Nucleic Acids Research, 1993, Vol. 21, No. 20 4824-4829
© 1993
MOLECULAR BIOLOGY |
In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells
Clinical Division Seattle,WA 98104 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center Seattle,WA 98104 2department of Radiation Oncology, University of Washington School of Medicine Seattle, WA 98195, USA
Received April 12, 1993. Revised August 13, 1993. Accepted August 13, 1993.
The IL-2 gene is a T cell specific gene that is expressed early during the activation-specific T lymphocyte development program. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assays have defined DNA/protein interactions at the IL-2 promoter c/s-elements in vitro. To determine if the transactivators documented in T cell nuclear extracts actually bind the IL-2 promoter in vivo, ligation mediated PCR (LMPCR) genomic footprinting was performed on the IL-2 promoter in both activated and non-activated T cells and HL60 promyelocytes, which do not express the IL-2 gene. The in vivo footprints indicate that the IL-2 gene transcription start site and TATA sequence are protected in both activated and resting T cells, prior to the appearance of detectable IL-2 steady state message. The distal NF-AT and the NFxB sites are each footprinted and the Oct/OAP site contains hypersensitive residues in the unstimulated T lymphocytes. Additional residues are protected in each of these sites after T cell activation. The proximal NF-AT site (NF-IL-2B) and the AP-1 site at -150 are protected in activated Jurkat T lymphocytes, but these two sites are not protected in activated Jurkat lymphocytes stably transfected a gene construct containing multiple NFAT binding sites.
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