T-cell nuclei contain a protein that binds upstream of the murine granulocyte-macrophage colony-stimulating factor gene.
Stimulation of a murine T-cell line (O18A) by Con A has been shown to cause a large increase in the cytoplasmic granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA level. Using run-on transcription experiments in isolated nuclei, we have shown that some of this response is from enhanced transcription of the GM-CSF gene. Changes in the transcription rate of this gene can be seen as early as 30 min after adding the Con A. With a DNA fragment mobility-shift assay on nuclear extracts from these cells we detected a protein that binds upstream of the murine GM-CSF gene. Partial purification and concentration of this protein by DEAE-Sephacel and phosphocellulose chromatography enabled us to examine its interaction with DNA in more detail. Competition and methylation interference experiments have shown that the protein binds to the sequence 3'-TCCATCAAGGGG-5' (positions -90 to -82). This sequence is contained within a region found to be involved in regulating inducible GM-CSF transcription in a human T-cell line [Miyatake, S., Seiki, M., Yoshida, M. & Arai, K. (1988) Mol. Cell. Biol. 8, 5581-5587].
James
R
R
Melbourne Tumor Biology Branch, Ludwig Institute for Cancer Research, Victoria, Australia.
Kazenwadel
J
J
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003115:Colony-Stimulating Factors / Q000235:genetics*; D003208:Concanavalin A / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation; D005796:Genes / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000235:genetics*; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D013601:T-Lymphocytes / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D003115:Colony-Stimulating Factors; D004268:DNA-Binding Proteins; D006133:Growth Substances; D009687:Nuclear Proteins; D003208:Concanavalin A; D016178:Granulocyte-Macrophage Colony-Stimulating Factor