Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.
Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site.
Watanabe
S
S
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Ishida
S
S
Koike
K
K
Arai
K
K
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D016259:Genes, myc; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D056465:Retinoblastoma-Binding Protein 1; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors; D007377:Interleukin-3; D009687:Nuclear Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases