Sd. Nimer et al., ADJACENT, COOPERATIVE ELEMENTS FORM A STRONG, CONSTITUTIVE ENHANCER IN THE HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE, Blood, 87(9), 1996, pp. 3694-3703
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.