ADJACENT, COOPERATIVE ELEMENTS FORM A STRONG, CONSTITUTIVE ENHANCER IN THE HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

Both copies of a repeated sequence CATT(A/T), located between bp -53 a nd -39 in the upstream region of the human GM-CSF gene, are required f or mitogen-inducible promoter activity in T lymphocytes. However, the proteins that recognize this region of the granulocyte-macrophage colo ny-stimulating factor (GM-CSF) promoter, and are responsible for its t ranscriptional regulatory activity, have not been clearly identified. Using transient transfection assays, we demonstrate that a 19-bp oligo nucleotide containing the CATT(A/T) repeats has strong constitutive en hancer activity in both T cell and non-T-cell lines, even though GM-CS F is not normally constitutively expressed by these cells. A 12-bp oli gonucleotide, containing only the sequence CATTAATCATTT, lacks enhance r activity indicating that the nucleotides surrounding these sequences are critical for this enhancer activity. The sequence TTTCCT, which c an bind members of the ets family of transcription factors, is located just 3' of these CATT(A/T) repeats, and mutagenesis of the CCT sequen ce abolishes (1) the constitutive (and mitogen inducible) enhancer act ivity of the 19-bp GM-CSF sequences, (2) the responsiveness to transac tivation by ets-1, and (3) the ability to specifically bind ets-1 and elf-1 in electrophoretic mobility shift assays (EMSA). We demonstrate that although T cells contain nuclear proteins capable of independentl y recognizing the ets binding site and the CATT(A/T) repeats in EMSAs, both of these regulatory elements are required for enhancer function, The strong constitutive activity of this 19-bp region suggests that n egative regulation of the GM-CSF promoter is critical for the restrict ed expression pattern of GM-CSF mRNA. (C) 1996 by The American Society of Hematology.