A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter

Abstract

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.

An Ordered Array of Cold Shock Domain Repressor Elements across Tumor Necrosis Factor-responsive Elements of the Granulocyte-Macrophage Colony-stimulating Factor Promoter

“…Domain 1 (Ϫ114 to Ϫ71) contains the CK-1 and CK-2 elements conserved in a number of cytokine genes and binds a number of transcription factors including NF-kB, Sp1, and the CD28-responsive complex (1)(2)(3)(4). This region is responsive to T cell receptor activators and costimulators (10 -12), is involved in response to TNF-␣ in fibroblasts (13,14), and is required for constitutive expression in juvenile myelomonocytic leukemia cells (15). The NF-kB site is critical for expression in these cell types.…”

“…Domain 2 (Ϫ70 to Ϫ31) binds CBF, AP1, ETS, and NFAT transcription factors (10, 16 -18). This region responds to TNF-␣ and interleukin (IL)-1 stimulation of fibroblast (13,14) and endothelial (19) cells and T cell receptor activation (10, 16 -18) and is required for constitutive expression in certain acute myeloid leukemia cell lines (20). The CBF, AP1, and ETS/NFAT transcription factor-binding sites have been shown to be essential for T cell receptor signaling in conjunction with the domain 1 NF-kB site (10,16,18), but it is not known which of these sites in domain 2 are required for activation in fibroblasts and endothelial cells.…”

“…In addition to the activators described above, we have identified nuclear complexes called NF-GMa, NF-GMb, and NFGMc that bind to domain 1 of the GM-CSF promoter (13,14,21,22). The NF-GMa complex was found to also bind to the CK-1/ CK-2 equivalent regions of the genes for two other myeloid growth factors, granulocyte colony-stimulating factor (G-CSF) and IL-3 (23,24).…”

“…In contrast, the NF-GMb/c complexes are implicated in repression. These complexes bound to two repressor sites in domain 1 that were functional in fibroblasts (3,4,13,14). NF-GMb contains two separate complexes, one containing a 42-kDa protein and the other containing a dimer of a 22-kDa protein, whereas NF-GMc represents the binding of a single 22-kDa protein.…”

“…We identified these proteins as cold shock domain (CSD) proteins (4,(27)(28)(29)(30)(31)(32) by cloning of factors contacting the repressor elements and by subsequent analysis of the NFGMb/c complexes (4,14). An interesting property of these proteins is that they bind to single-stranded DNA and in the case of the GM-CSF sites, to two repeated 5Ј-CCTG-3Ј elements on the noncoding (Ϫ) strand of domain 1 (13,14). CSD factors are expressed in all cell types, and consistently we have observed NF-GMb/c complexes in all cell types examined, including fibroblasts, endothelial cells, T cells, and myeloid cells (13,14).…”