Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.
Sakamoto
K M
KM
Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, UCLA School of Medicine 90024-1752.
Fraser
J K
JK
Lee
H J
HJ
Lehman
E
E
Gasson
J C
JC
D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D005786:Gene Expression Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D007377:Interleukin-3; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000242:Cyclic AMP