Characterization of the Human Granulocyte-Macrophage Colony-Stimulating Factor Promoter Region by Genetic Analysis: Correlation with DNase I Footprinting

Cytokine LD78 is a human counterpart of the mouse macrophage inflammatory protein 1 alpha/hematopoietic stem cell inhibitor. Promoters of the LD78 alpha and LD78 beta genes showed similar inducible activities in two leukemic cell lines, K562 and Jurkat, but the induction mechanisms differed between the two cell lines. Further characterization of the LD78 alpha promoter indicated that multiple positive and negative regulatory elements are present, some of which are differentially required for induction and repression of the promoter activity in different cells. One of the negative regulatory elements, ICK-1, functioned in both cell lines in the absence and presence of stimulation and was shown to be a recognition site for positive and negative transcriptional factors. This ICK-1 element contained a direct repeat, and similar repeats were also found in the negative regulatory elements of hematopoietic growth factor interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoters. Nuclear extracts from K562 and Jurkat cells formed several protein-DNA complexes with the LD78 alpha ICK-1 element, one of which was also observed with the IL-3 and GM-CSF ICK-1 elements. Results from in vivo and in vitro analyses suggested that the protein forming this complex functions as a negative factor. The binding affinity of this protein, ICK-1A, to the LD78 alpha ICK-1 element was low and was significantly affected by the incubation temperature and the salt concentration in the binding buffer. ICK-1B, another protein bound specifically by the LD78 alpha ICK-1 element, was shown to be a positive factor important for induction of the promoter. These results suggested that ICK-1A plays an important role in balanced expression of LD78, IL-3, and GM-CSF during hematopoietic cell growth and differentiation.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Characterization of Cytokine LD78 Gene Promoters: Positive and Negative Transcriptional Factors Bind to a Negative Regulatory Element Common to LD78, Interleukin-3, and Granulocyte-Macrophage Colony-Stimulating Factor Gene Promoters

Nomiyama

Hieshima

Hirokawa

et al. 1993

“…Combined with the data from the deletion and mutation analysis of the GM-CSF promoter (20,23), our data strongly Putative NF-KB-binding sites in the G (A) Schematic representation of a segment of th upstream promoter as described by Miyatake sequence motifs CLE 1, CLE 2, and the GC box; functional significance in inducible expression of are indicated. (B) Oligonucleotides used in this the sequences that are shown in alignment with ti double-stranded oligonucleotides contained Hind sequences on 5' and 3' ends, respectively.…”

mentioning

confidence: 66%

“…The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was previously shown to be activated by a TPA-phytohemagglutinin (PHA) or a TPA-calcium ionophore treatment mimicking T-cell activation and upon transient expression of tax1 (7,19,20,23). In a deletion and mutation analysis of the mouse GM-CSF promoter region, two short sequence elements between positions -113 and -73 upstream of the transcriptional start site that were sufficient to confer inducibility to the chloramphenicol transferase (CAT) gene were identified (20).…”

mentioning

confidence: 99%

NF-κΒ as Inducible Transcriptional Activator of the Granulocyte-Macrophage Colony-Stimulating Factor Gene

Schreck

Baeuerle

1990

The expression of the gene encoding the granulocyte-macrophage colony-stimulating factor (GM-CSF) is induced upon activation of T cells with phytohemagglutinin and active phorbolester and upon expression of tax1, a transactivating protein of the human T-cell leukemia virus type I. The same agents induce transcription from the interleukin-2 receptor alpha-chain and interleukin-2 genes, depending on promoter elements that bind the inducible transcription factor NF-kappa B (or an NF-kappa B-like factor). We therefore tested the possibility that the GM-CSF gene is also regulated by a cognate motif for the NF-kappa B transcription factor. A recent functional analysis by Miyatake et al. (S. Miyatake, M. Seiki, M. Yoshida, and K. Arai, Mol. Cell. Biol. 8:5581-5587, 1988) described a short promoter region in the GM-CSF gene that conferred strong inducibility by T-cell-activating signals and tax1, but no NF-kappa B-binding motifs were identified. Using electrophoretic mobility shift assays, we showed binding of purified human NF-kappa B and of the NF-kappa B activated in Jurkat T cells to an oligonucleotide comprising the GM-CSF promoter element responsible for mediating responsiveness to T-cell-activating signals and tax1. As shown by a methylation interference analysis and oligonucleotide competition experiments, purified NF-kappa B binds at positions -82 to -91 (GGGAACTACC) of the GM-CSF promoter sequence with an affinity similar to that with which it binds to the biologically functional kappa B motif in the beta interferon promoter (GGGAAATTCC). Two kappa B-like motifs at positions -98 to -108 of the GM-CSF promoter were also recognized but with much lower affinities. Our data provide strong evidence that the expression of the GM-CSF gene following T-cell activation is controlled by binding of the NF-kappa B transcription factor to a high-affinity binding site in the GM-CSF promoter.

“…Both PMA and A23187 are required to induce expression of GM-CSF; CsA or FK506 can completely inhibit this induction (4). After T-cell stimulation, GM-CSF mRNA can be detected in 2 to 4 h. The presence of this mRNA is largely due to transcription initiation (5,19,25) and requires the synthesis of specific factors, since cycloheximide (CHX), a protein synthesis inhibitor, blocks GM-CSF expression at the early stages of stimulation (27,29).…”

mentioning

confidence: 99%

The Granulocyte-Macrophage Colony-Stimulating Factor Promoter cis-Acting Element CLE0 Mediates Induction Signals in T Cells and is Recognized by Factors Related to AP1 and NFAT

Masuda

Tokumitsu

Tsuboi

et al. 1993

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.