Characterization of nuclear factors that bind to a critical positive regulatory element of the human granulocyte-macrophage colony-stimulating factor promoter.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates the proliferation, maturation, and functional activity of myeloid cells in peripheral blood and bone marrow. Expression of GM-CSF is tightly regulated and is limited to cells stimulated directly (T cells, macrophages) or indirectly (fibroblasts, endothelial cells) by immune challenge. Several studies of the transcriptional control of GM-CSF expression have elucidated a region of the GM-CSF promoter that mediates positive regulatory activity in a number of cell types. This region contains a direct repeat of the sequence CATTA/T that extends from nucleotides -37 to -48 upstream of the start of mRNA synthesis. Although specific DNA:protein interactions have been shown within this region, neither the nature nor the number of nuclear factors responsible for these interactions have been characterized. In this study, we use DNase I footprinting analysis to demonstrate that point mutations, which inactivate the GM-CSF promoter, disrupt DNA:protein interactions within this region. By combined electrophoretic mobility shift and ultraviolet cross-linking analysis, we have detected several protein species that bind specifically to the positive regulatory sequence.
Fraser
J K
JK
Department of Medicine, UCLA.
Tran
S
S
Nimer
S D
SD
Gasson
J C
JC
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004586:Electrophoresis; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D010835:Phytohemagglutinins / Q000494:pharmacology; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D013601:T-Lymphocytes / Q000648:ultrastructure; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D010835:Phytohemagglutinins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D003850:Deoxyribonuclease I