Abstract
The tumor necrosis factor-alpha-responsive region of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter (-114 to -31) encompasses binding sites for NF-kappaB, CBF, AP-1, ETS, and NFAT families of transcription factors. We show both here and previously that mutation of any one of these binding sites greatly reduces tumor necrosis factor-alpha induction of the GM-CSF promoter. Interspersed between these elements are sequences that when mutated lead to an increase in GM-CSF promoter activity. We have previously shown that two of these repressor elements bind proteins known as cold shock domain (CSD) factors and that overexpression of CSD proteins leads to repression of GM-CSF promoter activity in fibroblasts. CSD proteins are single strand DNA- and RNA-binding proteins that contact 5'-CCTG-3' sequences in the GM-CSF repressor elements. We show here that two newly identified repressor sequences in the proximal promoter can also bind CSD proteins. We have characterized the CSD-containing protein complexes that bind to the GM-CSF promoter and identified a novel protein related to mitochondrial single strand binding protein that forms part of one of these complexes. The four CSD-binding sites on the promoter occur in pairs on opposite strands of the DNA and appear to form an ordered array of binding elements. A similar ordered array of CSD sites are present in the promoters of the granulocyte colony-stimulating factor and interleukin-3 genes, implying a common mechanism for negative regulation of these myeloid growth factors.
Highlights
The tumor necrosis factor-␣-responsive region of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter (؊114 to ؊31) encompasses binding sites for NF-B, CBF, AP-1, ETS, and NFAT families of transcription factors
We have previously shown that two of these repressor elements bind proteins known as cold shock domain (CSD) factors and that overexpression of CSD proteins leads to repression of GM-CSF promoter activity in fibroblasts
Identification of Overlapping TNF-responsive Elements and Repressor Elements in the GM-CSF Domain 2 Region—We previously reported that a domain 2 reporter construct was responsive to TNF-␣ in fibroblasts and that this activity was repressed by overexpression of the CSD proteins, DbpB and DbpA, that were cloned as GM-CSF domain 1 repressor site-binding proteins [14]
Summary
Plasmid Constructs —The human GM-CSF promoter constructs pGM41 and pGM43 have previously been described and were constructed by cloning the oligonucleotides GM41 (Ϫ65 to Ϫ31) and GM43 (Ϫ114 to Ϫ31), respectively, into the pBLCAT2 reporter vector [13]. Single strand DNA probes for gel retardation assays were prepared by end-labeling coding (ϩ) or noncoding (Ϫ) strand oligonucleotides with [␥-32P]ATP and T4 polynucleotide kinase followed by gel purification. Gel Retardation Analysis and UV Cross-linking—Gel retardation assays were performed using 0.25 ng of single strand 32P-labeled oligonucleotide probe in a 10-l reaction mix of 0.5ϫ TM buffer [13, 14, 22] containing 200 mM KCl, 0.4 g of poly(dI-dC) and either 0.2 g of HS-enriched extract (HSGMa or HSGMb), 1.0 g of crude nuclear extract, 25 ng of recombinant CSD fusion protein (GST-DbpB), or 1 ng of affinity purified material. For UV cross-linking, crude nuclear extracts were bound to 32Plabeled single strand DNA probes in a 25-l retardation reaction and fractionated on a polyacrylamide gel as described above. The percentage of [14C]chloramphenicol conversion to acetylated forms via CAT activity in extracts was determined using PhosphorImager analysis (Molecular Dynamics)
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