In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells.
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 cis-elements in vitro. To determine if the trans-activators 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 NF kappa B 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.
Brunvand
M W
MW
Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Krumm
A
A
Groudine
M
M
D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013601:T-Lymphocytes / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D016328:NF-kappa B; D009687:Nuclear Proteins; D004247:DNA; D003850:Deoxyribonuclease I