Granulocyte-macrophage Colony-stimulating Factor Enhancer Research Articles

Runx1 binds as a dimeric complex to overlapping Runx1 sites within a palindromic element in the human GM-CSF enhancer

Runx1 is a developmentally regulated transcription factor that is essential for haemopoiesis. Runx1 can bind as a monomer to the core consensus sequence TGTGG, but binds more efficiently as a hetero-dimer together with the non-DNA binding protein CBFβ as a complex termed core binding factor (CBF). Here, we demonstrated that CBF can also assemble as a dimeric complex on two overlapping Runx1 sites within the palindromic sequence TGTGGCTGCCCACA in the human granulocyte macrophage colony-stimulating factor enhancer. Furthermore, we demonstrated that binding of Runx1 to the enhancer is rigidly controlled at the level of chromatin accessibility, and is dependent upon prior induction of NFAT and AP-1, which disrupt a positioned nucleosome in this region. We employed in vivo footprinting to demonstrate that, upon activation of the enhancer, both sites are efficiently occupied. In vitro binding assays confirmed that two CBF complexes can bind this site simultaneously, and transfection assays demonstrated that both sites contribute significantly to enhancer function. Computer modelling based on the Runx1/CBFβ/DNA crystal structure further revealed that two molecules of CBF could potentially bind to this class of palindromic sequence as a dimeric complex in a conformation whereby both Runx1 and CBFβ within the two CBF complexes are closely aligned.

Severe Food Allergy as a Variant of IPEX Syndrome Caused by a Deletion in a Noncoding Region of the FOXP3 Gene

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX; OMIM 304930) syndrome is a congenital syndrome characterized by autoimmune enteropathy, endocrinopathy, dermatitis, and other autoimmune phenomena. In the present work, we aimed to uncover the molecular basis of a distinct form of IPEX syndrome presenting at the edge of autoimmunity and severe allergy. The FOXP3 gene was sequenced, FOXP3 messenger RNA (mRNA) was quantified by real-time polymerase chain reaction (PCR), and protein expression in peripheral blood lymphocytes was analyzed by flow cytometry after intracellular staining. In coculture experiments (CD4(+)CD25(-) and CD4(+)CD25(+) cells), the functions of regulatory T cells were analyzed. Expression of interferon gamma and interleukin 2 and 4 mRNA within the inflamed intestinal mucosa was quantified by real-time PCR. Here, we describe a distinct familial form of IPEX syndrome that combines autoimmune and allergic manifestations including severe enteropathy, food allergies, atopic dermatitis, hyper-IgE, and eosinophilia. We have identified a 1388-base pair deletion (g.del-6247_-4859) of the FOXP3 gene encompassing a portion of an upstream noncoding exon (exon -1) and the adjacent intron (intron -1). This deletion impairs mRNA splicing, resulting in accumulation of unspliced pre-mRNA and alternatively spliced mRNA. This causes low FOXP3 mRNA levels and markedly decreased protein expression in peripheral blood lymphocytes of affected patients. Numbers of CD4(+)CD25(+)FOXP3(+) regulatory T cells are extremely low, and the CD4(+)CD25(+) T cells that are present exhibit little regulatory function. A new mutation within an upstream noncoding region of FOXP3 results in a variant of IPEX syndrome associating autoimmune and severe immunoallergic symptoms.

Granulocyte-macrophage colony-stimulating factor enhancer activation requires cooperation between NFAT and AP-1 elements and is associated with extensive nucleosome reorganization.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is activated by an NFAT-dependent enhancer forming an inducible DNase I hypersensitive (DH) site. The enhancer core comprising the DH site contains the GM330 and GM420 elements that bind NFAT and AP-1 cooperatively. Here we demonstrate that both elements are essential for enhancer activity and that Sp1 and AML1 sites in the enhancer become occupied in vivo only after activation. Chromatin structure analysis revealed that the GM-CSF enhancer core elements are divided between two adjacent nucleosomes that become destabilized and highly accessible after activation. Inducible chromatin reorganization was not restricted to the enhancer core but extended across a 3-kb domain of mobilized nucleosomes, within which the nucleosome repeat length was compressed from approximately 185 to 150 bp. The GM420 element is a high-affinity site that binds NFAT independently of AP-1 but depends on the linked AP-1 site for enhancer function. Nevertheless, just the NFAT motif from the GM420 element was sufficient to form a DH site within chromatin even in the absence of the AP-1 site. Hence, NFAT has the potential to cooperate with other transcription factors by promoting chromatin remodelling and increasing accessibility at inducible regulatory elements.

The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulatedin vivoby an inducible tissue-specific enhancer

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% +/- 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% +/- 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.

Familial Eosinophilia Maps to the Cytokine Gene Cluster on Human Chromosomal Region 5q31-q33

Familial eosinophilia (FE) is an autosomal dominant disorder characterized by peripheral hypereosinophilia of unidentifiable cause with or without other organ involvement. To localize the gene for FE, we performed a genomewide search in a large U.S. kindred, using 312 different polymorphic markers. Seventeen affected subjects, 28 unaffected bloodline relatives, and 8 spouses were genotyped. The initial linkage results from the genome scan provided evidence for linkage on chromosome 5q31-q33. Additional genotyping of genetic markers located in this specific region demonstrated significant evidence that the FE locus is situated between the chromosome 5q markers D5S642 and D5S816 (multipoint LOD score of 6.49). Notably, this region contains the cytokine gene cluster, which includes three genes-namely, those for interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF)-whose products play important roles in the development and proliferation of eosinophils. These three cytokine genes were screened for potential disease-specific mutations by resequencing of a subgroup of individuals from the present kindred. No functional sequence polymorphisms were found within the promoter, the exons, or the introns of any of these genes or within the IL-3/GM-CSF enhancer, suggesting that the primary defect in FE is not caused by a mutation in any one of these genes but, rather, is caused by another gene in the area.

Granulocyte-Macrophage Colony-stimulating Factor Gene Transcription Is Directly Repressed by the Vitamin D3Receptor: IMPLICATIONS FOR ALLOSTERIC INFLUENCES ON NUCLEAR RECEPTOR STRUCTURE AND FUNCTION BY A DNA ELEMENT

The primary function of activated T lymphocytes is to produce various cytokines necessary to elicit an immune response; these cytokines include interleukin-2 (IL-2), interleukin-4, and granulocyte-macrophage colony-stimulating factor (GMCSF). Steroid hormones and vitamin A and D3 metabolites act to repress the expression of cytokines. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) down-modulates activated IL-2 expression at the level transcription, through direct antagonism of the transactivating complex NFAT-1/AP-1 by the vitamin D3 receptor (VDR). We report here that GMCSF transcription in Jurkat T cells is also directly repressed by 1, 25-(OH)2D3 and VDR. Among four NFAT/AP-1 elements in the GMCSF enhancer, we have focused on one such element that when multimerized, is sufficient in mediating both activation by NFAT-1 and AP-1 and repression in response to 1,25-(OH)2D3. Although this element does not contain any recognizable vitamin D response elements (VDREs), high affinity DNA binding by recombinant VDR is observed. In contrast to VDR interactions with positive VDREs, this binding is independent of VDR's heterodimeric partner, the retinoid X receptor. Moreover, VDR appears to bind the GMCSF element as an apparent monomer in vitro. Protease digestion patterns of bound VDR, and receptor mutations affecting DNA binding and dimerization, demonstrate that the receptor binds to the negative site in a distinct conformation relative to a positive VDRE, suggesting that the DNA element itself acts as an allosteric effector of VDR function. This altered conformation may account for VDR's action as a repressing rather than activating factor at this locus.

Signals for activation of the GM-CSF promoter and enhancer in T cells.

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.

Regulation of Granulocyte-Macrophage Colony-Stimulating Factor and E-Selectin Expression in Endothelial Cells by Cyclosporin A and the T-Cell Transcription Factor NFAT

Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent.

Regulation of granulocyte-macrophage colony-stimulating factor and E- selectin expression in endothelial cells by cyclosporin A and the T- cell transcription factor NFAT

Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent.