Adjacent, cooperative elements form a strong, constitutive enhancer in the human granulocyte-macrophage colony-stimulating factor gene [published erratum appears in Blood 1996 Oct 1;88(7):2818]

Both copies of a repeated sequence CATT(A/T), located between bp -53 and -39 in the upstream region of the human GM-CSF gene, are required for mitogen-inducible promoter activity in T lymphocytes. However, the proteins that recognize this region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, and are responsible for its transcriptional regulatory activity, have not been clearly identified. Using transient transfection assays, we demonstrate that a 19-bp oligonucleotide containing the CATT(A/T) repeats has strong constitutive enhancer activity in both T cell and non-T-cell lines, even though GM-CSF is not normally constitutively expressed by these cells. A 12-bp oligonucleotide, containing only the sequence CATTAATCATTT, lacks enhancer activity indicating that the nucleotides surrounding these sequences are critical for this enhancer activity. The sequence TTTCCT, which can bind members of the ets family of transcription factors, is located just 3′ of these CATT(A/T) repeats, and mutagenesis of the CCT sequence abolishes (1) the constitutive (and mitogen inducible) enhancer activity of the 19-bp GM-CSF sequences, (2) the responsiveness to transactivation 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 independently 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 negative regulation of the GM-CSF promoter is critical for the restricted expression pattern of GM-CSF mRNA.

Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene.

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.