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Showing papers on "Transcription factor published in 1989"


Journal ArticleDOI
TL;DR: It is demonstrated that two different peptide hormones, or cytokines, stimulate the human immunodeficiency virus enhancer, and this effect is mediated by nuclear factor (NF) kappa B (nuclear factor that binds the kappa immunoglobulin light chain gene enhancer); this link between binding at the surface membrane and stimulation of a specific transcription factor should help define intermediates for these cytokine activation pathways.
Abstract: Binding of peptide hormones to surface membrane receptors leads to the transcription of specific genes within relevant target cells. How these signals are transduced to alter gene expression is largely unknown, but this mechanism probably involves a sequence of enzymatic steps that activate factors in the nucleus that modulate transcription. We now demonstrate that two different peptide hormones, or cytokines, stimulate the human immunodeficiency virus enhancer, and this effect is mediated by nuclear factor (NF) kappa B (nuclear factor that binds the kappa immunoglobulin light chain gene enhancer). These cytokines, tumor necrosis factor alpha and interleukin 1, act on multiple cell types and represent the only naturally occurring activators of this transcription factor among eight cytokines examined. Although NF-kappa B binding can be stimulated by phorbol 12-myristate 13-acetate, tumor necrosis factor alpha acts through an independent mechanism, inducing NF-kappa B binding in HT-2 cells, which did not show increased binding in response to phorbol 12-myristate 13-acetate, and causing superinduction in Jurkat T-lymphoma cells. Tumor necrosis factor alpha is also a more selective activator of T cells than phorbol 12-myristate 13-acetate, having no effect on lymphokine production in EL-4 cells at the same time it induces NF-kappa B. These findings suggest that human immunodeficiency virus gene expression can be induced in T cells without activating lymphokine secretion and that the role of these cytokines in the activation of latent human immunodeficiency virus infection deserves further clinical evaluation. Finally, this link between binding at the surface membrane and stimulation of a specific transcription factor should help define intermediates for these cytokine activation pathways.

1,559 citations


Journal ArticleDOI
TL;DR: DNA binding studies reveal two mechanisms for generating further diversity from the ATF proteins, and results help to explain how a single promoter element, an ATF site, can be present in a wide variety of promoters.
Abstract: An activating transcription factor (ATF)-binding site (consensus sequence 5'-GTGACGTACAG-3') is a promoter element present in a wide variety of viral and cellular genes. The two best-characterized classes of genes that contain ATF sites are E1A-inducible adenoviral genes and cAMP-inducible cellular genes. Here, we report the isolation of eight ATF cDNA clones, each of which is derived from a separate gene. All ATF cDNA clones examined contain a leucine zipper motif and are significantly similar to one another only within this region. The leucine zipper region of ATF proteins is also similar to that of the AP-1/c-jun family of transcription factors, whose DNA-binding site differs from the ATF-binding site at a single position. DNA binding studies reveal two mechanisms for generating further diversity from the ATF proteins. First, some, but not all, combinations of ATF proteins form heterodimers that efficiently bind to DNA. Second, although all ATF proteins bind to the ATF site, their precise interactions with DNA differ from one another, as evidenced by methylation interference analysis. Our results help to explain how a single promoter element, an ATF site, can be present in a wide variety of promoters.

951 citations


Journal ArticleDOI
TL;DR: TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.
Abstract: Expression of human immunodeficiency virus type 1 (HIV-1) can be activated in a chronically infected T-cell line (ACH2 cells) by a cytokine, human tumor necrosis factor alpha (TNF-alpha). TNF-alpha treatment of ACH2 cells resulted in an increase in steady-state levels of HIV RNA and HIV transcription. Gel mobility shift assays demonstrated that the transcriptional activation of the HIV long terminal repeat (LTR) by TNF-alpha was associated with the induction of a nuclear factor(s) binding to the NF-kappa B sites in the LTR. Deletion of the NF-kappa B sites from the LTR eliminated activation by TNF-alpha in T cells transfected with plasmids in which the HIV LTR directed the expression of the bacterial chloramphenicol acetyltransferase gene. Thus, TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.

763 citations


Journal ArticleDOI
16 Feb 1989-Nature
TL;DR: It is demonstrated that TNF-α also stimulates collagenase gene transcription; this stimulation is mediated by an element of the gene that is responsive to the transcription factor AP-1, the major component of which (jun/AP-1) is encoded by the jun gene.
Abstract: Tumour necrosis factor-alpha (TNF-alpha) is secreted by macrophages in response to inflammation, infection and cancer. Sublethal doses of recombinant TNF-alpha to rats causes cachexia, anaemia and inflammation. TNF-alpha plays a major part in tissue inflammation and remodelling by stimulating production of collagenase. Cellular responses to TNF-alpha are initiated by binding to high-affinity cell surface receptors. TNF-alpha then profoundly affects gene regulation, stimulating the fos, myc, interleukin-1 and interleukin-6 genes and inhibiting the type I collagen gene. Here we demonstrate that TNF-alpha also stimulates collagenase gene transcription; this stimulation is mediated by an element of the gene that is responsive to the transcription factor AP-1, the major component of which (jun/AP-1) is encoded by the jun gene; and that TNF-alpha stimulates prolonged activation of jun gene expression. This prolonged induction of jun contrasts with its transient activation by the phorbol ester TPA and provides a physiological example of the ability of jun/AP-1 to stimulate its own transcription. This may be a key mechanism for mediating at least some of the biological effects of TNF-alpha.

698 citations


Journal ArticleDOI
04 May 1989-Nature
TL;DR: It is reported that HIV gene expression in the monocyte lineage is regulated byNF-kB, the same transcription factor known to stimulate the HIV enhancer in activated T cells9; however, control of NF-kB and HIV in monocytes differs from that observed in T cells.
Abstract: THE latent period of AIDS is influenced by factors which activate human immunodeficiency virus (HIV) replication in different cell types. Although monocytic cells may provide a reservoir for virus production in vivo1–8, their regulation of HIV transcription has not been defined. We now report that HIV gene expression in the monocyte lineage is regulated by NF-kB, the same transcription factor known to stimulate the HIV enhancer in activated T cells9; however, control of NF-kB and HIV in monocytes differs from that observed in T cells. NF-kB-binding activity appears during the transition from promonocyte to monocyte in U937 cells induced to differentiate in vitro and is present constitutively in mature monocytes and macrophages. In a chronically infected pro-monocytic cell, Ul, differentiation is associated with HIV-1 replication as well as NF-kB binding activity. These findings suggest that NF-kB binding activity is developmentally regulated in the monocyte lineage, and that it provides one signal for HIV activation in these cells.

582 citations


Journal ArticleDOI
TL;DR: CpG methylation of the CRE consensus sequences resulted in loss of specific factor binding, as well as loss of transcriptional activity in vitro and in vivo, in both cell types, suggesting that the inactivity of methylated promoters can, at least in some cases, be explained by their inability to bind specific transcription factors.
Abstract: In mammals and other vertebrates, cytosine methylation in CpG sites is often negatively correlated with gene activity. Because methylation of the promoter region is most crucial for this effect, the simplest hypothesis is that CpG methylation interferes with the binding of specific transcription factors. We have examined this hypothesis with two different transcription factor-binding sites that contain a CpG dinucleotide, namely the cAMP-responsive element (CRE; 5'-TGACGTCA) and the Sp1-binding site (5'-GTGAGGCGGTGAGACT). We have reported previously that CpG methylation of the Sp1-binding site affected neither factor binding nor transcription in HeLa cells, which may be related to the fact that Sp1 is typically associated with promoters of housekeeping genes. In contrast, CREs are often associated with promoters of cell type-specific genes. A synthetic oligonucleotide containing two tandem CREs derived from the gene encoding the human glycoprotein hormone alpha-subunit was cloned upstream of a reporter gene. Transcription of this gene was dependent on the CRE sequences in both PC12 and HeLa cells. Bandshift and methylation interference assays show that similar, if not the same, factor(s) bind to the CRE in both cell lines, even though induction by cAMP was only observed in PC12 cells. CpG methylation of the CRE consensus sequences (TGACGTCA) resulted in loss of specific factor binding, as well as loss of transcriptional activity in vitro and in vivo, in both cell types. This suggests that the inactivity of methylated promoters can, at least in some cases, be explained by their inability to bind specific transcription factors.

579 citations


Journal ArticleDOI
08 Sep 1989-Cell
TL;DR: It is demonstrated that the cDNA encodes the specific Eryf1 binding activity found in erythrocytes, and the evidence suggests, furthermore, that transition metal ions are unusually tightly bound, or may not be necessary for the sequence-specific DNA binding of Ery f1.

576 citations


Journal ArticleDOI
TL;DR: Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver.
Abstract: Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCD1) genes. DNase I footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCD1 promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCD1 gene promoters.

558 citations


Journal ArticleDOI
05 May 1989-Cell
TL;DR: Stimulation of transcription of reporter genes by the progesterone receptor was inhibited in transfected HeLa cells by co-expressing the estrogen receptor (ER) in an ER-dose- and estrogen-dependent manner, and it is proposed that these observations reflect competition for a functionally limiting transcription factor(s).

551 citations


Journal ArticleDOI
TL;DR: Zif268 synthesized in Escherichia coli bound to two sites upstream of the zif268 gene and to sites in the promoter regions of other genes, resulting in the following consensus sequence for a Zif268 high-affinity binding site: GCGTGGGGCG.
Abstract: Zif268, a zinc finger protein whose mRNA is rapidly activated in cells exposed to growth factors or other signaling agents, is thought to play a role in regulating the genetic program induced by extracellular ligands. We report that Zif268 has one of the characteristics of a transcriptional regulator, namely, sequence-specific binding to DNA. Zif268 synthesized in Escherichia coli bound to two sites upstream of the zif268 gene and to sites in the promoter regions of other genes. The nucleotide sequences responsible for binding were defined by DNase I footprinting, by methylation interference experiments, and by use of synthetic oligonucleotides. From these results we derived the following consensus sequence for a Zif268 high-affinity binding site: GCGTGGGGCG.

543 citations


Journal ArticleDOI
22 Dec 1989-Science
TL;DR: The data suggest that the proenkephalin gene may be a physiological target for Fos and Jun in the hippocampus and indicate that these proto-oncogene transcription factors may play a role in neuronal responses to stimulation.
Abstract: Fos and Jun form a heterodimeric complex that associates with the nucleotide sequence motif known as the AP-1 binding site. Although this complex has been proposed to function as a transcriptional regulator in neurons, no specific target gene has yet been identified. Proenkephalin mRNA increased in the hippocampus during seizure just after an increase in c-fos and c-jun expression was detected. Fos-Jun complexes bound specifically to a regulatory sequence in the 5' control region of the proenkephalin gene. Furthermore, c-fos and c-jun stimulated transcription from this control region synergistically in transactivation assays. These data suggest that the proenkephalin gene may be a physiological target for Fos and Jun in the hippocampus and indicate that these proto-oncogene transcription factors may play a role in neuronal responses to stimulation.


Journal ArticleDOI
TL;DR: Immunoprecipitation studies showed that fos B as c‐fos protein, forms a complex in vitro with c‐jun and jun B proteins in the absence of a target binding sequence, suggesting thatfos B protein plays a role in control of gene expression.
Abstract: We have identified a gene, fos B, encoding a nuclear protein of 338 amino acids presenting a 70% homology with c-fos, whose expression is activated during G0/G1 transition. Growth factor stimulation of quiescent cells leads to a rapid and transient accumulation of fos B mRNA, with kinetics similar to those of c-fos. The induction of fos B mRNA levels is in part due to a dramatic increase in the transcription of the gene. The half-life of fos B mRNA is in the order of 10-15 min. Both transcriptional activation and mRNA stability are substantially increased in the presence of protein synthesis inhibitors. Immunoprecipitation studies showed that fos B as c-fos protein, forms a complex in vitro with c-jun and jun B proteins in the absence of a target binding sequence. Gel retardation assays demonstrated that fos B protein positively influences the binding of c-jun and jun B proteins to an AP-1 binding consensus sequence, suggesting that fos B protein plays a role in control of gene expression.

Journal ArticleDOI
01 Dec 1989-Cell
TL;DR: Evidence is presented that distally and proximally bound Sp1 can stimulate transcription synergistically and a DNA binding-deficient mutant of Sp1 that retains glutamine-rich domains can interact with proximatically bound Sp 1 to superactivate transcription.

Journal ArticleDOI
TL;DR: It is concluded that multiple nonconvergent signal transduction pathways control early response gene expression and that the diversity and specificity of cellular response to environmental change can be accounted for by the differential combinatorial induction of a relatively small number of early response genes.
Abstract: A set of early response genes has been identified whose transcription in fibroblasts is rapidly induced in response to growth factors. Prototype members of this group, c-fos and c-jun, encode products that form a heterodimer and have been implicated in the regulation of gene expression and cell growth. It is thought that other early response genes also encode critical mediators of the cell's response to external stimuli. We have used PC12 pheochromocytoma cells as a model system to test the hypothesis that different extracellular signals induce distinct patterns of expression of early response genes. Our results indicate that membrane depolarization, induced either by potassium chloride or by the neurotransmitter analog nicotine, activates a program of gene expression distinct from that activated by nerve growth factor or epidermal growth factor. Notably, c-fos and c-jun activation can be dissociated; whereas c-jun is coinduced with c-fos and jun-B after growth factor stimulation, membrane depolarization activates c-fos and jun-B without stimulating c-jun. Fos may therefore form transcription complexes with alternative cofactors under different stimulation conditions. nur/77 and zif/268, which encode putative transcription factors, also show markedly different responses to growth factors and depolarization. We conclude that multiple nonconvergent signal transduction pathways control early response gene expression. Our findings also indicate that the diversity and specificity of cellular response to environmental change can be accounted for by the differential combinatorial induction of a relatively small number of early response genes.

Journal ArticleDOI
TL;DR: Surprisingly, NF-kappaB could only be inactivated by I kappaB when p65 was bound, and it would appear that one function of p65 is to make NF-cappaB susceptible to inhibition by IKappaB.
Abstract: The NF-kappaB transcription factor was affinity-purified from deoxycholate (DOC)-treated cytosol of HeLa cells and shown to contain both a 50-kappaD polypeptide (p50) with a DNA-binding specificity identical to that of nuclear NF-kappaB and a 65-kappaD protein (p65) lacking DNA binding activity. Electrophoretically purified p50, after renaturation, gave rise to a protein-DNA complex that migrated faster than that made by native NF-kappaB. Reconstitution of p50 and p65 together produced a protein that combined with DNA to form a complex with electrophoretic mobility indistinguishable from that of the complex formed by nuclear extracts and DOC-treated cytosolic fractions. Sedimentation and gel filtration analyses indicate that alone, the p50 protein exists as a dimer; two molecules of p65 bind to it to form a heterotetramer. Unlike I kappaB, the specific inhibitor of NF-kappaB, p65 displayed no inhibitor activity and was not released from NF-kappaB by DOC. p65 did not change the DNA binding specificity or the stimulatory effect of GTP on the p50 homodimer. Surprisingly, NF-kappaB could only be inactivated by I kappaB when p65 was bound. It would appear that one function of p65 is to make NF-kappaB susceptible to inhibition by I kappaB.

Journal ArticleDOI
13 Jul 1989-Nature
TL;DR: It is concluded that this superfamily of gene regulators contains proteins which bind and activate distal promoter elements of eukaryotic genes, and COUP-TF is the first member of this family that has been shown to function in a cell–free transcription system.
Abstract: THE COUP (chicken ovalbumin upstream promoter) transcription factor (COUP-TF) exists in a number of different tissues and is essential for expression of the chicken ovalbumin gene1-3. It binds to the ovalbumin promoter and, in conjunction with a second protein (S300-II), stimulates initiation of transcription in vitro. COUP-TF also binds specifically to the rat insulin promoter element4,5, although the two binding sites share little sequence similarity. Here we report the isolation of a human complementary DNA clone encoding COUP-TF. Comparison of the amino-acid sequence of COUP-TF with known sequences reveals that it is a member of the steriod/thyroid hormone/vitamin receptor super-family6. Consequently, it is the first member of this family that has been shown to function in a cell–free transcription system7,8. We conclude that this superfamily of gene regulators contains proteins which bind and activate distal promoter elements of eukaryotic genes.

Journal ArticleDOI
TL;DR: Tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes and binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays.
Abstract: tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays. tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.

Journal ArticleDOI
TL;DR: Cytoplasmically localized transcription factor precursors serve as second messengers to translate directly an extracellular signal into specific transcriptional activity in the nucleus to stimulate transcription of a defined set of genes.
Abstract: The signal transduction pathway through which interferon-alpha (IFN alpha) stimulates transcription of a defined set of genes involves activation of DNA-binding factors specific for the IFN alpha-stimulated response element (ISRE). IFN-stimulated gene factor-3 (ISGF3), the positive regulator of transcription, was derived in response to IFN alpha treatment from preexisting protein components that were activated first in the cell cytoplasm prior to appearance in the nucleus. Nuclear translocation of ISGF3 required several minutes and could be inhibited by NaF. Formation of active ISGF3 was mimicked in vitro by mixing cytoplasmic extracts from IFN alpha-stimulated cells with extracts of cells treated to contain high amounts of the unactivated factor. Active ISGF3 was found to be formed from association of two latent polypeptide precursors that were distinguished biochemically by differential sensitivity to N-ethyl maleimide. One precursor was modified in response to IFN alpha occupation of its cell-surface receptor, thus enabling association with the second subunit. The resulting complex then was competent for nuclear translocation and binding to ISRE. Cytoplasmically localized transcription factor precursors thus serve as second messengers to translate directly an extracellular signal into specific transcriptional activity in the nucleus.

Journal ArticleDOI
21 Apr 1989-Cell
TL;DR: The wide variety of cell types in which β-interferon can be induced and the divergent set of gene induction processes involving NF-κB suggest that this transcription factor plays a broad role in gene regulation as a mediator of inducible signal transduction.

Journal ArticleDOI
TL;DR: It is speculated that C/EBP may play a general role in establishing and maintaining the differentiated, nonproliferative state.
Abstract: An expression vector capable of encoding full-length CCAAT/enhancer-binding protein (C/EBP) has been constructed and tested in transient transfection assays for its capacity to activate transcription from the promoter of the serum albumin gene When tested in cultured hepatoma cells, the C/EBP expression vector achieved potent trans-activation of the albumin promoter Less substantial activation was observed when the same experiment was conducted using cultured mouse fibroblasts Expression vectors that encoded defective forms of C/EBP failed to activate the albumin promoter Moreover, mutated variants of the albumin promoter that lack the C/EBP-binding site failed to be trans-activated The data are consistent with the interpretation that C/EBP is a bona fide transcription factor During the course of these experiments it was noted also that C/EBP is more than an order of magnitude less concentrated in cultured hepatoma cells than it is in adult liver cells Given these findings, we speculate that C/EBP may play a general role in establishing and maintaining the differentiated, nonproliferative state

Journal ArticleDOI
TL;DR: Cl cloning and analysis of a cDNA encoding a third member of the murine jun family, jun-D, are reported here, which appears to be regulated differently than c-jun and jun-B.
Abstract: The protooncogene c-jun encodes a component of the transcription factor AP-1. Both murine c-jun and a related gene (jun-B) are rapidly activated in BALB/c3T3 cells by serum growth factors. We report here the cloning and analysis of a cDNA encoding a third member of the murine jun family, jun-D. The amino acid sequence encoded by jun-D has two extensive regions of homology with the other Jun proteins. One homology region includes the DNA-binding domain and sequences required for dimer formation and interaction with the Fos oncoprotein; the other includes the acidic sequence thought to be involved in gene activation. All three jun mRNAs are present in a variety of murine tissues and cell lines. In resting 3T3 cells, jun-D is expressed at a higher level compared to c-jun and jun-B, and its transcription is stimulated only slightly by serum growth factors. Thus, jun-D appears to be regulated differently than c-jun and jun-B.

Journal ArticleDOI
08 Sep 1989-Cell
TL;DR: A sensitive polymerase chain reaction assay is developed for measuring the fraction of rearranged immunoglobulin kappa genes in a cell population and is able to detect kappa gene rearrangement in cell lines that do not produce a functional heavy chain gene product (mu protein).

Journal ArticleDOI
TL;DR: The results suggest that the tissue‐specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.
Abstract: A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds at least three different factors, TTF-1, TTF-2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL-5. TTF-1 and TTF-2 are FRTL-5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid-differentiated function, while UFA is present in all cell lines tested. TTF-1 has been extensively purified. It binds to the rat thyroglobulin promoter at three different sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF-1 in vitro and promoter function in vivo. This suggests that the tissue-specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.

Journal ArticleDOI
01 Sep 1989-Neuron
TL;DR: Alterations in the levels and composition of transcription factors may represent one of the molecular mechanisms underlying neuronal adaptation in the brain.

Journal ArticleDOI
TL;DR: It is shown that the transcription factor NF‐kappa B, which binds to the 18 bp repeat, plays a central role in enhancer activation in infected human fibroblasts and that activation is mediated by the product of the viral gene ie1.
Abstract: The expression of cytomegalovirus alpha (immediate early) genes is under control of an enhancer that carries signals for strong constitutive expression as well as response elements for transactivation by viral proteins. We have used synthetic oligonucleotides representing the 16, 18 and 19 bp repeat elements within the enhancer to investigate the role of virus-induced cellular transcription factors in enhancer activation. We show that the transcription factor NF-kappa B, which binds to the 18 bp repeat, plays a central role in enhancer activation in infected human fibroblasts and that activation is mediated by the product of the viral gene ie1. The simian immunodeficiency virus kappa B site can functionally substitute for the 18 bp element in transient transactivation assays and can also compete efficiently for specific binding to the 18 bp repeat element in vitro. Point mutations in the NF-kappa B site within the 18 bp element disrupt ie1-mediated transactivation and binding. We have found that the characteristics of the 18 bp binding factor from human fibroblasts are indistinguishable from NF-kappa B induced by phorbol ester plus mitogen treatment of T lymphocytes, as determined by gel mobility shift assay as well as protection of the binding site from chemical cleavage. Furthermore, T cell stimulation mediates activation of the viral enhancer via kappa B sites, an observation that may be important in the interaction of cytomegalovirus with the naturally infected human host. Thus, NF-kappa B plays a central role as a target for enhancer activation via viral and cellular factors.

Journal ArticleDOI
TL;DR: The muscle creatine kinase gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes and the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1) is designated.
Abstract: The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

Journal ArticleDOI
TL;DR: The possible role of precise chromatin organization in glucocorticoid induction is discussed on the basis of the nucleosome phasing found in the LTR region of mouse mammary tumour virus.

Journal ArticleDOI
TL;DR: The autoinduction of expression from the alpha promoter-enhancer by the most abundant alpha gene product, a 491-amino-acid nuclear phosphoprotein referred to as ie1 is described, which strongly implicated ie1 in the transcriptional transactivation of thealpha promoter through its enhancer.
Abstract: The expression of alpha (immediate-early) genes of cytomegalovirus is regulated via a complex enhancer that consists of several different repeat elements. We describe here the autoinduction of expression from the alpha promoter-enhancer by the most abundant alpha gene product, a 491-amino-acid nuclear phosphoprotein referred to as ie1. We defined the 18-base-pair repeat element within the alpha enhancer as the signal through which ie1 acts to regulate gene expression. This element contains an NF kappa B site that may play an important role in ie1 autoregulation. Our analysis, which relied on deletions through the enhancer as well as reconstitution of responsiveness to a promoter with synthetic 18-base-pair repeats, strongly implicated ie1 in the transcriptional transactivation of the alpha promoter through its enhancer.

Journal ArticleDOI
TL;DR: It is demonstrated that Fra-1 contributes to the DNA-binding activity ascribed to transcription factor AP-1, using proteins synthesized in reticulocyte lysates.
Abstract: fra-1 encodes a serum-inducible protein (Fra-1) that is antigenically related to Fos. We have characterized Fra-1 expression in serum-stimulated cells using antibodies raised against several regions of this protein. Fra-1, expressed transiently in COS cells or in serum-stimulated rat fibroblasts, undergoes extensive post-translational modification, primarily by phosphorylation of serine residues. It is present in both the nucleus and the cytoplasm and participates in a protein complex with Jun. Using proteins synthesized in reticulocyte lysates, we have shown that Fra-1, like Fos, binds to the AP-1 recognition element cooperatively with Jun. A truncated Fra-1 protein that contains the leucine zipper region but not an adjacent basic amino acid domain, complexes with Jun in vitro but fails to bind AP-1 oligonucleotides. These results demonstrate that Fra-1 contributes to the DNA-binding activity ascribed to transcription factor AP-1.