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GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements
Abstract
Granulocyte-macrophage colony stimulating factor (GM-CSF) plays a key role in myeloid cell function and is rapidly and transiently
expressed in T cells in response to immune or inflammatory stimuli. Induction of GM-CSF gene expression is accompanied by
changes in chromatin structure across the proximal promoter region of the gene. We show that the promoter remodelling and
subsequent gene transcription occurs with distinct signal and transcription factor requirements. Activation of the protein
kinase C (PKC) signalling pathway is sufficient to induce changes in chromatin structure across the promoter, but both the
PKC and calcium signalling pathways are required for efficient gene transcription. Although NFAT transcription factors contribute
to GM-CSF gene transcription, they are not required for promoter remodelling. However, the presence of the nuclear factor-κB
transcription factor, c-Rel, in the nucleus is strongly correlated with and required for the events of chromatin remodelling.
... The phenotype and antigen specificity of 5C.C7 TCR-transgenic Rag -/mice has been described previously (288,328). In brief, the 5C.C7 TCR is specific for moth cytochrome C (MCC [88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] ) oligopeptide presented in the context of the class II MHC molecule I-E k . ...
... 5x10 4 5C.C7 TCR-transgenic CD4+ T cells were co-cultured in 96-well round-bottom plates with 5x10 4 mitomycin C-treated Chinese hamster ovary (CHO) antigen presenting cells (APCs) engineered to express I-E k and a CD28 ligand. APCs were prepulsed for five hours with 5x10 -4 µM moth cytochrome C oligonucleotide (MCC[88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] prior to start of coculture. T cells were removed from initial co-culture conditions at indicated times (X-axis) by gentle pipetting and co-cultured with CHO APCs prepulsed with 5x10 -4 µM MCC under varying conditions of TCR monostimulation, CD28 costimulation, and CD28+Ctla-4 costimulation (blue, green, and red curves respectively). ...
... Sort protocols for in vitro-stimulated T cells at varying timesSorting protocols used to isolate CD4+ populations homogenous for activation status. 5x10 4 5C.C7 TCR-transgenic CD4+ T cells were co-cultured in96- well round-bottom plates with 5x10 4 mitomycin C-treated Chinese hamster ovary (CHO) antigen presenting cells (APCs) engineered to express surface ligands for TCR, CD28and Ctla-4 in varying combination that were prepulsed for five hours with 5x10 -4 µM moth cytochrome C oligonucleotide (MCC[88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] ). 5C.C7 lymphocyte activation, asmeasured by responder frequencey (CD69 upregulation) correlates with antigen prepulse concentration and is enhanced or inhibited by CD28 and Ctla-4 ligands respectively. ...
Immunologic self-tolerance is maintained by both central and peripheral mechanisms. Furthermore, regulation of mature lymphocyte responses is governed by inhibitory as well as stimulatory signals. TCR recognition of cognate peptide bound to MHC molecules provides the initial stimulus leading to T lymphocyte activation and determines the antigen specificity of any subsequent response. However, lymphocytes must discriminate between foreign and self antigens presented by self-MHC molecules to maintain self tolerance and avoid pathological autoimmunity. Consequently, TCR ligation alone is reported to result in abortive activation, T cell anergy, apoptosis, and tolerance. Under normal physiological conditions, costimulatory signals modify lymphocyte responsiveness to TCR ligation to prevent autoimmunity while enabling robust responses to foreign antigen. Members of the CD28/B7 superfamily provide the critical secondary signals essential for normal immune cell function.
CD28 is an essential positive costimulatory molecule with critical functions in thymic development, lineage commitment, and regulation of peripheral lymphocyte responses to antigenic stimuli. CD28 ligation by APC-expressed B7 molecules alters proximal signaling events subsequent to MHC/TCR interactions, and initiates unique signaling pathways that alter mRNA stability and gene transcription. Furthermore, CD28 signaling is required for regulatory T cell development and function. Thus, CD28 has a central role in both potentiating lymphocyte activation mediated by TCR engagement and regulating peripheral tolerance. In contrast, Ctla-4 mediates an inhibitory signal upon binding B7 molecules on an antigen-presenting cell. Its importance in governing lymphocyte responses is manifested in the fatal lymphoproliferative disorder seen in Ctla-4-/- mice. The lymphocyte proliferation is polyclonal, antigen and CD28 dependent, and arises from defects in peripheral CD4+ T cell regulation. The high percentage of peripheral T lymphocytes expressing activation markers is accompanied by lymphocyte infiltration into numerous non-lymphoid tissues and results in death by 3-4 weeks. While still controversial, Ctla-4 signaling has been reported to be essential for induction of peripheral T lymphocyte tolerance in vivo and in some model systems is proposed to regulate both T lymphocyte anergy induction and the immune suppressive effects of some regulatory T cells in the prevention of autoimmunity.
Signaling pathways activated by TCR ligation and CD28 costimulation have been extensively characterized. In contrast, the mechanisms mediating Ctla-4 maintenance of tolerance remain largely unknown. Ctla-4 gene expression is tightly controlled during T cell development and activation, and its intracellular localization and expression on the cell surface is regulated by numerous pathways and intermediates. While a tailless Ctla-4 mutant is capable of inhibiting T cell activation, recent studies have shown that a ligand independent form of Ctla-4 is also capable of providing an inhibitory signal to T lymphocytes. In conjunction with the strictly controlled expression kinetics and the perfect amino acid homology between the intracellular domains of mouse and human Ctla-4, this data suggests that Ctla-4 may participate in the modulation or initiation of intracellular signaling pathways.
Positive and negative costimulatory receptors on the T cell modify lymphocyte responses by altering both quantitative and qualitative aspects of the lymphocyte response including threshold of activation, cytokine secretion, and memory responses. Positive costimulation augments T cell responses, in part, by downregulating the expression of genes that actively maintain the quiescent phenotype. This study was initiated to determine the role of Ctla-4 ligation in modifying the global gene expression profile of stimulated T cells and to determine if the Ctla-4 mediated maintenance of T cell tolerance was achieved, in part, by altering the transcription of quiescence genes necessary for the prevention of T cell activation subsequent to TCR and CD28 stimulation.
Previous studies investigating the influence of Ctla-4 ligation on transcriptional profiles of activated lymphocytes detected only quantitative alterations in the transcriptional regulation initiated by CD28 signaling. In contrast, our data suggests that quantitative effects of Ctla-4 ligation that differentially influence pathways acting downstream of stimulatory receptors results in a stable and qualitatively unique phenotype detectable at the level of the transcriptome. Thus, the cumulative effect of Ctla-4 signaling is unique and not constrained to reversing alterations in expression initiated by CD28. In addition, Ctla-4 ligation can be shown to influence T lymphocyte responsiveness and the resulting global expression profile within 4 hours after stimulation and prior to detectable Ctla-4 surface expression. In a subpopulation of T cells, TCR stimulation activates pathways that result in commitment to activation with 2-6 hours. In contrast, CD28 signaling must be maintained for 12-16 hours to ensure maximal responses at the population level. The period of sensitivity to Ctla-4 inhibition of activation is more constrained and does not extend beyond 12 hours. Together, these data support a potential role for Ctla-4 in modification of the early transcriptional response and may explain various alterations in phenotype resulting from Ctla-4 ligation that have been reported in secondary responses.
Identification of genes involved in lymphocyte activation, maintenance of selftolerance, and attenuation of immune responses opens the door to therapeutic manipulation of the pathways implicated. CD28 costimulation results in general amplification of TCR-initiated transcriptional responses, and specifically alters the expression profile of a subset of genes. In contrast, Ctla-4 ligation directly and specifically alters the expression of a select group of genes when ligated, and results in minimal suppression of the global CD28-mediated costimulatory transcriptional response. Ctla-4 regulated genes comprise a heterogeneous family, but include known quiescence factors, transcriptional regulators, and various determinants of cell cycle progression and senescence. The role of Ctla-4 in maintaining self-tolerance indicates that targeted manipulation of these gene products presents a novel therapeutic opportunity, and suggests that the mechanisms involved in Ctla-4-mediated maintenance of peripheral T cell tolerance and regulation of immune responsiveness is more nuanced than previously thought. In addition, this study provides the most comprehensive description of global gene expression during primary lymphocyte activation yet available. The integration of statistical and bioinfomatics analyses with large scale data mining tools identifies genes not previously characterized in lymphocytes and can direct future work by predicting potentially interacting gene products and pathways.
... The NF-kB transcription factors are required for activation of both the GM-CSF and IL-2 cytokine genes in response to T cell activation [10]. However, c-Rel, but not RelA, is required for chromatin remodeling events that accompany activation of both the GM-CSF [11] and IL-2 [5] genes. These chromatin remodeling events involve depletion of histones specifically from the promoter region of the gene [11,12,13]. ...
... However, c-Rel, but not RelA, is required for chromatin remodeling events that accompany activation of both the GM-CSF [11] and IL-2 [5] genes. These chromatin remodeling events involve depletion of histones specifically from the promoter region of the gene [11,12,13]. Alterations to the NF-kB binding sites have been shown to prevent chromatin remodeling events in a transgenic mouse model [14] and inhibition of NF-kB translocation in mouse T cell lines also prevents promoter chromatin remodeling [11,15]. ...
... These chromatin remodeling events involve depletion of histones specifically from the promoter region of the gene [11,12,13]. Alterations to the NF-kB binding sites have been shown to prevent chromatin remodeling events in a transgenic mouse model [14] and inhibition of NF-kB translocation in mouse T cell lines also prevents promoter chromatin remodeling [11,15]. Furthermore, GM-CSF gene expression [16,17] and promoter chromatin remodeling [11] is inhibited in primary CD4 + T cells from c-Rel 2/2 knockout mice. ...
The role of the Nuclear Factor κB (NF-κB) transcription factor family in T cell function has been well described. The c-Rel family member is of particular importance in initiating T cell responses to antigen and regulating activation of inflammatory cytokine genes, including the Interleukin-2 (IL-2) and Granulocyte macrophage colony stimulating factor (GM-CSF) genes. c-Rel is required for chromatin remodeling of these gene promoters, which involves depletion of histones from the promoters in response to T cell activating signals. These chromatin remodeling events precede transcriptional activation of the genes. The subsequent down-regulation of cytokine gene expression is important in the termination of an immune response and here we examine this process at the murine GM-CSF and IL-2 genes. We show that the cytokine mRNA levels rapidly return to basal levels following stimulus removal and this is associated with reassembly of histones onto the promoter. Histone reassembly at the GM-CSF and IL-2 promoters occurs concomitantly with depletion of RelA, c-Rel and RNA polymerase II from the promoters. Furthermore we show that transcriptional down-regulation and chromatin reassembly is dependent on depletion of c-Rel from the nucleus, and that this is regulated by the nuclear translocation of the NF-κB inhibitor, IκBα. The nuclear activation of c-Rel therefore not only regulates the initiation of GM-CSF and IL-2 gene activation in response to T cell activation, but also the termination of these gene responses following the removal of the activating signal.
... Regulation of the GM-CSF gene is tightly controlled [33,34], and its inducible expression is preceded by the establishment of a transcriptionally competent chromatin environment involving histone acetylation at the gene promoter [35]. Following stimulation, inducible GM-CSF gene expression is then accompanied by eviction of a nucleosome upstream of the transcription start site, reflected by increased nuclease accessibility at this location [35][36][37][38][39][40][41][42]. ...
... Here we demonstrate that RUNX1 is also required for the inducible expression of the GM-CSF gene in myeloid cells. Furthermore, we show that inducible transcriptional activation of GM-CSF gene expression is both preceded by and dependent upon histone acetylation within the promoter [37]. Our data suggest that RUNX1 contributes to both the generation of a hyperacetylated chromatin environment at the GM-CSF promoter prior to gene activation, and the transcriptional activation of the promoter in response to immune stimuli. ...
... Total RNA was isolated using Tri-Reagent (Sigma), digested with DNase I, and reverse transcribed with SuperScript III RNase H − reverse transcriptase (200 U/mL, Invitrogen). Amplification and quantification of cDNA by PCR in the presence of SYBR GreenER was performed on the Rotor-Gene 2000 real-time cycler (Corbett Research) using the SYBR GreenER TM qPCR SuperMix (Invitrogen) under the conditions described previously [37]. PCR was conducted using human GM-CSF mRNA primers (forward 5 -GGATGTGGCTGCAGAGCCTGC-3 ; reverse 5 -CTGGATGGCATTCACAT-3 ), human RUNX1 mRNA primers (forward 5 -CACCTACCACAGAGCCATCA-3 ; reverse 5 -CTCGGAAAAGGACAAGCTCC-3 ) and human RUNX1-ETO mRNA primers (forward 5 -AATCACAGTGGATGGGCCC-3 ; reverse 5 -TGCGTCTTCACATCCACAGG-3 ). ...
The RUNX1 gene, which is essential for normal hematopoiesis, is frequently rearranged by the t(8;21) chromosomal translocation in acute myeloid leukemia. The resulting RUNX1-ETO fusion protein contributes to leukemic progression by directing aberrant association of transcriptional cofactors and epigenetic modifiers to RUNX1 target genes. For example, the GM-CSF gene is activated by RUNX1, but is repressed by RUNX1-ETO. Here we show that RUNX1 normally cooperates with the histone acetyltransferase, CBP, to regulate GM-CSF expression at two levels. Firstly, it directs the establishment of a competent chromatin environment at the GM-CSF promoter prior to gene activation. It then participates in the transcriptional activation of the promoter in response to immune stimuli. In contrast, RUNX1-ETO, which cannot associate with CBP, is unable to transactivate the GM-CSF promoter and is associated with the generation of a repressive chromatin environment at the promoter.
... Nuclear extracts were prepared by a modification of the method of Schreiber et al. (34), as described previously (35). Nuclear proteins were separated by SDS-PAGE through 12% polyacrylamide, transferred onto nitrocellulose membrane and subjected to western blot analysis using anti-HA (Sigma-Aldrich), anti-AcH3 (Upstate Biotech, USA), anti-Sp1, anti-p65, anti-cRel and anti-BRG1 (Santa Cruz Biotechnology, USA) antibodies and the corresponding peroxidase-conjugated secondary antibodies (DAKO, Denmark). ...
... Total RNA was isolated using Tri-Reagent (Sigma) and reverse transcribed using Superscript II Reverse Transcriptase (Life Technologies, USA), and SYBR Green PCR amplification performed on the Rotor-Gene 2000 real-time cycler (Corbett Research, Australia) using the QuantiTect SYBR Green PCR kit (Qiagen, USA), as described previously (35) PCR was conducted using GM-CSF primer set +II and in parallel using the GAPDH primers (Table 1) to normalize for differences in cDNA synthesis and RNA input. To correlate the threshold (Ct) values from the amplification plots to copy number, a standard curve was generated using the mouse GM-CSF plasmid AOGM (31) and pCR2.1-GAPDH ...
... To correlate the threshold (Ct) values from the amplification plots to copy number, a standard curve was generated using the mouse GM-CSF plasmid AOGM (31) and pCR2.1-GAPDH plasmid (35). ...
Granulocyte macrophage-colony stimulating factor (GM-CSF) is produced by T cells, but not B cells, in response to immune signals. GM-CSF gene activation in response to T-cell stimulation requires remodelling of chromatin associated with the gene promoter, and these changes do not occur in B cells. While the CpG methylation status of the murine GM-CSF promoter shows no correlation with the ability of the gene to respond to activation, we find that the basal chromatin environment of the gene promoter influences its ability to respond to immune signals. In unstimulated T cells but not B cells, the GM-CSF promoter is selectively marked by enrichment of histone acetylation, and association of the chromatin-remodelling protein BRG1. BRG1 is removed from the promoter upon activation concomitant with histone depletion and BRG1 is required for efficient chromatin remodelling and transcription. Increasing histone acetylation at the promoter in T cells is paralleled by increased BRG1 recruitment, resulting in more rapid chromatin remodelling, and an associated increase in GM-CSF mRNA levels. Furthermore, increasing histone acetylation in B cells removes the block in chromatin remodelling and transcriptional activation of the GM-CSF gene. These data are consistent with a model in which histone hyperacetylation and BRG1 enrichment at the GM-CSF promoter, generate a chromatin environment competent to respond to immune signals resulting in gene activation.
... Conversely, chromatin regions in a relatively open configuration are digested and cannot be amplified by primers spanning the site. Undenatured DNA was prepared and evaluated by PCR with or without EpiQuik nuclease digestion, using primers spanning 2195 to 278 bases (encompassing NF-kB/Sp1/AP-1 sites) relative to the transcription start site (17). Accessibility of the chromatin region in question is then determined as the difference in cycle threshold (Ct) for PCR of digested minus the Ct for PCR of undigested samples. ...
... Compared with AEC in normoxia, hypoxia resulted in decreased accessibility of this binding region in the promoter GM-CSF proximal promoter (Fig. 5D). Interestingly, this region also includes binding sites for AP-1 and Sp1, transcription factors that have been found to cooperate with NF-kB for promotion of GM-CSF transcription in other systems (17). Together, these data suggest that hypoxia may decrease activation of NF-kB while also decreasing accessibility of the promoter in a region targeted by multiple transcription factors, thereby reducing GM-CSF gene transcription in AEC. ...
Pulmonary innate immune responses involve a highly regulated multicellular network to defend the enormous surface area of the lung. Disruption of these responses renders the host susceptible to pneumonia. Alveolar epithelial cells (AEC) are a critical source of innate immune molecules such as GM-CSF, which determine the functional maturation of alveolar macrophages. In many pulmonary diseases, heterogeneous ventilation leads to regional hypoxia in the lung. The effect of hypoxia on AEC innate immune function is unknown. We now report that exposure of primary murine AEC to hypoxia (1% oxygen) for 24 h results in significant suppression of key innate immune molecules, including GM-CSF, CCL2, and IL-6. This exposure did not cause toxicity but did induce stabilization of hypoxia-inducible factor 1α protein (HIF-1α) and shift to glycolytic metabolism. Focusing on GM-CSF, we found that hypoxia greatly decreased the rate of GM-CSF transcription. Hypoxia both decreased NF-κB signaling in AEC and induced chromosomal changes, resulting in decreased accessibility in the GM-CSF proximal promoter of target sequences for NF-κB binding. In mice exposed to hypoxia in vivo (12% oxygen for 2 d), lung GM-CSF protein expression was reduced. In vivo phagocytosis of fluorescent beads by alveolar macrophages was also suppressed, but this effect was reversed by treatment with GM-CSF. These studies suggest that in critically ill patients, local hypoxia may contribute to the susceptibility of poorly ventilated lung units to infection through complementary effects on several pathways, reducing AEC expression of GM-CSF and other key innate immune molecules.
... GM-CSF production by T cells is regulated cell-intrinsically through the action of transcription factors. Factors that regulate its transcription include AP-1 family members, NFAT, NF-κB family members, including c-Rel and NF-κB1, and RUNX1 [14][15][16][17][18][19] . Recently, Codarri et al. reported a requirement for RORγt for maximal production of GM-CSF by T H 17 cells, although a second report observed no reduction in GM-CSF secretion by Rorc −/− T H cells in vitro 10,11 . ...
... These seemingly conflicting observations have been reconciled by the recent finding that GM-CSF, produced by both T H 1 and T H 17 cells, serves a non-redundant pro-inflammatory role in EAE development [8][9][10][11][12] . Control of Csf2 expression involves AP-1, NFAT, Runx1, NF-κB, histone acetylation, and BRG1 recruitment [14][15][16][17][18][19]45 . c-Rel-and NF-κB1-deficient T cells produce less GM-CSF, but these factors may be non-selective in their impact on cytokine production 17,18 . ...
TH1 and TH17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Pathogenic TH cells in EAE must produce the pro-inflammatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). TH cell pathogenicity in EAE is also regulated by cell-intrinsic production of the immunosuppressive cytokine interleukin 10 (IL-10). Here we demonstrate that mice deficient for the basic helix-loop-helix (bHLH) transcription factor Bhlhe40 (Bhlhe40(-/-)) are resistant to the induction of EAE. Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10. In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(-/-) TH1 and TH17 cells, and these cells show increased production of IL-10. Blockade of IL-10 receptor in Bhlhe40(-/-) mice renders them susceptible to EAE. These findings identify Bhlhe40 as a critical regulator of autoreactive T-cell pathogenicity.
... 24 Indeed, GM-CSF plays an essential role in myeloid cell development and is a direct transcriptional target of NFAT. 25 Other downstream targets of NFAT in innate immune cells 26 include early growth response 2 and 3 (Egr2, Egr3), and cyclo-oxygenase-2 (Cox2), which are involved in angiogenesis and inflammation. ...
... 10,103 NFAT regulation of immune homeostasis is mediated by growth factors, such as GM-CSF and IL-2, which are transcribed after NFAT translocation. 25 Homeostasis of innate immune cells is a tightly regulated process that depends on growth factors, including GM-CSF 104 and Flt3-L. 105 Systemic administration of calcineurin/ NFAT inhibitors may therefore profoundly impact innate immune homeostasis in treated patients. ...
The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway mediates multiple adaptive T-cell functions, but recent studies have shown that calcineurin/NFAT signaling also contributes to innate immunity and regulates the homeostasis of innate cells. Myeloid cells, including granulocytes and dendritic cells, can promote inflammation, regulate adaptive immunity, and are essential mediators of early responses to pathogens. Microbial ligation of pattern-recognition receptors, such as TLR4, CD14, and dectin 1, is now known to induce the activation of calcineurin/NFAT signaling in myeloid cells, a finding that has provided new insights into the molecular pathways that regulate host protection. Inhibitors of calcineurin/NFAT binding, such as cyclosporine A and FK506, are broadly used in organ transplantation and can act as potent immunosuppressive drugs in a variety of different disorders. There is increasing evidence that these agents influence innate responses as well as inhibiting adaptive T-cell functions. This review focuses on the role of calcineurin/NFAT signaling in myeloid cells, which may contribute to the various unexplained effects of immunosuppressive drugs already being used in the clinic.
... For Western blotting of NFjB, 25 cm 2 flasks of confluent OEC, AC, and SC cultures were treated for 1 h with E. coli or control broth as mentioned earlier, in the absence of Hoechst blue. Nuclear extracts were prepared according to the procedure used by Brettingham-Moore et al. (2005), with some modifications. Briefly, flasks were rinsed twice with PBS-containing 0.5 mM EDTA and the cells were lifted with weak trypsin (0.01%; Sigma). ...
... Nuclear and cytoplasmic protein fractions were resolved by SDS-PAGE through 12% polyacrylamide, transferred onto nitrocellulose membrane and subjected to Western blot analysis using rabbit anti-NFjB p65 and rabbit anti-SP-1 (both 1:1,000; Santa Cruz Biotechnology) and peroxidase-conjugated anti-rabbit secondary antibodies (1:2,000; DakoCytomation). SP-1 is a transcription factor localized to the nucleus and was used as a loading control (Brettingham-Moore et al., 2005). Proteins were visualized using the SuperSignal West Pico Chemiluminescent kit (Pierce Biotechnology, Rockford, IL), according to the manufacturer's instructions. ...
The primary olfactory nerves provide uninterrupted conduits for neurotropic pathogens to access the brain from the nasal cavity, yet infection via this route is uncommon. It is conceivable that olfactory ensheathing cells (OECs), which envelope the olfactory nerves along their entire length, provide a degree of immunological protection against such infections. We hypothesized that cultured OECs would be able to mount a biologically significant response to bacteria and pathogen-associated molecular patterns (PAMPs). The response of OECs to Escherichia coli (E. coli) and various PAMPs was compared to that of Schwann cells (SCs), astrocytes (ACs), and microglia (MG). A subset of OECs displayed nuclear localization of nuclear factor kappaB), an inflammatory transcription factor, after treatment with E. coli (20% +/- 5%), lipopolysacchride (33% +/- 9%), and Poly I:C (25% +/- 5%), but not with peptidoglycan or CpG oligonucleotides. ACs displayed a similar level of activation to these treatments, and in addition responded to peptidoglycan. The activation of OECs and ACs was enhanced by coculture with MG (56% +/- 16% and 85% +/- 13%, respectively). In contrast, SCs did not respond to any treatment or to costimulation by MG. Immunostaining for the chemokine Gro demonstrated a functional response that was consistent with NF kappaB activation. OECs expressed mRNA for Toll-like receptors (TLRs) 2 and 4, but only TLR4 protein was detected by Western blotting and immunohistochemistry. The results demonstrate that OECs possess the cellular machinery that permits them to respond to certain bacterial ligands, and may have an innate immune function in protecting the CNS against infection.
... The promoters of many of these genes have potential c-Rel binding sites, implying a direct effect on gene transcription. We have recently shown that chromatin remodeling across the promoter regions of IL-2 and GM-CSF does not occur following the activation of CD4 ϩ T cells from c-Rel Ϫ/Ϫ mice (9,15). Moreover, the histone loss that accompanies this chromatin remodeling is not observed in c-Rel Ϫ/Ϫ T cells (16). ...
... Several of these genes, GM-CSF, IL-2, ICAM-1, IP-10, Bcl2l1, and CD40, have previously been described as NF-B target genes either in single genes studies or expression profiling studies (9,12,20,(43)(44)(45). We have previously shown that GM-CSF and IL-2 are c-Rel target genes where c-Rel binds to and activates transcription through the CD28RE of the promoter (9,15,18). c-Rel-containing NF-B complexes have also been shown to bind to the ICAM-1, Bcl2l1, and IL-4 promoters in specific cell types (12,43,46). However, the involvement of c-Rel in the regulation of the IP-10, IL-13, Iigp1, and CD40 genes has not previously been reported. ...
It is well established that the NF-kappaB family of transcription factors serves a major role in controlling gene expression in response to T cell activation, but the genome-wide roles of individual family members remain to be determined. c-Rel, a member of the NF-kappaB family, appears to play a specific role in T cell function because T cells from c-Rel(-/-) animals are defective in their response to immune signals. We have used expression profiling to identify sets of genes that are affected by either deletion or overexpression of c-Rel in T cells. Very few of these genes exhibit a strong requirement for c-Rel; rather, c-Rel appears to modulate the expression of a large number of genes in these cells. The sets of c-Rel-affected genes are significantly enriched for genes containing consensus NF-kappaB/Rel sites in their proximal promoter regions. In addition, their promoters contain a higher average density of NF-kappaB/Rel sites compared with all genes represented on the microarrays. A transcriptional module comprised of two closely spaced c-Rel consensus sites is found with higher frequency in the c-Rel-affected gene sets and may represent an important control module for genes regulated by c-Rel or other NF-kappaB family members. We confirmed the importance of these findings on a subgroup of genes by using quantitative PCR to monitor gene expression as well as in vitro c-Rel/DNA binding assays and luciferase reporter assays. The c-Rel-regulated genes identified here support a role for c-Rel in inflammatory responses as well as in the promotion of cell growth and survival.
... However, the exact mechanisms through which GM-CSF-production is regulated within T cells are less clear. IL-1b and IL-23 cytokine signaling are required for in vivo GM-CSF production during EAE and the transcription factors c-Rel, NF-kB1, RUNX1, RORgt, Bhlhe40 have been proposed to affect T cell Csf2 expression (22)(23)(24)(25)(26)(27)(28)(29)(30). Of the aforementioned transcription factors, Bhlhe40 is particularly noteworthy as it is induced within T cells upon TCR stimulation and Bhlhe40 -/mice have been demonstrated to be deficient in GM-CSF production in T cells in vivo (31,32). ...
T-bet-expressing Th17 (T-bet ⁺ RORγt ⁺ ) cells are associated with the induction of pathology during experimental autoimmune encephalomyelitis (EAE) and the encephalitic nature of these Th17 cells can be explained by their ability to produce GM-CSF. However, the upstream regulatory mechanisms that control Csf2 (gene encoding GM-CSF) expression are still unclear. In this study, we found that Th17 cells dynamically expressed GATA3, the master transcription factor for Th2 cell differentiation, during their differentiation both in vitro and in vivo . Early deletion of Gata3 in three complimentary conditional knockout models by Cre-ERT2, hCd2 Cre and Tbx21 Cre , respectively, limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells. These results indicate that early GATA3-dependent gene regulation is critically required to generate a de novo encephalitogenic Th17 response. Furthermore, a late deletion of Gata3 via Cre-ERT2 in the adoptive transfer EAE model resulted in a cell intrinsic failure to induce EAE symptoms which was correlated with a substantial reduction in GM-CSF production without affecting the generation and/or maintenance of T-bet-expressing Th17 cells. RNA-Seq analysis of Gata3- sufficient and Gata3 -deficient CNS-infiltrating CD4 ⁺ effector T cells from mixed congenic co-transfer recipient mice revealed an important, cell-intrinsic, function of GATA3 in regulating the expression of Egr2 , Bhlhe40 , and Csf2 . Thus, our data highlights a novel role for GATA3 in promoting and maintaining the pathogenicity of T-bet-expressing Th17 cells in EAE, via putative regulation of Egr2, Bhlhe40, and GM-CSF expression.
... GM-CSF is a NFAT target and calcineurin impairment was associated with an anti-inflammatory or immature DC phenotype. [243][244][245] Hence, calcineurin and NFAT activity should be analysed to further elucidate the mode of action of Anx. ...
Apoptotic cells mediate the development of tolerogenic dendritic cells (DC) and thus facilitate the induction and maintenance of peripheral tolerance. Our group investigated the influence of apoptotic cells on DC and identified the cell surface exposure of the evolutionary conserved annexin core domain (Anx) as a specific signal which antagonises Toll-like receptor (TLR) signalling. This study examined whether the tolerogenic capacity of Anx can be exploited to downregulate antigen-specific immune responses to the model antigen ovalbumin (Ova). The treatment of bone marrow-derived dendritic cells (BMDC) with soluble Anx prior to Ova administration or the treatment with beads harbouring Anx as well as Ova attenuated the response of Ova-specific OT-II Tcells. The co-culture of treated DC and Tcells resulted in an anergy-like phenotype characterised by reduced proliferation and cytokine secretion. The anti-inflammatory effects of Anx are mediated through DC by yet unknown mechanisms. Anx did not lead to the tolerogenic DC phenotype described to be induced by apoptotic cells and also the previously reported suppression of anti-inflammatory cytokines by Anx was dispensable for the effect on Tcells. However, this study revealed enhanced production od reactive oxygen species (ROS) in BMDC in response to Anx which might affect the DC/Tcell interactions. Although the underlying mechanisms remain to be elucidated, this study demonstrates that Anx can be used as a tool to generate a particle-based antigen delivery system that promotes antigen-specific immunosuppression. Such Anx-particles may be a new therapeutic approach for the treatment of autoimmune disease.
... Accordingly, the observed activation-induced differentially expressed genes in TXNIP KO clones, namely EGR2 [312], GMCSF [313], TNFA [314], IFNG [315,316], GZMB [317] as well as the above-mentioned CD95L and IL2 are all reported to be controlled by NFAT. ...
T cell receptor (TCR) engagement and subsequent signalling are a prerequisite to initiate a T cell immune response. In recent years, our group investigated the underlying mechanisms of TCR signalling and identified Thioredoxin-interacting protein (TXNIP) as a possible regulator of a T cell immune response. This study examined the role of TXNIP in TCR signalling. Following TCR stimulation, TXNIP expression was reduced due to accelerated proteasomal degradation as well as decreased protein synthesis. Since TXNIP is a negative regulator of Trx, activation-induced downregulation of TXNIP expression resulted in increased Trx activity. Using TXNIP knockout (KO) T cells as a model system, this study revealed that TXNIP has an impact on gene expression upon TCR engagement. By analysing stimulation-induced whole genome expression of TXNIP KO cells in comparison to control cells, this study demonstrated that TXNIP acts as a transcriptional inhibitor. TXNIP affects transcription of CD95L, GMCSF, GZMB, IFNG, IL2, TNFA and EGR2. These genes are involved in T cell activation, differentiation, cytokine signalling as well as cell death and designated as NFκB, AP1 as well as NFAT targets. Thus, TXNIP might control gene expression by regulating the activity of one or various transcription factors. Expression of CD95L upon TCR stimulation mediates activation-induced cell death (AICD) in apoptosis-sensitive T cells. In accordance with increased stimulation-induced CD95L expression, AICD was enhanced in TXNIP KO cells compared to control cells. This result underlines the important role of TXNIP in TCR signalling. Taken together, this study demonstrates that TXNIP is involved in TCR signalling by acting as transcriptional inhibitor. Hence, TXNIP might be considered as a potential therapeutic target to shape T cell responses e.g. in autoimmune or tumour diseases.
... Furthermore, GM-CSF regulates the release of neutrophils from the bone marrow(22). It had been revealed that NFAT contribute to the transcriptions of IL-3 and GM-CSF(21,23,24). In addition, it has been discovered that NFAT activation regulates the induction of cyclooxygenase-2 (COX-2) (14), which is required for the production of key inflammatory mediators including prostaglandins. ...
Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506) are broadly used in organ transplantations as immune suppressants. As the calcineurin/NFAT signaling pathway has been identified as critical pathway in the interleukin-2 (IL-2) production of T cells, inhibition of T-cell derived IL-2 has been considered the major mechanism of calcineurin inhibitors. However, there is increasing evidence that NFAT transcription factor is involved in multiple functions of dendritic cells and innate immune cells as well. NFAT expression is not restricted to T cells, and IL-2 can be produced in dendritic cells and macrophages through the calcineurin/NFAT pathway. Furthermore, it has been discovered that NFAT regulates expressions of several inflammatory mediators, including TNF-α and cyclooxygenase-2 in innate immune cells. Therefore, calcineurin inhibitors may have much broader effects in the transplant recipients than previously being considered. In this review, we reviewed recently discovered roles of NFAT pathway in dendritic cells and innate immune cells, and discussed positive and negative implications of calcineurin inhibitors' broader effects with a focus on islet xenotransplantation. © 2016, Chonnam National University Medical School. All rights reserved.
... Another region (-54~-31 bp) within the proximal promoter of Csf2 that contains NFAT, AP-1, and ETS1 binding sites also regulates GM-CSF promoter activity [5,51,52]. Although the calcium/NFAT pathway is not necessary for promoter remodeling, both PKC/NF-κB1 and calcium/NFAT pathways are required for efficient Csf2 transcription [53]. An inducible enhancer located at 3k bp upstream of transcription start site of Csf2 contains binding sites for NFAT and AP-1 proteins, and integrates multiple signals for the control of GM-CSF expression as well [54,55]. ...
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by a variety of cells and plays an important role in the inflammatory response in infection as well as in autoimmunity. Recent progress has indicated that CD4+ T cell-derived GM-CSF has a prominent and non-redundant function in mediating autoimmune neuroinflammation. Thus, there is increased interest on studying the regulation of GM-CSF production by T helper cells, which could translate to the development of novel therapeutics for autoimmune diseases such as multiple sclerosis. This review focuses on our current understanding of the regulation and function of T cell-derived GM-CSF.
... Nuclear extracts were prepared as previously described [19]. Protein concentrations were determined by Bradford Assay (Bio-Rad, USA). ...
Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.
Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.
Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).
These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.
... mRNA was reverse-transcribed to cDNA in a solution containing first strand buffer (Invitrogen), 5 units/µl Superscript III reverse transcriptase (Invitrogen), 0.01 M dithiothreitol (Invitrogen) and 1.125 mM dNTP (Promega) in a total volume of 20 µl.The reaction was incubated at 42°C for 50 minutes followed by enzyme heatinactivation at 70°C for 15 minutes. Real-time PCR was conducted to quantify concentration of various cDNA transcripts using Quantitect SYBR green PCR master mix (Qiagen) on the Corbett Rotorgene-6000 as described by(Brettingham-Moore et al. 2005). The initial melt phase was set to 95°C for 900 seconds followed by cycling which consisted of 95°C for 15 seconds and 60°C for 60 seconds then acquiring data to cycling A, Channel 1. PCR cycling was conducted for 45 cycles and melt curves were generated from 60°C to 95°C holding for 5 seconds at every 1°C increment and acquiring data to cycling A, Channel 1. Primers (sets are listed in table 3.1) were used at a final concentration of 1 µM. ...
... Brain and liver samples were homogenised by Ultra-Turrax mechanical homogenizer (IKA) in TRI reagent (Sigma) and RNA was extracted according to manufacturer's protocol. Reverse transcription with the Superscript-III reverse transcriptase system (Invitrogen) and quantitative PCR with Quantitect SYBR green (Qiagen) was conducted according to the method of Brettingham-Moore et al.[37]. Oligonucleotide primers are detailed in table 1. ...
Unlabelled:
Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II(-/-)) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II(-/-) mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver.
Conclusion:
MT-I/II is up-regulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver.
... RNA was isolated from TRI-reagent according to the manufacturer's protocol. Reverse transcription with the Superscript-III reverse transcriptase system (Invitrogen) and quantitative PCR with Quantitect SYBR green (Qiagen, Hilden, Germany) was conducted according to the method of Brettingham- Moore et al. [29]. Oligonucleotide primers are detailed in table 1. ...
Metallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system.
Wild type and MT-I/II(-/-) mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay.
Comparison of MT-I/II(-/-) mice with wild type controls following cryolesion brain injury revealed that the MT-I/II(-/-) mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II(-/-) mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II(-/-) mice that was independent of brain injury.
These results contribute to the evidence that MT-I/II(-/-) mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II(-/-) mice after brain injury relative to wild type mice.
... NF-κB binding sites [55,56] and vitamin D responsive elements (VDRE) [57] have been identified in the GM-CSF promoter region. The downstream activation of NF-κB by TNFα and its subsequent binding to the GM-CSF promoter has been shown to be important in the expression of the protein in activated T-lymphocytes. ...
Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.
... Previous reports demonstrated that regulatory elements in the IL-3 and GM-CSF locus are regulated by the NF-κB pathway (Brettingham-Moore et al., 2005;Holloway et al., 2003). Additionally, c-rel-deficient mice have significantly reduced IL-3 and GM-CSF gene expression (Gerondakis et al., 1996). ...
We investigated gene regulation at the IL-3/GM-CSF gene cluster. We found BRG1, a SWI/SNF remodeling ATPase, bound a distal element, CNSa. BRG1 binding was strongest in differentiated, stimulated T helper cells, paralleling IL-3 and GM-CSF expression. Depletion of BRG1 reduced IL-3 and GM-CSF transcription. BAF-specific SWI/SNF subunits bound to this locus and regulated IL-3 expression. CNSa was in closed chromatin in fibroblasts, open chromatin in differentiated T helper cells, and moderately open chromatin in naïve (undifferentiated) T helper cells; BRG1 was required for the most open state. CNSa increased transcription of a reporter in an episomal expression system, in a BRG1-dependent manner. The NF-κB subunit RelA/p65 bound CNSa in activated T helper cells. Inhibition of NF-κB blocked BRG1 binding to CNSa, chromatin opening at CNSa, and activation of IL-3 and GM-CSF. Together, these findings suggest CNSa is a distal enhancer that binds BRG1 and NF-κB.
... To confine the DNase I hypersensitive site to more narrow regions, a
recently described CHART-PCR assay was performed to further locate the DNase I
hypersensitive site between nucleotides -270 and +230, which is about 6.5 and
6.0 kb from the probe used in DNase I hypersensitivity assays. CHART-PCR
evaluates the accessibility of genomic DNA to nuclease (such as DNase I,
restriction enzyme, or micrococcal nuclease) by comparing the quantity of
intact DNA from a nuclease-treated sample to that of an untreated sample
(21-27). Chromatin recovered from both DNase I-treated and untreated cells was
quantitated by real-time PCR. ...
Fibroblast growth factor receptor 2 (FGFR2) plays an important regulatory role in bone development. However, the regulatory mechanisms controlling FGFR2 expression remain poorly understood. Here we have identified a role for the nuclear factor Y (NF-Y) in constitutive activation of FGFR2. A unique DNase I hypersensitive site was detected in the region encompassing nucleotides -270 to +230 after scanning a large range covering 33.3 kilobases around the transcription start site of FGFR2. Using a PCR-based chromatin accessibility assay, an open chromatin conformation was detected around the proximal 5' fragment of FGFR2 gene. Deletion constructs of the 5'-flanking region of FGFR2 were fused to a luciferase reporter gene. After transient transfection in C3H10T1/2, ME3T3-E1, and C2C12 as well as primary osteoblasts, a minimal region -86/+139 that is highly homologous to the human sequence and bears a CCAAT box was identified as the core promoter. Electrophoretic mobility shift assay supershift and chromatin immunoprecipitation demonstrated that the CCAAT box was the binding site for NF-Y. Deletion of NF-Y consensus sequence resulted in the total loss of NF-Y promoter activity. Overexpression of NF-Y protein and transfection of NF-Y small interfering RNAs in the cells substantially changed the promoter activity. Moreover, NF-Y small interfering RNAs greatly inhibited the endogenous FGFR2 transcription level and the chromatin accessibility and H3 acetylation across the promoter. Taken together, our results demonstrate that interaction of NF-Y at the CCAAT box is pivotal to FGFR2 gene transcription partly through the construction of a local open chromatin configuration across the promoter.
Significance
The production of proinflammatory cytokines, particularly granulocyte-macrophage colony-stimulating factor, by CD4 ⁺ T cells is a key process for amplifying immune responses but can also lead to harmful tissue damage in pathologies like multiple sclerosis and Covid-19. Correctly controlling the expression of proinflammatory cytokines is therefore of major interest. However, the pathogenic signature of CD4 ⁺ T cells relies on a transcriptional program that is thus far poorly understood. Here, we identified the transcription factor Ikaros as an essential transcriptional repressor of proinflammatory cytokine gene expression. Our work identifies a critical molecular pathway regulating the pathogenic program of CD4 ⁺ T cells and brings new perspectives for potential therapies of autoimmune and inflammatory diseases.
T cells (or T lymphocytes) exhibit a myriad of functions in immune responses, ranging from pathogen clearance to autoimmunity, cancer and even non-lymphoid tissue homeostasis. Therefore, deciphering the molecular mechanisms orchestrating their specification, function and gene expression pattern is critical not only for our comprehension of fundamental biology, but also for the discovery of novel therapeutic targets. Among the master regulators of T-cell identity, the functions of the NF-κB family of transcription factors have been under scrutiny for several decades. However, a more precise understanding of their pleiotropic functions is only just emerging. In this review we will provide a global overview of the roles of NF-κB in the different flavors of mature T cells. We aim at highlighting the complex and sometimes diverging roles of the five NF-κB subunits in health and disease.
Integrins are transmembrane adhesion receptors that play an important role in hematopoiesis by facilitating interactions between hematopoietic cells and extracellular matrix components of the bone marrow and hematopoietic tissues. These interactions are important in regulating the function, proliferation and differentiation of hematopoietic cells, as well as their homing and mobilization in the bone marrow. Not surprisingly altered expression and function of integrins plays a key role in the development and progression of cancer including leukemias. However, the regulation of integrin gene expression is not well characterized and the mechanisms by which integrin genes are disrupted in cancer remain unclear. Here we demonstrate for the first time that a key regulator of hematopoiesis, RUNX1, binds to and regulates the promoters of both the ITGA6 and ITGB4 genes in myeloid cells. The ITGA6 and ITGB4 integrin genes form the α6β4 integrin receptor. However our data indicates that RUNX1 functions differently at these two promoters. RUNX1 regulates ITGA6 through a consensus RUNX1 binding motif in its promoter. In contrast, although the ITGB4 promoter is also activated by RUNX1, it does so in the absence of a recognized consensus RUNX1 binding motif. Further, our data suggest that regulation of ITGB4 may involve interactions between the promoter and upstream regulatory elements. This article is protected by copyright. All rights reserved
The immune system is exquisitely poised to identify, respond to, and eradicate pathogens from the body, as well as to produce a more rapid and augmented response to a subsequent encounter with the pathogen. These cellular responses rely on the highly coordinated and rapid activation of gene expression programs as well as the ability of the cell to retain a memory of the initial gene response. It is clear that chromatin structure and epigenetic mechanisms play a crucial role in determining these gene responses, and in fact the immune system has proved an instructive model for investigating the multifaceted mechanisms through which the chromatin landscape contributes to gene expression programs. These mechanisms include modifications to the DNA and histone proteins, the positioning, composition, and remodeling of nucleosomes, as well as the formation of higher-order chromatin structures. Moreover, it is now apparent that epigenetic mechanisms also provide an instrument by which cells can retain memory of the initial transcriptional response, “priming” the genome so that it can respond more quickly to subsequent exposure to the signal. Here, we use the immune system as a model to demonstrate the complex interplay between transcription factors and the chromatin landscape required to orchestrate precise gene responses to external stimuli and further to demonstrate how these interactions can establish memory of past transcriptional events. We focus on what we have learnt from the immune system and how this can inform our understanding of other cellular systems.
Las citocinas IL-12 p70 e IL-23 polarizan la respuesta inmune a los tipos Th1 y Th17. Estas citocinas comparten una subunidad, la IL-12 p40 (gen IL12/23B) y difieren en una subunidad específica, la IL-12 p35 (gen IL12A) en la IL-12 p70, y la IL-23 p19 (gen IL23A) en la IL-23. La regulación del balance IL-12 p70/IL-23 es relevante en infecciones fúngicas invasivas y enfermedades autoinmunes. La transcripción de IL23A se regula por una combinación de c-Rel con coactivadores como ATF2, que se activa por fosforilación complementaria, dependiente de PKC y MAPKs, de las Thr-71 y Thr-69. Los receptores de patrones fúngicos Dectin-1 y Dectin-2 inducen la producción de LTB4, cisteinil-leucotrienos y PAF que contribuyen a la fosforilación de ATF2. La transcripción de IL12A se reprime por el estímulo fúngico zimosano mediante la desacetilación dependiente de NAD+ de las histonas del promotor de IL12A por SIRT1, reduciendo la accesibilidad de c-Rel.
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The engagement of the receptors for fungal patterns induces the expression of cytokines, the release of arachidonic acid and the production of PGE2 in human dendritic cells (DC), but few data are available about other lipid mediators that may modulate DC function. The combined antagonism of leukotriene (LT) B4, cysteinyl-LT, and platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn -glycero-3-phosphocholine) inhibited IL23A mRNA expression in response to the fungal surrogate zymosan and to a lower extent TNFA (tumor necrosis factor-α) and CSF2 (granulocyte macrophage colony-stimulating factor) mRNA. The combination of lipid mediators and the lipid extract of zymosan-conditioned medium increased the induction of IL23A by LPS (bacterial lipopolysaccharide), thus suggesting that unlike LPS, zymosan elicits the production of mediators at a concentration enough for optimal response. Zymosan induced the release of LTB4, LTE4, 12-hydroxyeicosatetraenoic acid (12-HETE), and PAF C16:0. DC showed a high expression and detectable Ser663 phosphorylation of 5-lipoxygenase in response to zymosan, and a high expression and activity of LPCAT1/2 (lysophosphatidylcholine acyltransferase 1 and 2), the enzymes that incorporate acetate from acetyl-CoA into choline-containing lysophospholipids to produce PAF. Pharmacological modulation of the arachidonic acid cascade and the PAF receptor inhibited the binding of P-71Thr-ATF2 (activating transcription factor 2) to the IL23A promoter mirroring their effects on the expression of IL23A mRNA and IL-23 protein. These results indicate that LTB4, cysteinyl-LT, and PAF, acting through their cognate G protein-coupled receptors, contribute to the phosphorylation of ATF2 and play a central role in IL23A promoter trans -activation and the cytokine signature induced by fungal patterns.
Objective
To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. Methods
Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte–macrophage colony-stimulating factor–knockout (GM-CSF−/−) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5–6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. ResultsIn both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)2D3 to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF−/− mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. Conclusion
These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.
c-Rel is a member of the nuclear factor κB (NF-κB) transcription factor family. Unlike other NF-κB proteins that are expressed in a variety of cell types, high levels of c-Rel expression are found primarily in B and T cells, with many c-Rel target genes involved in lymphoid cell growth and survival. In addition to c-Rel playing a major role in mammalian B and T cell function, the human c-rel gene (REL) is a susceptibility locus for certain autoimmune diseases such as arthritis, psoriasis, and celiac disease. The REL locus is also frequently altered (amplified, mutated, rearranged), and expression of REL is increased in a variety of B and T cell malignancies and, to a lesser extent, in other cancer types. Thus, agents that modulate REL activity may have therapeutic benefits for certain human cancers and chronic inflammatory diseases.
Tumor necrosis factor (TNF) induces expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but lymphotoxin
β (LTβ) does not. Here we report that priming of cells with agonistic LTβ receptor antibody synergistically enhanced TNF-induced
GM-CSF expression. The LTβ priming process was not due to an increase in TNF-mediated nuclear translocation of p65, p65 DNA
binding, or NF-κB transactivational activity. The synergistic effect of LTβ priming was not observed with other TNF-responsive
genes such as Ccl2 or RelB, which suggested that this effect was not a general increase in TNF signaling. Furthermore, RelB
and p65 were both independently recruited to the GM-CSF promoter when cells were primed with LTβ followed by TNF treatment.
As a consequence, an increase in both chromatin accessibility and the recruitment of RNA polymerase II were observed to the
GM-CSF promoter. Taken together, these findings suggested that LTβ signaling amplified TNF-mediated GM-CSF expression by facilitating
chromatin access and the co-recruitment of RNA polymerase II to increase gene transcription. Moreover, the novel priming process
described here underscores the complexity of the interactions between the classical and alternative NF-κB signaling pathways.
Nuclear factor kappaB (NFκB) is a key transcriptional regulator of inflammatory genes. We investigated the modulatory effects of olfactory ensheathing cells (OECs), microglia and meningeal fibroblasts on translocation of NFκB to astrocyte nuclei. The percentage of activated astrocytes in co-cultures with OECs was significantly less than for co-cultures with microglia (p<0.001) and fibroblasts (p<0.05). Phorbol myristate acetate (PMA) and calcium ionophore stimulation of p65 NFκB translocation to nuclei provided an in vitro model of astrocyte inflammatory activation. Soluble factors released by OECs significantly moderated the astrocytic NFκB translocation induced by either PMA/calcium ionophore or microglia-derived factors (p<0.001). Insulin-like growth factor-1 may contribute to these effects, since it is expressed by OECs and also significantly moderated the astrocytic NFκB translocation (p<0.05), albeit insufficiently to fully account for the OEC-induced moderation (p<0.01). Olfactory ensheathing cells significantly moderated the increased transcription of the pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor in the activated astrocytes (p<0.01). These results suggest that transplanted OECs could improve neural repair after CNS injury by moderating astrocyte activation.
Olfactory ensheathing cells (OECs) have been investigated extensively as a therapy to promote repair in the injured CNS, with variable efficacy in numerous studies over the previous decade. In many studies that report anatomical and functional recovery, the beneficial effects have been attributed to the ability of OECs to cross the PNS-CNS boundary, their production of growth factors, cell adhesion molecules and extracellular matrix proteins that promote and guide axon growth, and their ability to remyelinate axons. In this brief review, we focus on the interaction between OECs and astrocytes in vivo and in vitro, in the context of how OECs may be overcoming the deleterious effects of the glial scar. Drawing from a selection of different experimental models of spinal injury, we discuss the morphological alterations of the glial scar associated with OEC transplants, and the in vitro research that has begun to elucidate the interaction between OECs and the cell types that compose the glial scar. We also discuss recent research showing that OECs bear properties of immune cells and the consequent implication that they may modulate neuroinflammation when transplanted into CNS injury sites. Future studies in unraveling the molecular interaction between OECs and other glial cells may help explain some of the variability in outcomes when OECs are used as transplants in CNS injury and more importantly, contribute to the optimization of OECs as a cell-based therapy for CNS injury. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.
Interleukin-6 (IL-6), involved in cancer-related inflammation, acts as an autocrine and paracrine growth factor, which promotes angiogenesis, metastasis, and subversion of immunity, and changes the response to hormones and to chemotherapeutics. We explored transcription mechanisms involved in differential IL-6 gene expression in breast cancer cells with different metastatic properties. In weakly metastatic MCF7 cells, histone H3 K9 methylation, HP1 binding, and weak recruitment of AP-1 Fra-1/c-Jun, NF-kappaB p65 transcription factors, and coactivators is indicative of low chromatin accessibility and gene transcription at the IL-6 gene promoter. In highly metastatic MDA-MB231 cells, strong DNase, MNase, and restriction enzyme accessibility, as well potent constitutive transcription of the IL-6 gene promoter, coincide with increased H3 S10 K14 phosphoacetylation and promoter enrichment of AP-1 Fra-1/c-Jun and NF-kappaB p65 transcription factors and MSK1, CBP/p300, Brg1, and Ezh2 cofactors. Complementation, silencing, and kinase inhibitor experiments further demonstrate involvement of AP-1 Fra-1/c-Jun and NF-kappaB p65/RelB members, but not of the alpha estrogen receptor in promoting chromatin accessibility and transcription across the IL-6 gene promoter in metastatic breast cancer cells. Finally, the natural withanolide Withaferin A was found to repress IL-6 gene transcription in metastatic breast cancer cells upon dual inhibition of NF-kappaB and AP-1 Fra-1 transcription factors and silencing of IL-6 promoter chromatin accessibility.
Controlled expression of cytokine genes is an essential component of an immune response and is crucial for homeostasis. In order to generate an appropriate response to an infectious condition, the type of cytokine, as well as the cell type, dose range and the kinetics of its expression are of critical importance. The nuclear factor-kappaB (NF-kappaB) family of transcription factors has a crucial role in rapid responses to stress and pathogens (innate immunity), as well as in development and differentiation of immune cells (acquired immunity). Although quite a number of genes contain NF-kappaB-responsive elements in their regulatory regions, their expression pattern can significantly vary from both a kinetic and quantitative point of view, reflecting the impact of environmental and differentiative cues. At the transcription level, selectivity is conferred by the expression of specific NF-kappaB subunits and their respective posttranslational modifications, and by combinatorial interactions between NF-kappaB and other transcription factors and coactivators, that form specific enhanceosome complexes in association with particular promoters. These enhanceosome complexes represent another level of signaling integration, whereby the activities of multiple upstream pathways converge to impress a distinct pattern of gene expression upon the NF-kappaB-dependent transcriptional network. Today, several pieces of evidence suggest that the chromatin structure and epigenetic settings are the ultimate integration sites of both environmental and differentiative inputs, determining proper expression of each NF-kappaB-dependent gene. We will therefore discuss in this review the multilayered interplay of NF-kappaB signaling and epigenome dynamics, in achieving appropriate gene expression responses and transcriptional activity.
Understanding the transcriptional regulation of an important class of innate and adaptive immune system effector molecules, the cytokines, is increasingly important given the promise cytokine regulation holds for treating various autoimmune and inflammatory diseases. Studies defining the mechanisms regulating cytokine transcription initially focused on identifying the cis-acting elements and trans-acting factors that activate cytokine promoters and enhancers. In the past, these studies were largely completed in the absence of constraints instituted by cellular chromatin. Over the past decade it has become obvious that changes in chromatin accessibility critically control, rather than simply correlate with, the transcriptional activation of most genes, including cytokines. Hence candidate transcriptional activators are being re-evaluated for potency in the context of cellular chromatin. Several distinct mechanisms for manipulating the generally repressive context of chromatin have been identified for cytokine genes. Most recently, single nucleotide polymorphisms in cytokine transcriptional regulatory elements have been shown to play measurable roles in regulating cytokine levels in the context of naturally selected haplotypes. Overall, subtle differences in DNA sequence and nucleoprotein complex composition, including protein post-translational modification, come together in cell type-specific combinations to explain the normal variation in cytokine transcription throughout the human populace.
The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.
We describe a novel biphasic regulation of Il2 transcription in naive CD4(+) T cells. Few ( approximately 5%) CD4(+) T cells transcribe Il2 within 6 h of anti-TCR-beta plus anti-CD28 stimulation (early phase). Most naive CD4(+) T cells do not initiate Il2 transcription until after an additional approximately 12 h of T cell stimulation (late phase). In comparison, essentially all previously activated (Pre-Ac) CD4(+) T cells that transcribe Il2 do so with an early-phase response. Late-phase Il2 expression mostly requires c-Rel, CD28, and TNFR signaling. In contrast, early-phase transcription is only partly c-Rel and CD28 dependent and TNFR independent. There was also increased stable DNA accessibility at the Il2 locus and elevated c-Rel expression in resting Pre-Ac CD4(+) cells. Upon T cell activation, a faster and greater increase in DNA accessibility as well as c-Rel nuclear expression were observed in Pre-Ac CD4(+) cells relative to naive CD4(+) T cells. In addition, both acetylated histone H3 and total H3 decreased at the Il2 locus upon rechallenge of Pre-Ac CD4(+) T cells, whereas increased acetylated histone H3 with no change in total H3 was observed following activation of naive CD4(+) T cells. We propose a model in which nucleosome disassembly facilitates rapid initiation of Il2 transcription in CD4(+) T cells, and suggest that a threshold level of c-Rel must be reached for Il2 promoter activity in both naive and Pre-Ac CD4(+) T cells. This is provided, at least partially, by TNFR signaling during priming, but not during recall.
The GM-CSF gene is expressed following activation of T cells. The proximal promoter and an upstream enhancer have previously been characterized using transfection and reporter assays in T cell lines in culture. A 10.5-kb transgene containing the entire human GM-CSF gene has also been shown to display inducible, position-independent, copy number-dependent transcription in mouse splenocytes. To determine the role of individual promoter elements in transgene function, mutations were introduced into the proximal promoter and activity assessed following the generation of transgenic mice. Of four mutations introduced into the transgene promoter, only one, in an NF-kappaB/Sp1 region, led to decreased induction of the transgene in splenocytes or bone marrow-derived macrophages. This mutation also affected the activity of reporter gene constructs stably transfected into T cell lines in culture, but not when transiently transfected into the same cell lines. The mutation alters the NF-kappaB family members that bind to the NF-kappaB site as well as reducing the binding of Sp1 to an adjacent element. A DNase I hypersensitive site that is normally generated at the promoter following T cell activation on the wild-type transgene does not appear in the mutant transgene. These results suggest that the NF-kappaB/Sp1 region plays a critical role in chromatin remodeling and transcription on the GM-CSF promoter in primary T cells.
T-cell activation results in the production of multiple lymphokines. Efficient lymphokine gene expression appears to require both T-cell antigen receptor (TCR) signal transduction and an uncharacterized second or costimulatory signal. CD28 is a T-cell differentiation antigen that can generate intracellular signals that synergize with those of the TCR to increase T-cell activation and interleukin-2 (IL-2) gene expression. In these studies, we have examined the effect of CD28 signal transduction on granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and gamma interferon (IFN-gamma) promoter activity. Stimulation of CD28 in the presence of TCR-like signals increases the activity of the GM-CSF, IL-3, and IFN-gamma promoters by three- to sixfold. As previously demonstrated for the IL-2 promoter, the IL-3 and GM-CSF promoters contain distinct elements of similar sequence which specifically bind a CD28-induced nuclear complex. Mutation of the CD28 response elements in the IL-3 and GM-CSF promoters abrogates the CD28-induced activity without affecting phorbol ester- and calcium ionophore-induced activity. UV cross-linking indicates that the CD28-induced nuclear complex contains polypeptides of approximately 35, 36, and 44 kDa. These studies indicate that the TCR and CD28-regulated signal transduction pathways coordinately regulate the transcription of several lymphokines and that the influence of CD28 signals on transcription is mediated by a common complex.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) are pleiotropic hemopoietic growth factors whose genes are closely linked and induced in T lymphocytes in a cyclosporin A (CsA)-sensitive fashion. Since we found that the human GM-CSF and IL-3 proximal promoters were not sufficient to account for the observed regulation of these genes, we mapped DNase I hypersensitive sites across the GM-CSF/IL-3 locus in the Jurkat human T-cell line to identify additional regulatory elements. We located an inducible DNase I hypersensitive site, 3 kb upstream of the GM-CSF gene, that functioned as a strong CsA-sensitive enhancer of both the GM-CSF and IL-3 promoters. Binding studies employing Jurkat cell nuclear extracts indicated that four sites within the enhancer associate with the inducible transcription factor AP1. Three of these AP1 elements lie within sequences that also associate with factors resembling the CsA-sensitive, T cell-specific transcription factor NFAT. We provide additional evidence suggesting that an AP1-like factor represents one of the components of NFAT. We propose that the intergenic enhancer described here is required for the correctly regulated activation of both GM-CSF and IL-3 gene expression in T cells and that it mediates the CsA sensitivity of the GM-CSF/IL-3 locus.
The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.
Activation of helper T cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GM-CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFkappaB, AP1 and ETS-like binding motifs have been identified in the promoter region of the GM-CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GM-CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GM-CSF reporter construct in unstimulated Jurkat cells, providing that either NFkappaB or AP1 transcription factors are supplied by co-transfection. We confirm that binding of endogenous NFkappaB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFkappaB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are interdependent. Our results suggest that constitutive ETS1, and inducible NFkappaB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells.
Known transcription factor-DNA interactions in the minimal enhancer of the murine interleukin-2 gene (IL-2) do not easily explain the T cell specificity of IL-2 regulation. To seek additional determinants of cell type specificity, in vivo methodologies were employed to examine chromatin structure 5′ and 3′ of the 300 bp IL-2 proximal promoter/enhancer region. Restriction enzyme accessibility revealed that until stimulation the IL-2 proximal promoter/enhancer exists in a closed conformation in resting T and non-T cells alike. Within this promoter region,
DMS and DNase I genomic footprinting also showed no tissue-specific differences prior to stimulation. However, DNase I footprinting
of the distal −600 to −300 bp region revealed multiple tissue-specific and stimulation-independent DNase I hypersensitive
sites. Gel shift assays detected T cell-specific complexes binding within this region, which include TCF/LEF or HMG family
and probable Oct family components. Upon stimulation, new DNase I hypersensitive sites appeared in both the proximal and distal
enhancer regions, implying that there may be a functional interaction between these two domains. These studies indicate that
a region outside the established IL-2 minimal enhancer may serve as a stable nucleation site for tissue-specific factors and as a potential initiation site for
activation-dependent chromatin remodeling.
The nucleosome, which is the primary building block of chromatin, is not a static structure: It can adopt alternative conformations. Changes in solution conditions or changes in histone acetylation state cause nucleosomes and nucleosomal arrays to behave with altered biophysical properties. Distinct subpopulations of nucleosomes isolated from cells have chromatographic properties and nuclease sensitivity different from those of bulk nucleosomes. Recently, proteins that were initially identified as necessary for transcriptional regulation have been shown to alter nucleosomal structure. These proteins are found in three types of multiprotein complexes that can acetylate nucleosomes, deacetylate nucleosomes, or alter nucleosome structure in an ATP-dependent manner. The direct modification of nucleosome structure by these complexes is likely to play a central role in appropriate regulation of eukaryotic genes.
The SWI/SNF complex is a 2 MDa multi-subunit DNA-dependent ATPase that contributes to the regulation of gene transcription by altering chromatin structure. Recent studies have revealed that the SWI/SNF complex is targeted to promoters via direct interactions with transcription activators and have provided insights into mechanisms by which the complex alters nucleosome structure and contributes to the remodeling of chromatin.
The structure of chromatin and its remodeling following activation are important aspects of the control of inducible gene transcription. The IL-2 gene is induced in a cell specific-manner in T cells following an antigenic stimulus. We show, using a novel real-time PCR assay, that significant chromatin remodeling of the IL-2 proximal promoter region occurred upon stimulation of both the murine EL-4 T cell line and primary CD4(+) T cells. Chromatin remodeling appears to be limited to the first 300 bp of the proximal promoter region as measured by micrococcal nuclease and restriction enzyme accessibility. Time course studies indicated that chromatin remodeling was observed at 1.5 h postinduction and was maintained for up to 16 h. The remodeling is reversible upon removal of the stimulus. The region immediately upstream from the transcription start site, however, remains accessible for up to 16 h. Upon restimulation, remodeling occurs much more rapidly, consistent with a more rapid rise in IL-2 mRNA levels. Using a number of pharmacological inhibitors we show that remodeling is dependent on the presence of specific transcription factors, but not on the modification of histones. The development of this novel chromatin accessibility assay based on real-time PCR has allowed rapid, sensitive, and quantitative measurements on the IL-2 gene following cellular activation in both T cell lines and primary cells.
Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a key cytokine in myelopoiesis and aberrant expression is associated with chronic inflammatory disease and myeloid leukemias. This aberrant expression is often associated with constitutive nuclear factor (NF)-kappaB activation. To investigate the relationship between NF-kappaB and GM-CSF transcription in a chromatin context, we analyzed the chromatin structure of the GM-CSF gene in T cells and the role of NF-kappaB proteins in chromatin remodeling. We show here that chromatin remodeling occurs across a region of the GM-CSF gene between -174 and +24 upon T cell activation, suggesting that remodeling is limited to a single nucleosome encompassing the proximal promoter. Nuclear NF-kappaB levels appear to play a critical role in this process. In addition, using an immobilized template assay we found that the ATPase component of the SWI/SNF chromatin remodeling complex, brg1, is recruited to the GM-CSF proximal promoter in an NF-kappaB-dependent manner in vitro. These results suggest that chromatin remodeling across the GM-CSF promoter in T cells is a result of recruitment of SWI/SNF type remodeling complexes by NF-kappaB proteins binding to the CD28 response region of the promoter.
Histone modification is emerging as a major regulatory mechanism for modulating gene expression by altering the accessibility of transcription factors to DNA. This study unravels the relationship between histone H3 modifications and LDL receptor induction, focusing also on routes by which phosphorylation is mediated in human hepatoma HepG2 cells. We show that while histone H3 is constitutively acetylated at LDL receptor chromatin, 12-O -tetradecanoylphorbol-13-acetate (TPA) causes rapid hyperphosphorylation of histone H3 on serine 10 (histone H3-Ser10), despite global reduction in its phosphorylation levels. Ser10 hyperphosphorylation precedes LDL receptor induction and is independent of the p42/44MAPK, p38MAPK, pp90RSK, or MSK-1 cascade. Interestingly, inhibition of protein kinase C (PKC) blocks Ser10 hyperphosphorylation and also compromises LDL receptor induction by TPA. Consistent with its role, recombinant purified PKC phosphorylate purified histone H3-Ser10.
Collectively, our findings highlight a novel role for PKC in regulating histone H3-Ser10 phosphorylation and suggest that histone modification provides numerous regulatory opportunities to set the overall range of control attainable for LDL receptor gene induction.
Nature Reviews Immunology 2, 725–734 (2002) On page 728 of this article, the following incorrect sentence was published: 'The complex of transforming-growth-factor-β-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1) and TAB2 was thought to be an integral component that relays the signals to IKK complexes downstream of TRAF6 (REF.
Although the immediate receptors (immunophilins) of the immunosuppressants cyclosporin A (CsA) and FK506 are distinct, their similar mechanisms of inhibition of cell signaling suggest that their associated immunophilin complexes interact with a common target. We report here that the complexes cyclophilin-CsA and FKBP-FK506 (but not cyclophilin, FKBP, FKBP-rapamycin, or FKBP-506BD) competitively bind to and inhibit the Ca(2+)- and calmodulin-dependent phosphatase calcineurin, although the binding and inhibition of calcineurin do not require calmodulin. These results suggest that calcineurin is involved in a common step associated with T cell receptor and IgE receptor signaling pathways and that cyclophilin and FKBP mediate the actions of CsA and FK506, respectively, by forming drug-dependent complexes with and altering the activity of calcineurin-calmodulin.
The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.
Granulocyte-macrophage (GM)-CSF and IL-3 are hemopoietic growth factors whose genes are closely linked in both humans and mice. In humans, the GM-CSF and IL-3 genes are regulated by a cyclosporin A-inhibitable enhancer located 3 kb upstream of the GM-CSF gene that is inducible by signals that mimic TCR activation. To search for a murine homologue of this enhancer we probed mouse genomic DNA and located a 400-bp element 2 kb upstream of the mouse GM-CSF gene that was 76% homologous with the human GM-CSF enhancer. Like the human GM-CSF enhancer, this element formed a cyclosporin A-inhibitable DNase I-hypersensitive site in the murine T cell line EL4 upon activation with phorbol ester and calcium ionophore. Transient transfection assays showed that this homologue of the human enhancer acted as an inducible enhancer of the thymidine kinase promoter, the mouse IL-3 promoter, and the human GM-CSF promoter. We observed, however, that the mouse GM-CSF promoter was significantly more active than the human GM-CSF promoter and found that it supported a level of activity equivalent to the combination of the human GM-CSF promoter and the human GM-CSF enhancer. Consequently, the activity of mouse GM-CSF promoter was not significantly elevated in the presence of the mouse GM-CSF enhancer. Because the mouse GM-CSF enhancer is considerably less active than its human homologue we suggest that the mouse GM-CSF gene has evolved with less dependence upon the upstream enhancer for its activation.
The human granulocyte-macrophage CSF (GM-CSF) gene is expressed in T cells in response to TCR activation that can be mimicked by treatment of the cells with PMA and Ca2+ ionophore. The gene contains a proximal functional promoter region (-620 to +34), as well as a powerful enhancer located 3 kb upstream, both of which are involved in the response of the gene to TCR activation. The proximal promoter contains a region termed CLEO (-54 to -31) that consists of a purine-rich element abutting an activator protein-1 (AP-1)-like site, as well as an upstream nuclear factor-kappa B (NF-kappa B) site (-85 to -76) and a CK-1 element (-101 to -92). We show in this work that mutations in either the purine-rich region of the CLEO element or the NF-kappa B site result in reduced PMA/Ca2+ activation of a 620-bp human GM-CSF promoter-luciferase reporter construct in Jurkat T cells by 65% and 50%, respectively. The major inducible protein complex that binds to the human CLEO (hCLEO) element is an AP-1-like complex that is inducible by PMA alone, but shows increased binding in response to PMA together with Ca2+ ionophore. Although the binding of this complex is not cyclosporin-sensitive, promoter induction is inhibited by cyclosporin treatment. A second weak inducible complex resembling nuclear factor of activated T cells (NF-AT) was also observed binding to the hCLEO region. By using recombinant proteins, we confirmed that AP-1, NF-ATp, and a higher order NF-ATp/AP-1 complex could all form with the hCLEO element, and we have also defined the sequence requirements for binding of each of these complexes. We found that expression of a constitutively active form of calcineurin could substitute for Ca2+ ionophore and synergize with PMA to activate the GM-CSF promoter, and conversely that mutant-activated Ras could substitute for PMA and cooperate with Ca2+ ionophore. Co-expression of Ras and calcineurin, however, did not activate the GM-CSF promoter, but required the additional expression of NF-kappa B p65. These results imply that at least three signals are required to activate the GM-CSF proximal promoter, and that the signals impinge on distinct transcription factors that bind to the hCLEO and NF-kappa B regions of the promoter.
Antigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The granulocyte-macrophage colony-stimulating factor requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (IL-2) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the CD28 cell surface molecule on T cells, lead to activation through a distinct region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the NF-kappa B family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase GM-CSF expression. We have found that the HTLV-1 transactivator protein, tax, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the GM-CSF promoter through the CK-1/CD28RE region and also activates nuclear factor-kappa B binding to this region. However, other transcription factors or coactivators of NF-kappa B are required for tax activation but these remain to be identified. The CK-1/CD28RE of GM-CSF shows a high degree of similarity to the IL-2 CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both GM-CSF and IL-2 respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells.
NF-kappa B is a ubiquitous transcription factor. Nevertheless, its properties seem to be most extensively exploited in cells of the immune system. Among these properties are NF-kappa B's rapid posttranslational activation in response to many pathogenic signals, its direct participation in cytoplasmic/nuclear signaling, and its potency to activate transcription of a great variety of genes encoding immunologically relevant proteins. In vertebrates, five distinct DNA binding subunits are currently known which might extensively heterodimerize, thereby forming complexes with distinct transcriptional activity, DNA sequence specificity, and cell type- and cell stage-specific distribution. The activity of DNA binding NF-kappa B dimers is tightly controlled by accessory proteins called I kappa B subunits of which there are also five different species currently known in vertebrates. I kappa B proteins inhibit DNA binding and prevent nuclear uptake of NF-kappa B complexes. An exception is the Bcl-3 protein which in addition can function as a transcription activating subunit in th nucleus. Other I kappa B proteins are rather involved in terminating NF-kappa B's activity in the nucleus. The intracellular events that lead to the inactivation of I kappa B, i.e. the activation of NF-kappa B, are complex. They involve phosphorylation and proteolytic reactions and seem to be controlled by the cells' redox status. Interference with the activation or activity of NF-kappa B may be beneficial in suppressing toxic/septic shock, graft-vs-host reactions, acute inflammatory reactions, acute phase response, and radiation damage. The inhibition of NF-kappa B activation by antioxidants and specific protease inhibitors may provide a pharmacological basis for interfering with these acute processes.
CD28 response elements (CD28REs) within cytokine promoters are variant NF-kappaB-binding sites and are essential for transcription in response to CD28 receptor activation in T cells. We show that the CK-1 element (CD28RE) within the GM-CSF promoter binds the RelA and c-Rel transcription factors in response to CD28 activation. We further show that the high mobility group protein HMG I(Y) can bind to the CD28REs of both GM-CSF and IL-2 and that this binding is critical for c-Rel, but not RelA, binding. A second NF-kappaB site in the GM-CSF promoter that binds p50 and RelA, but neither c-Rel nor HMG I(Y), failed to respond to CD28 activation. Expression of HMG I or c-Rel antisense RNA inhibited CD28 activation of the IL-2 and GM-CSF promoters, implying that HMG I(Y) enhancement of c-Rel binding plays an important role in the activity of the CD28REs.
The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo.
Activation of the Saccharomyces cerevisiae PHO5 gene is accompanied by the disruption of four positioned nucleosomes at the promoter. The chromatin transition requires a DNA-binding protein, Pho4, and its transactivation domain. The mechanism of nucleosome disruption and the contribution of the nucleosomes to PHO5 regulation are reviewed.
As targets for the immunosuppressive drugs cyclosporin A and FK506, transcription factors of the NFAT (nuclear factor of activated T cells) family have been the focus of much attention. NFAT proteins, which are expressed in most immune-system cells, play a pivotal role in the transcription of cytokine genes and other genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a primary target for inhibition by cyclosporin A and FK506. Calcineurin controls the translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells by interacting with an N-terminal regulatory domain conserved in the NFAT family. The DNA-binding domains of NFAT proteins resemble those of Rel-family proteins, and Rel and NFAT proteins show some overlap in their ability to bind to certain regulatory elements in cytokine genes. NFAT is also notable for its ability to bind cooperatively with transcription factors of the AP-1 (Fos/Jun) family to composite NFAT:AP-1 sites, found in the regulatory regions of many genes that are inducibly transcribed by immune-system cells. This review discusses recent data on the diversity of the NFAT family of transcription factors, the regulation of NFAT proteins within cells, and the cooperation of NFAT proteins with other transcription factors to regulate the expression of inducible genes.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the many cytokines produced following T-cell activation. It is also produced in a variety of other cell types, in particular following activation by inflammatory mediators. Changes in the rate of transcription are important in the control of GM-CSF expression in T cells and in fibroblasts and endothelial cells. The GM-CSF gene contains two distinct transcriptional control regions. These are the proximal promoter consisting of the first 120 bp from the transcription start site and an enhancer located approximately 3 kb upstream from the proximal promoter. Distinct regions of the proximal promoter respond to a wide array of signals such as phorbol myristate acetate (PMA) and Ca2+ ionophore or phytohemaglutinin (PHA), CD28 activation, human T leukemia virus (HTLV)-1 tax, TNF, and interleukin 1 (IL-1). The transcription factors that mediate these responses have mainly been defined, with the major inducible proteins being the NF-kappa B/rel and AP-I families of transcription factors. In contrast to the promoter, the enhancer responds only to PMA and Ca2+ ionophore signals and binds NFAT/AP-1 complexes that appear to mediate its function.
Perhaps the best characterized example of an activator-induced chromatin transition is found in the activation of the
Saccharomyces cerevisiae
acid phosphatase gene
PHO5
by the basic helix-loop-helix (bHLH) transcription factor Pho4. Transcription activation of the
PHO5
promoter by Pho4 is accompanied by the remodeling of four positioned nucleosomes which is dependent on the Pho4 activation domain but independent of transcription initiation. Whether the requirements for transcription activation through the TATA sequence are different from those necessary for the chromatin transition remains a major outstanding question. In an attempt to understand better the ability of Pho4 to activate transcription and to remodel chromatin, we have initiated a detailed characterization of the Pho4 activation domain. Using both deletion and point mutational analysis, we have defined residues between positions 75 and 99 as being both essential and sufficient to mediate transcription activation. Significantly, there is a marked concordance between the ability of mutations in the Pho4 activation domain to induce chromatin opening and transcription activation. Interestingly, the requirements for transcription activation within the Pho4 activation domain differ significantly if fused to a heterologous bHLH-leucine zipper DNA-binding domain. The implications for transcription activation by Pho4 are discussed.
Nucleosomes are important for gene regulation, but comprehensive studies of nucleosome positioning, remodeling, and transcription factor binding at inducible mammalian promoters have not been reported. We have analyzed the IL-12 p40 promoter, which is induced in macrophages by bacterial products. High-resolution micrococcal nuclease analyses revealed that a positioned nucleosome, nucleosome 1, spans the promoter, with three positioned nucleosomes further upstream. Upon activation, nucleosome 1 was rapidly and selectively remodeled in a protein synthesis-dependent manner. In primary macrophages, IFNgamma synergistically enhanced p40 expression, but little effect on remodeling or promoter occupancy was observed. These results suggest that remodeling complexes are selectively targeted to a single, promoter-encompassing nucleosome and that IFNgamma influences an event that is independent or downstream of remodeling.
Here, we show that the IFN-beta enhanceosome activates transcription by directing the ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. The enhanceosome is assembled in the nucleosome-free enhancer region of the IFN-beta gene, leading to the modification and remodeling of a strategically positioned nucleosome that masks the TATA box and the start site of transcription. Initially, the GCN5 complex is recruited, which acetylates the nucleosome, and this is followed by recruitment of the CBP-PolII holoenzyme complex. Nucleosome acetylation in turn facilitates SWI/SNF recruitment by CBP, resulting in chromatin remodeling. This program of recruitment culminates in the binding of TFIID to the promoter and the activation of transcription.
Lipopolysaccharide (LPS) induction of the gene encoding interleukin 12 p40 requires remodeling of a promoter-encompassing nucleosome and the Toll-like receptor (TLR)-mediated activation of a c-Rel-containing complex. Analysis of TLR4-mutant mice revealed that remodeling requires TLR signaling. However, Rel proteins and other proteins required for transcription of an integrated p40 promoter were insufficient for remodeling. c-Rel was also unnecessary for remodeling, as remodeling was observed in c-Rel-/- macrophages, which lack p40 transcripts. These results suggest that remodeling requires TLR signaling pathways that diverge from the c-Rel activation pathways. The factors that stimulate remodeling may represent, therefore, newly identified targets of TLR signaling and of agents that regulate inflammatory responses and TH1 development.
Here, we show that a nucleosome obstructing transcription from the IFN-beta promoter slides in vivo in response to virus infection, thus exposing the previously masked TATA box and the initiation site, a requirement for transcriptional activation. Our experiments also revealed that this mode of chromatin remodeling is a two-step reaction. First, the enhanceosome recruits the SWI/SNF chromatin-remodeling complex that modifies the nucleosome to allow binding of TBP. Second, DNA bending is induced by TBP binding, and the nucleosome slides to a new position. Experiments with other DNA binding proteins demonstrated a strong correlation between the ability to bend DNA and nucleosome sliding, suggesting that the sliding is induced by the bend.
The controlled expression of cytokine genes is an essential component of an immune response. The specific types of cytokines as well as the time and place of their production is important in generating an appropriate immune response to an infectious agent. Aberrant expression is associated with pathological conditions of the immune system such as autoimmunity, atopy and chronic inflammation. Cytokine gene transcription is generally induced in a cell-specific manner. Over the last 15 years, a large amount of information has been generated describing the transcriptional controls that are exerted on cytokine genes. Recently, efforts have been directed at understanding how these genes are transcribed in a chromatin context. This review will discuss the mechanisms by which cytokine genes become available for transcription in a cell-restricted manner as well as the mechanisms by which these genes sense their environment and activate high level transcription in a transient manner. Particular attention will be paid to the role of chromatin in allowing transcription factor access to appropriate genes.
It has been a long-standing challenge to decipher the principles that enable cells to both organize their genomes into compact chromatin and ensure that the genetic information remains accessible to regulatory factors and enzymes within the confines of the nucleus. The discovery of nucleosome remodeling activities that utilize the energy of ATP to render nucleosomal DNA accessible has been a great leap forward. In vitro, these enzymes weaken the tight wrapping of DNA around the histone octamers, thereby facilitating the sliding of histone octamers to neighboring DNA segments, their displacement to unlinked DNA, and the accumulation of patches of accessible DNA on the surface of nucleosomes. It is presumed that the collective action of these enzymes endows chromatin with dynamic properties that govern all nuclear functions dealing with chromatin as a substrate. The diverse set of ATPases that qualify as the molecular motors of the nucleosome remodeling process have a common history and are part of a superfamily. The physiological context of their remodeling action builds on the association with a wide range of other proteins to form distinct complexes for nucleosome remodeling. This review summarizes the recent progress in our understanding of the mechanisms underlying the nucleosome remodeling reaction, the targeting of remodeling machines to selected sites in chromatin, and their integration into complex regulatory schemes.
Transcriptional activation of the IFN-beta gene in response to virus infection requires the assembly of an enhanceosome, which instructs a recruitment program of chromatin modifiers/remodelers and general transcription factors to the promoter. This program culminates with sliding of a nucleosome blocking the core promoter to a downstream position, a prerequisite for transcriptional activation. We show that delivery of this nucleosome to the same downstream position to create an accessible IFN-beta core promoter prior to enhanceosome assembly results in major changes in the gene expression program with regard to the temporal pattern and the signal specificity of the transcriptional response. Thus, the identity of a gene expression program is achieved and maintained by the dynamic interplay between specific enhanceosomes and specific local chromatin structure.
The nuclear factor-kappaB (NF-kappaB)/REL family of transcription factors has a central role in coordinating the expression of a wide variety of genes that control immune responses. There has been intense scientific activity in the NF-kappaB field owing to the involvement of these factors in the activation and regulation of key molecules that are associated with diseases ranging from inflammation to cancer. In this review, we focus on our current understanding of NF-kappaB regulation and its role in the immune system and inflammatory diseases. We also discuss the role of NF-kappaB proteins as potential therapeutic targets in clinical applications.
IL-2 gene transcription occurs in an activation-dependent manner in T cells responding to TCR and CD28 activation. One of the critical events leading to increased IL-2 transcription is an alteration in chromatin structure across the 300-bp promoter region of the gene. We initially showed that IL-2 gene transcription in CD4(+) primary T cells is dependent on the NF-kappaB family member, c-Rel, but not RelA. We found that c-Rel is essential for global changes in chromatin structure across the 300-bp IL-2 promoter in response to CD3/CD28 in primary CD4(+) T cells, but not in response to pharmacological signals, paralleling the requirement for c-Rel in IL-2 mRNA and protein accumulation. Interestingly, measurement of activation-induced localized accessibility changes using restriction enzyme digestion revealed that accessibility close to the c-Rel binding site in the CD28RR region of the promoter is specifically dependent on c-Rel. In contrast, restriction enzyme sites located at a distance from the CD28RR behave independently of c-Rel. These results suggest a nonredundant role for c-Rel in generating a correctly remodeled chromatin state across the IL-2 promoter and imply that the strength of the signal determines the requirement for c-Rel.
Rapid detectionofoctamerbindingproteinswith‘mini-extracts’,preparedfrom a small number of cells
- E Schreiber
- P Matthias
- M M Muller
- W Schaffner
Schreiber,E., Matthias,P., Muller,M.M. and Schaffner,W. (1989) Rapid detectionofoctamerbindingproteinswith‘mini-extracts’,preparedfrom a small number of cells. Nucleic Acids Res., 17, 6419