An ordered array of cold shock domain repressor elements across tumor necrosis factor-responsive elements of the granulocyte-macrophage colony-stimulating factor promoter

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-kappa B, 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.