen Lyases↗
The regulation of c-fos: too much is never enough.
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DNA-Binding Proteins↗
Escape from redox regulation enhances the transforming activity of Fos.
Fos and Jun form dimeric complexes that bind to DNA sequences containing activator protein 1 (AP-1) sites and regulate gene expression. The in vitro DNA-binding activity of these proteins is sensitive to reduction-oxidation (redox). Reduction of a single conserved cysteine residue, located in the DNA-binding domain, either by reducing agents or by a nuclear redox factor (Ref-1), is required for AP-1 DNA-binding activity. Replacing the critical cysteine with serine results in a protein that can bind to DNA in vitro even under oxidizing conditions. To determine whether redox control affects the function of Fos in vivo, we have constructed, and compared the properties of, retroviral vectors expressing either a truncated Fos protein (F118-211) or a truncated Fos protein in which the critical cysteine was replaced by serine (FC154S). In infected chicken embryo fibroblasts (CEFs), both vectors expressed similar levels of Fos protein, which formed heterodimers with Jun at equivalent efficiencies. However, extracts from cells expressing FC154S exhibited a threefold increase in AP-1 DNA-binding activity compared with cells expressing F118-211. Furthermore, this enhanced binding activity was resistant to treatment with the oxidizing agent diamide. Infection of CEFs by virus expressing FC154S resulted in increased numbers of transformed colonies and an increase in colony size compared with those obtained following infection by virus expressing Fos 118-211. These results suggest that redox regulation may limit the total level of functional Fos-Jun complexes in vivo and that escape from this control enhances transforming activity.
Animals↗
Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity.
Fos and Jun form a heterodimeric complex that regulates gene transcription by binding to the activator protein-1 (AP-1) DNA sequence motif. Previously, we demonstrated that the DNA-binding activity of Fos and Jun is regulated in vitro by a novel redox (reduction-oxidation) mechanism. Reduction of a conserved cysteine (cys) residue in the DNA-binding domains of Fos and Jun by chemical reducing agents or by a nuclear redox factor stimulates DNA-binding activity. Here, we describe purification and characterization of a 37 kDa protein (Ref-1) corresponding to the redox factor. Although Ref-1 does not bind to the AP-1 site in association with Fos and Jun, it partially copurifies with a subset of AP-1 proteins. Purified Ref-1 protein stimulates AP-1 DNA-binding activity through the conserved Cys residues in Fos and Jun, but it does not alter the DNA-binding specificity of Fos and Jun. Ref-1 may represent a novel redox component of the signal transduction processes that regulate eukaryotic gene expression.
Cell Nucleus↗
Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme.
The DNA binding activity of Fos and Jun is regulated in vitro by a post-translational mechanism involving reduction-oxidation. Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun. Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref-1) recently purified from Hela cells, stimulates AP-1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity. Here we demonstrate that the protein product of the ref-1 gene stimulates the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kappa B, Myb and members of the ATF/CREB family. Furthermore, immunodepletion analysis indicates that Ref-1 is the major AP-1 redox activity in Hela nuclear extracts. Interestingly, Ref-1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity. However, the redox and DNA repair activities of Ref-1 can, in part, be distinguished biochemically. This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes.
Amino Acid Sequence↗
UV cross-linking of distinct proteins to the PRDII domain of the interferon-beta promoter.
The human interferon-beta gene (IFN-beta) is transcriptionally activated in a variety of cell types, mediated in part by a decameric element, designated the PRDII domain (-55 to -64) which is the target for the transcription factor NF-kappa B. In the present study, we have examined factor binding to the PRDII domain in three cell lines (HeLa, U937, Jurkat) following treatment with known inducers of IFN-beta transcription and NF-kappa B binding activity. UV cross-linking analysis has revealed that in all 3 cell types two inducible proteins of 58 kD and 69 kD and one constitutive protein of 48 kD bind to the PRDII domain. These experiments suggest that multiple proteins may interact with this element to regulate IFN-beta gene transcription.
Base Sequence↗
Multiple protein-DNA interactions within the human interferon-beta regulatory element.
High efficiency nuclear protein extracts were prepared from uninduced and recombinant interferon (IFN-alpha 2-primed Sendai virus-induced HeLa S3 cells and analyzed for DNA binding proteins specific for the human interferon-beta regulatory element (IRE). Analysis of protein-DNA interactions by a gel electrophoresis DNA binding assay resolved three complexes (designated A, B, and C) specific for the IRE probe (-79 to -36) in uninduced and induced cells. Competition studies using greater than a 100-fold molar excess of unlabeled DNA fragments representing different IFN-beta promoter sequences localized the DNA binding domain to the region -79 to -64 (P1 probe), a region previously shown to be essential for virus-inducible gene expression. Other adjacent IFN-beta segments including the negative regulatory element (-65 to -30) as well as the SV40 enhancer, c-fos serum-responsive element, and IFN-alpha 1 promoter fragment (-131 to -71) were unable to compete for the IRE binding protein. The 16-base pair enhancer core (P1 probe) specifically bound similar amounts of protein from uninduced and induced extracts; a synthetic tetrahexamer, AAGTGA, competed efficiently for the binding to the P1 region. Competition studies indicated that adjacent upstream IFN-beta DNA sequences -94 to -78 (P5) also contained a sequence motif capable of binding the P1 protein. In vitro transcription of the IFN-beta template in the HeLa nuclear extracts was partially inhibited when increasing amounts of competitor IRE fragment were added to the reaction. These results demonstrate that multiple DNA sequence motifs within the IFN-beta regulatory element interact to bind transcription regulatory proteins which are present in normal and virus-infected HeLa cells.
Base Sequence↗
Induction of human interferon gene expression is associated with a nuclear factor that interacts with the NF-kappa B site of the human immunodeficiency virus enhancer.
The relationship between transcription of alpha and beta interferon (IFN-alpha and IFN-beta) genes and the interaction of IFN promoter-binding transcription factors has been examined in monoblastoid U937 cells following priming with recombinant IFN-alpha 2 (rIFN-alpha 2) and Sendai virus induction. Pretreatment of U937 cells with rIFN-alpha 2 prior to Sendai virus infection increased the mRNA levels of IFN-alpha 1, IFN-alpha 2, and IFN-beta as well as the final yield of biologically active IFN. Analysis of nuclear protein-IFN promoter DNA interactions by electrophoretic mobility-shift assays demonstrated increased factor binding to IFN-alpha 1 and IFN-beta regulatory domains, although no new induction-specific complexes were identified. On the basis of competition electrophoretic mobility-shift assay results, factors interacting with the IFN-alpha 1 and IFN-beta promoters appear to be distinct DNA-binding proteins. U937 factor binding was localized to the P2 domain (-64 to -55) of the IFN-beta regulatory element, a sequence motif with 80% homology to the recognition site of transcription factor NF-kappa B. Protein-DNA interactions within the IFN-beta P2 domain were, in fact, specifically competed by either excess homologous P2 fragment or the human immunodeficiency virus enhancer element which contains two duplicated NF-kappa B recognition sites. Hybrid promoter-chloramphenicol acetyltransferase fusion plasmids, containing either the IFN-beta regulatory element or the human immunodeficiency virus enhancer element linked to the simian virus 40 promoter, were analyzed for virus and phorbol ester inducibility in epithelial and lymphoid cells, respectively. In the 293 cell line, both plasmids were constitutively expressed but not virus inducible, while in Jurkat cells, chloramphenicol acetyltransferase activity from these plasmids was induced by tumor-promoting agent treatment. These experiments suggest that induction of IFN gene expression may be controlled in part by transcription regulatory proteins binding to an NF-kappa B-like site within the IFN-beta promoter.
Base Sequence↗
Modulation of interferon gene transcription by positive and negative cellular factors.
In vivo competition experiments were designed to identify the role of trans-acting cellular factors in the virus-inducible activation of the interferon-beta promoter. Co-transfection of a constant amount of IFN-beta/CAT test gene and increasing amounts of competitive DNA containing different IFN regulatory domains into human epithelioid 293 cells identified a low abundance, positive cellular factor(s) that interacts with the IFN regulatory region. Competition of the factor decreases virus-induced and constitutive level expression of the IFN-beta promoter, and also partially inhibits expression from the SV40 promoter. Negative regulatory effects produced by factors interacting with the IFN upstream region (-135 to -202) and with the SV40 enhancer were also observed.
Cell Line↗
trans activation of type 1 interferon promoters by simian virus 40 T antigen.
A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.
Adenovirus Early Proteins↗
Identification of a nuclear DNA binding protein associated with the interferon-beta upstream regulatory region.
Nuclear protein extracts were prepared from uninduced myeloid leukemic KG-1 cells and analyzed for interferon-specific DNA binding by a gel electrophoresis DNA binding assay. A protein was detected that bound specifically to a 163-base pair upstream region of the IFN-beta promoter. A series of competition studies were performed to assess whether this was a general DNA binding protein; binding of this factor was competed from radiolabeled beta 163 fragment only by an excess of cold unlabeled beta 163 or a 67-base pair fragment (beta 67) derived from beta 163. Other promoter and enhancer transcriptional domains including 1) c-fos serum responsive element, 2) c-fos promoter, 3) SV40 enhancer, 4) IFN-alpha 1 promoter, and 5) plasmid pAT153 did not compete effectively for binding of the protein. These results indicate that this protein is not a general enhancer or promoter binding factor and may be unique to IFN-beta. Analysis of beta 67 DNA sequences revealed no homology to known recognition sequences for Sp1 or CTF transcription factors.
Cell Line↗
Transient expression of the beta interferon promoter in human cells.
A human transient expression assay was used to examine the inducible transcriptional activation of beta interferon (IFN-beta) and IFN-alpha 1 promoters in a homologous cellular environment. Use of 293 cells, an adenovirus DNA-transformed human embryonic kidney cell line, permitted Sendai virus-inducible expression of IFN-beta-CAT hybrid gene. Introduction of the simian virus 40 (SV40) enhancer 5' or 3' to the IFN-CAT gene increased basal (uninduced) levels of chloramphenicol acetyltransferase (CAT) activity; in one construct the SV40 enhancer--IFN-beta regulatory region combination increased the induced CAT activity 50- to 100-fold, suggesting that this may be a generally useful inducible enhancer-promoter combination. No expression from the IFN-alpha-CAT hybrid gene was detected in 293 cells, indicating that human epithelioid cells lack a factor required for expression of the IFN-alpha promoter. However, when the IFN-alpha regulatory region was combined with the SV40 enhancer, a low level of inducible CAT activity was detected in the human transient system.
Cell Line↗