Showing papers on "Transcription factor published in 1993"
TL;DR: It is demonstrated that Hif-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed, providing evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to Hypoxia.
Abstract: Transcription of the human erythropoietin (EPO) gene is activated in Hep3B cells exposed to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whose DNA binding activity is induced by hypoxia in Hep3B cells, and HIF-1 binds at a site in the EPO gene enhancer that is required for hypoxic activation of transcription. In this paper, we demonstrate that HIF-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed. The composition of the HIF-1 DNA binding complex and its isolated DNA binding subunit and the mechanism of HIF-1 activation appear to be similar or identical in EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells and mutations that eliminate HIF-1 binding eliminate inducibility. These results provide evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia.
1,405 citations
TL;DR: It is suggested that the mdm2 gene is a target for activation by wt p53, and the induction of mDM2 expression by t p53 activity is at the mRNA level, suggesting a direct involvement of p53 in the process.
Abstract: We have recently characterized a 95 kDa protein, p95, which exhibits enhanced binding to temperature-sensitive p53 (ts-p53) when cells are shifted down to 325 degrees C, a temperature at which ts-p53 possesses wild-type (wt)-like activities In the present study we show that p95 is a product of the mdm2 putative proto-oncogene The enhanced complex formation of mdm2 with ts-p53 in cells maintained at 325 degrees C is due to an elevation in total mdm2 protein levels following the temperature shift We further demonstrate that the induction of mdm2 expression by t p53 activity is at the mRNA level The induction occurs with very rapid kinetics and does not require de novo protein synthesis, suggesting a direct involvement of p53 in the process Based on these data and on recent findings implicating p53 as a transcription factor, we suggest that the mdm2 gene is a target for activation by wt p53 In view of the ability of mdm2 to act as a specific antagonist of p53 activity, this induction process may serve to tightly autoregulate p53 activity in living cells
1,331 citations
TL;DR: It is shown that AP‐1 is an antioxidant‐responsive transcription factor that is induced in HeLa cells upon treatment with the antioxidants pyrrolidine dithiocarbamate and N‐acetyl‐L‐cysteine, and upon transient expression of the antioxidative enzyme thioredoxin.
Abstract: We show that AP-1 is an antioxidant-responsive transcription factor. DNA binding and transactivation by AP-1 were induced in HeLa cells upon treatment with the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC), and upon transient expression of the antioxidative enzyme thioredoxin. While PDTC and NAC enhanced DNA binding and transactivation of AP-1 in response to phorbol ester, the oxidant H2O2 suppressed phorbol ester activation of the factor. H2O2 on its own was only a weak inducer of AP-1. Activation of AP-1 by PDTC was dependent on protein synthesis and involved transcriptional induction of c-jun and c-fos genes. Transcriptional activation of c-fos by PDTC was conferred by the serum response element, suggesting that serum response factor and associated proteins function as primary antioxidant-responsive transcription factors. In the same cell line, the oxidative stress-responsive transcription factor NF-kappa B behaved in a manner strikingly opposite to AP-1. DNA binding and transactivation by NF-kappa B were strongly activated by H2O2, while the antioxidants alone were ineffective. H2O2 potentiated the activation of NF-kappa B by phorbol ester, while PDTC and NAC suppressed PMA activation of the factor. PDTC did not influence protein kinase C (PKC) activity and PKC activation by PMA, indicating that the antioxidant acted downstream of and independently from PKC.
1,308 citations
TL;DR: Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B, and association of the newly synthesized I k Kappa B alpha with p65 restores intracellular inhibition of NF -kappaB DNA binding activity and prolongs the survival of this labile inhibitor.
Abstract: The eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression. Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B. Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway.
1,137 citations
TL;DR: In this article, a 1,749-bp fragment from the 5'-flanking region of the iNOS gene was cloned from a mouse genomic library, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it.
Abstract: Inducible nitric oxide synthase (iNOS) can be expressed by many types of mammalian cells in response to diverse signals acting synergistically, including cytokines and microbial products. We previously showed that induction of iNOS in mouse macrophages by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) was at the transcriptional level. From a mouse genomic library, we now cloned a 1,749-bp fragment from the 5'-flanking region of the iNOS gene, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it. The mRNA initiation site is preceded by a TATA box and at least 22 oligonucleotide elements homologous to consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines or bacterial products. These include 10 copies of IFN-gamma response element; 3 copies of gamma-activated site; 2 copies each of nuclear factor-kappa B, IFN-alpha-stimulated response element, activating protein 1, and tumor necrosis factor response element; and one X box. Plasmids in which all or the downstream one half or one third of this region of iNOS were linked to a reporter gene encoding chloramphenicol acetyltransferase were transfected into cells of the RAW264.7 macrophage-like line. All these constructs conferred inducibility of the iNOS promoter by LPS, but only the construct containing all 1,749 bp conferred synergistic inducibility by IFN-gamma plus LPS.
1,134 citations
TL;DR: Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, howIFN-Gamma augments the inflammatory response to LPS.
Abstract: The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.
1,083 citations
TL;DR: It is demonstrated that overexpression of the E2F1 complementary DNA can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus El A gene.
Abstract: SEVERAL lines of evidence implicate the E2F transcription factor as an important component of cell proliferation control. First, E2F binding sites are found in the promoters of genes responsive to proliferation signals and the level of E2F binding activity increases at a time when many of these genes are activated1–3. Second, the tumour suppressor protein Rb, as well as the related p107 protein, complexes with E2F2–7, resulting in an inhibition of E2F transcriptional activity3,8–12. Third, oncogenic products of the DNA tumour viruses can dissociate these E2F complexes4,13. We provide here direct evidence that E2F is involved in cellular proliferation control. Specifically, we demonstrate that overexpression of the E2F1 complementary DNA14,15 can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus El A gene. Moreover, microinjection of the E2F1 cDNA into quiescent cells can induce S-phase entry, whereas two E2F1 mutants, which are unable to transactivate the DHFR and TK promoters, are unable to induce S phase. We conclude that the E2F transcription factor plays an important role in progression into S phase and that this probably coincides with its capacity to stimulate transcription.
978 citations
TL;DR: The synergistic activation of the IL-6 promoter by transcription factors NF-IL6 and NF-kappa B and the possible interplay between various viral gene products and these two factors in the process of viral infection and constitutive cytokine production is discussed.
Abstract: Single binding sites for transcription factors NF-IL6 and NF-kappa B are present in the promoter of the interleukin (IL) 6 gene. Previous studies of internally deleted promoter mutants demonstrated that these two sites are important for the transcriptional regulation of this gene. In this report, we describe the synergistic activation of the IL-6 promoter by transcription factors NF-IL6 and NF-kappa B. Cotransfection of NF-IL6 with the NF-kappa B p65 subunit resulted in strong synergistic activation of an IL-6 promoter-reporter construct. Both the NF-IL6 and NF-kappa B binding sites in the IL-6 promoter were required for synergistic activation. Similar synergistic activation was observed in the IL-8 promoter, which also contains both NF-IL6 and NF-kappa B binding sites. Furthermore, we demonstrated that NF-IL6 and the NF-kappa B p65 subunit directly associated via the basic leucine-zipper domain of NF-IL6 and the Rel homology domain of p65. Since the promoters of many other genes involved in the inflammatory and acute-phase responses also contain binding sites for NF-IL6 and NF-kappa B, the cooperation between these two factors may have an important role in these responses. We also discuss the possible interplay between various viral gene products and these two factors in the process of viral infection and constitutive cytokine production.
943 citations
TL;DR: It is demonstrated that HSF1 can be activated in the absence of physiological stress and support is provided for a model of regulation ofHSF1 and HSF2 activity by a titratable negative regulatory factor.
Abstract: The existence of multiple heat shock factor (HSF) genes in higher eukaryotes has promoted questions regarding the functions of these HSF family members, especially with respect to the stress response. To address these questions, we have used polyclonal antisera raised against mouse HSF1 and HSF2 to examine the biochemical, physical, and functional properties of these two factors in unstressed and heat-shocked mouse and human cells. We have identified HSF1 as the mediator of stress-induced heat shock gene transcription. HSF1 displays stress-induced DNA-binding activity, oligomerization, and nuclear localization, while HSF2 does not. Also, HSF1 undergoes phosphorylation in cells exposed to heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity, oligomerization, and transcriptional activity. These results demonstrate that HSF1 can be activated in the absence of physiological stress and also provide support for a model of regulation of HSF1 and HSF2 activity by a titratable negative regulatory factor.
885 citations
TL;DR: It is demonstrated that induction of both Hif-1 and erythropoietin RNA is inhibited by the protein kinase inhibitor 2-aminopurine, consistent with the proposed function of HIF-1 as a physiologic regulator of gene expression that responds to changes in cellular oxygen tension.
875 citations
TL;DR: How several Ets-related proteins fit into this scheme, and how their activity is regulated both post- and pre-translationally are discussed are discussed.
Abstract: Interest in the Ets proteins has grown enormously over the last decade. The v-ets oncogene was originally discovered as part of a fusion protein expressed by a transforming retrovirus (avian E26), and later shown to be transduced from a cellular gene. About 30 related proteins have now been found in species ranging from flies to humans, that resemble the vEts protein in the so-called 'ets domain'. The ets domain has been shown to be a DNA-binding domain, that specifically interacts with sequences containing the common core trinucleotide GGA. Furthermore, it is involved in protein-protein interactions with co-factors that help determine its biological activity. Many of the Ets-related proteins have been shown to be transcription activators, like other nuclear oncoproteins and anti-oncoproteins (Jun, Fos, Myb, Myc, Rel, p53, etc.). However, Ets-like proteins may have other functions, such as in DNA replication and a general role in transcription activation. Ets proteins have been implicated in regulation of gene expression during a variety of biological processes, including growth control, transformation, T-cell activation, and developmental programs in many organisms. Signals regulating cell growth are transmitted from outside the cell to the nucleus by growth factors and their receptors. G-proteins, kinases and transcription factors. We will discuss how several Ets-related proteins fit into this scheme, and how their activity is regulated both post- and pre-translationally. Loss of normal control is often associated with conversion to an oncoprotein. vEts has been shown to have different properties from its progenitor, which might explain how it has become oncogenic. Oncogene-related products have been implicated in the control of various developmental processes. Evidence is accumulating for a role for Ets family members in Drosophila development, Xenopus oocyte maturation, lymphocyte differentiation, and viral infectious cycles. An ultimate hope in studying transformation by oncoproteins is to understand how cells become cancerous in humans, which would lead to more effective treatments. vEts induces erythroblastosis in chicken. Cellular Ets-family proteins can be activated by proviral insertion in mice and, most interestingly, by chromosome translocation in humans. We are at the beginning of understanding the multiple facets of regulation of Ets activity. Future work on the Ets family promises to provide important insights into both normal control of growth and differentiation, and deregulation in illness.
TL;DR: Findings indicate that the t(2;13) generates a potentially tumorigenic fusion transcription factor consisting of intact PAX3 DNA binding domains, a truncated fork head DNA binding domain and C–terminal FKHR regions.
Abstract: We have examined the structure and expression of the products associated with the t(2;13)(q35;q14) translocation associated with alveolar rhabdomyosarcoma. The chromosome 13 gene (FKHR) is identified as a member of the fork head domain family of transcription factors characterized by a conserved DNA binding motif. Polymerase chain reaction analysis demonstrates that a 5'PAX3-3' FKHR chimaeric transcript is expressed in all eight alveolar rhabdomyosarcomas investigated. Immunoprecipitation experiments detect the predicted fusion protein. These findings indicate that the t(2;13) generates a potentially tumorigenic fusion transcription factor consisting of intact PAX3 DNA binding domains, a truncated fork head DNA binding domain and C-terminal FKHR regions.
TL;DR: Interferon-gamma stimulates transcription of specific genes by inducing tyrosine phosphorylation of a 91-kilodalton cytoplasmic protein ( STAT for signal transducer and activator of transcription).
Abstract: Interferon-gamma (IFN-gamma) stimulates transcription of specific genes by inducing tyrosine phosphorylation of a 91-kilodalton cytoplasmic protein (termed STAT for signal transducer and activator of transcription). Stat91 was phosphorylated on a single site (Tyr701), and phosphorylation of this site was required for nuclear translocation, DNA binding, and gene activation. Stat84, a differentially spliced product of the same gene that lacks the 38 carboxyl-terminal amino acids of Stat91, did not activate transcription, although it was phosphorylated and translocated to the nucleus and bound DNA. Thus, Stat91 mediates activation of transcription in response to IFN-gamma.
TL;DR: The interaction between the lymphoid-specific factor NFATp and the ubiquitous transcription factors Fos and Jun provides a novel mechanism for combinatorial regulation of interleukin-2 gene transcription, which integrates the calcium-dependent and the protein-kinase C-dependent pathways of T-cell activation.
Abstract: TRANSCRIPTION of lymphokine genes in activated T cells is inhibited by the immunosuppressive agents cyclosporin A and FK506, which act by blocking the phosphatase activity of calcineurin1–3. NFAT, a DNA-binding protein required for interleukin-2 gene transcription, is a potential target for calcineurin, cyclosporin A and FK5064–11. NFAT contains a subunit (NFATp) which is present in unstimulated T cells and which forms a complex with Fos and Jun proteins in the nucleus of activated T cells9,11. Here we report that NFATp is a DNA-binding phosphoprotein of relative molecular mass ∼ 120,000 and is a substrate for calcineurin in vitro. Purified NFATp forms DNA–protein complexes with recombinant Jun homodimers or Jun–Fos heterodimers; the DNA-binding domains of Fos and Jun are essential for the formation of the NFATp–Fos–Jun–DNA complex. The interaction between the lymphoid-specific factor NFATp and the ubiquitous transcription factors Fos and Jun provides a novel mechanism for combinatorial regulation of interleukin-2 gene transcription, which integrates the calcium-dependent and the protein-kinase C-dependent pathways of T-cell activation.
TL;DR: The tentative conclusion is drawn that the specificity of the hormonal response in different cells results from a combination of developmental restrictions both in the accessibility of genomic sequence and in the repertoire of regulatory proteins present in each particular cell.
Abstract: GENE regulation by steroid hormones is mediated by binding of the hormone ligand to the corresponding receptor that triggers a complex set of interactions of the hormone receptors with each other, with DNA in chromatin, and with a variety of other proteins. In this review we attempt to summarize what is known about these interactions using as the main example the regulation of mouse mammary tumor virus transcription by glucocorticoids and progestins. We describe in some detail the interaction of monomers and homodimers of the steroid receptors with their recognition sequences, and the molecular mechanism used to discriminate between the responsive elements for glucocorticoids/progestins and estrogens. We then review the interactions between homologous and heterologous hormone receptors on complex hormone regulatory regions, before devoting some attention to the synergistic and inhibitory interactions of hormone receptors with other transcription factors. Finally we briefly summarize some of the possible m...
TL;DR: Treatment of Hep3B cells with desferrioxamine (DFX) induced HIF-1 activity and EPO RNA expression with kinetics similar to the induction of Hif-1 by hypoxia or cobalt chloride, suggesting the existence of a common Hypoxia signal-transduction pathway leading to HIF -1 induction in different cell types.
TL;DR: The tissue expression pattern of EKLF, in conjunction with its function as a transcriptional activator, strongly suggests that the EKlF protein may be intimately involved in establishment and/or maintenance of the erythroid cell phenotype.
Abstract: We describe a novel erythroid cell-specific cDNA (EKLF [erythroid Kruppel-like factor]) isolated by enriching for genes expressed in a mouse erythroleukemia cell line but not expressed in a mouse monocyte-macrophage cell line. The complete cDNA sequence is predicted to encode a protein of approximately 38,000 Da that contains a proline-rich amino domain and three TFIIIA-like zinc fingers within the carboxy domain. Additional sequence analyses reveal that the EKLF zinc fingers are most homologous to the Kruppel family of transcription factors and also allow us to predict potential DNA-binding target sites for the EKLF protein. On the basis of this prediction, we show that EKLF is able to bind the sequence CCA CAC CCT, an essential element of the beta-globin promoter. Its tissue distribution establishes that the EKLF transcript is expressed only in bone marrow and spleen, the two hematopoietic organs of the mouse, and analysis of murine cell lines indicates that EKLF expression is limited to erythroid and mast cell lines. Cotransfection assays establish that EKLF transcriptionally activates a target promoter that contains its DNA-binding site. The tissue expression pattern of EKLF, in conjunction with its function as a transcriptional activator, strongly suggests that the EKLF protein may be intimately involved in establishment and/or maintenance of the erythroid cell phenotype.
TL;DR: The crystal structure of the fully specific complex between the DNA-binding domain from the estrogen receptor and DNA reveals how the protein recognizes its own half site sequence rather than that of the related glucocorticoid receptor, which differs by only two base pairs.
TL;DR: It is concluded that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor that plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.
Abstract: We report the cDNA cloning and characterization of mouse GATA-4, a new member of the family of zinc finger transcription factors that bind a core GATA motif. GATA-4 cDNA was identified by screening a 6.5-day mouse embryo library with oligonucleotide probes corresponding to a highly conserved region of the finger domains. Like other proteins of the family, GATA-4 is approximately 50 kDa in size and contains two zinc finger domains of the form C-X-N-C-(X17)-C-N-X-C. Cotransfection assays in heterologous cells demonstrate that GATA-4 trans activates reporter constructs containing GATA promoter elements. Northern (RNA) analysis and in situ hybridization show that GATA-4 mRNA is expressed in the heart, intestinal epithelium, primitive endoderm, and gonads. Retinoic acid-induced differentiation of mouse F9 cells into visceral or parietal endoderm is accompanied by increased expression of GATA-4 mRNA and protein. In vitro differentiation of embryonic stem cells into embryoid bodies is also associated with increased GATA-4 expression. We conclude that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor. On the basis of its tissue distribution, we speculate that GATA-4 plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.
TL;DR: It is demonstrated that c-Myc is a potent transactivator of ODC promoter-reporter gene constructs in fibroblasts that requires the CACGTG repeat, suggesting that ODC is regulated by Myc at the level of transcription initiation.
Abstract: Constitutive c-myc expression suppresses cell cycle arrest, promotes entry into S phase, and results in the growth factor-independent expression of ornithine decarboxylase (ODC; EC 4.1.1.17). The ODC gene contains a conserved repeat of the Myc binding site, CACGTG, in intron 1. In this report, we demonstrate that c-Myc is a potent transactivator of ODC promoter-reporter gene constructs in fibroblasts that requires the CACGTG repeat. These sites conferred Myc responsiveness on heterologous promoter constructs, suggesting that ODC is regulated by Myc at the level of transcription initiation. Analysis of deletion and point mutants of c-myc revealed that domains required for transactivation of the ODC promoter did not include the leucine zipper of the Myc protein. This suggests that Myc may interact with transcription factors other than Max to transactivate the ODC gene.
TL;DR: In this review, Peter Barnes and Ian Adcock discuss recent studies that have increased the understanding of glucocorticoid mechanisms and that may lead to improved anti-inflammatory therapies in the future.
TL;DR: In vivo transactivation assays suggest that Myc-Max and Mad-Max complexes have opposing functions in transcription and that Max plays a central role in this network of transcription factors.
TL;DR: Two unrelated receptors may activate a common nuclear signal transduction pathway that, through differential use of latent cytoplasmic proteins, permits these receptors to regulate both common and unique sets of genes.
Abstract: Growth factors and cytokines act through cell surface receptors with different biochemical properties Yet each type of receptor can elicit similar as well as distinct biological responses in target cells, suggesting that distinct classes of receptors activate common gene sets Epidermal growth factor, interferon-gamma, and interleukin-6 all activated, through direct tyrosine phosphorylation, latent cytoplasmic transcription factors that recognized similar DNA elements However, different ligands activated different patterns of factors with distinct DNA-binding specificities in the same and different cells Thus, unrelated receptors may activate a common nuclear signal transduction pathway that, through differential use of latent cytoplasmic proteins, permits these receptors to regulate both common and unique sets of genes
TL;DR: It is demonstrated that the bZIP regions of c‐Fos and c‐Jun are capable of physically interacting with NF‐kappa B p65 through the Rel homology domain, which supports a combinatorial mechanism of gene regulation involving the unexpected cross‐coupling of two different classes of transcription factors to form novel protein complexes exhibiting potentiated biological activity.
Abstract: NF-kappa B and AP-1 represent distinct mammalian transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is typically composed of two DNA binding subunits, NF-kappa B p50 and NF-kappa B p65, which share structural homology with the c-rel proto-oncogene product. Similarly, the AP-1 transcription factor complex is comprised of dimers of the c-fos and c-jun proto-oncogene products or of closely related proteins. We now demonstrate that the bZIP regions of c-Fos and c-Jun are capable of physically interacting with NF-kappa B p65 through the Rel homology domain. This complex of NF-kappa B p65 and Jun or Fos exhibits enhanced DNA binding and biological function via both the kappa B and AP-1 response elements including synergistic activation of the 5' long terminal repeat of the human immunodeficiency virus type 1. These findings support a combinatorial mechanism of gene regulation involving the unexpected cross-coupling of two different classes of transcription factors to form novel protein complexes exhibiting potentiated biological activity.
TL;DR: Activity of these regions in vivo requires an erythroid-specific nuclear factor that binds AP-1-like recognition sites and its tissue-specific component has been characterized by complementary DNA cloning as a new basic region–leucine zipper protein which dimerizes with a ubiquitous partner to form native NF-E2.
Abstract: Expression of globin genes in developing erythroid cells is controlled by upstream locus control regions. Activity of these regions in vivo requires an erythroid-specific nuclear factor (NF-E2) that binds AP-1-like recognition sites. Its tissue-specific component (p45 NF-E2) has been characterized by complementary DNA cloning as a new basic region-leucine zipper protein which dimerizes with a ubiquitous partner to form native NF-E2.
TL;DR: The expression of PDGF-B and other pathophysiologically relevant genes in vascular endothelium appears to be regulated, in part, by shear-stress-induced transcription factors interacting with a common promoter element.
Abstract: The endothelial lining of blood vessels is constantly exposed to fluid mechanical forces generated by flowing blood. In vitro application of fluid shear stresses to cultured endothelial cells influences the expression of multiple genes, as reflected by changes in their steady-state mRNA levels. We have utilized the B chain of platelet-derived growth factor (PDGF-B) as a model to investigate the mechanisms of shear-stress-induced gene regulation in cultured bovine aortic endothelial cells (BAECs). Northern blot analysis revealed elevated endogenous PDGF-B transcript levels in BAECs, after exposure to a physiological level of laminar shear stress (10 dynes/cm2; 1 dyne = 100 mN) for 4 h. A transfected reporter gene, consisting of a 1.3-kb fragment of the human PDGF-B promoter coupled to chloramphenicol acetyltransferase (CAT), indicated a direct effect on transcriptional activity. Transfection of a series of PDGF-B-CAT deletion mutants led to the characterization of a cis-acting component within the PDGF-B promoter that was necessary for shear-stress responsiveness. In gel-shift assays, overlapping oligonucleotide probes of this region formed several protein-DNA complexes with nuclear extracts prepared from both static and shear-stressed BAECs. A 12-bp component (CTCTCAGAGACC) was identified that formed a distinct pattern of complexes with nuclear proteins extracted from shear-stressed BAECs. This shear-stress-responsive element does not encode binding sites for any known transcription factor but does contain a core binding sequence (GAGACC), as defined by deletion mutation in gel-shift assays. Interestingly, this putative transcription factor binding site is also present in the promoters of certain other endothelial genes, including tissue plasminogen activator, intercellular adhesion molecule 1, and transforming growth factor beta 1, that also are induced by shear stress. Thus, the expression of PDGF-B and other pathophysiologically relevant genes in vascular endothelium appears to be regulated, in part, by shear-stress-induced transcription factors interacting with a common promoter element.
TL;DR: It is demonstrated here with human T lymphocytes and monocytes that different stimuli, including tumor necrosis factor alpha and phorbol 12-myristate 13-acetate, cause rapid degradation of I kappa B-alpha, with concomitant activation of NF-kappa B, followed by a dramatic increase in I k Kappa B- alpha mRNA and protein synthesis.
Abstract: The NK-kappa B transcription factor complex is sequestered in the cytoplasm by the inhibitory protein I kappa B-alpha (MAD-3). Various cellular stimuli relieve this inhibition by mechanisms largely unknown, leading to NF-kappa B nuclear localization and transactivation of its target genes. It is demonstrated here with human T lymphocytes and monocytes that different stimuli, including tumor necrosis factor alpha and phorbol 12-myristate 13-acetate, cause rapid degradation of I kappa B-alpha, with concomitant activation of NF-kappa B, followed by a dramatic increase in I kappa B-alpha mRNA and protein synthesis. Transfection studies reveal that the I kappa B-alpha mRNA and the encoded protein are potently induced by NF-kappa B and by homodimers of p65 and of c-Rel. We propose a model in which NF-kappa B and I kappa B-alpha mutually regulate each other in a cycle: saturating amounts of the inhibitory I kappa B-alpha protein are destroyed upon stimulation, allowing rapid activation of NF-kappa B. Subsequently, I kappa B-alpha mRNA and protein levels are quickly induced by the activated NF-kappa B. This resurgence of I kappa B-alpha protein acts to restore an equilibrium in which NF-kappa B is again inhibited.
TL;DR: It is shown that a counterpart of SNF2/SWI2 also exists in mice and humans, and that the human protein, designated hbrm, is a 180 kDa nuclear factor that can function as a transcriptional activator when fused to a heterologous DNA binding domain.
Abstract: Several of the SNF and SWI genes of Saccharomyces cerevisiae code for proteins believed to assist transcriptional activators by relieving nucleosome repression. One of these proteins, SNF2/SWI2, has a homologue in Drosophila, a regulator of homeotic genes known as brahma or brm. In this report, we show that a counterpart of SNF2/SWI2 also exists in mice and humans. The human protein, designated hbrm, is a 180 kDa nuclear factor that can function as a transcriptional activator when fused to a heterologous DNA binding domain. The mouse homologue of hbrm is expressed in all mouse organs tested while hbrm was detected in some but not all investigated human cell lines. In cells failing to express the endogenous gene, transfected hbrm cooperates with the glucocorticoid receptor (GR) in transcriptional activation. However, hbrm had no effect on the activity of several other transcription factors, including the homeoprotein HNF-1. The co-operation between hbrm and GR required the DNA binding domain of GR and two separated regions of the hbrm protein, including a domain with homology to known helicases.
TL;DR: The IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.
Abstract: The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.