Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene.
The structure of chromatin and its remodeling following activation are important aspects of the control of inducible gene transcription. The IL-2 gene is induced in a cell specific-manner in T cells following an antigenic stimulus. We show, using a novel real-time PCR assay, that significant chromatin remodeling of the IL-2 proximal promoter region occurred upon stimulation of both the murine EL-4 T cell line and primary CD4(+) T cells. Chromatin remodeling appears to be limited to the first 300 bp of the proximal promoter region as measured by micrococcal nuclease and restriction enzyme accessibility. Time course studies indicated that chromatin remodeling was observed at 1.5 h postinduction and was maintained for up to 16 h. The remodeling is reversible upon removal of the stimulus. The region immediately upstream from the transcription start site, however, remains accessible for up to 16 h. Upon restimulation, remodeling occurs much more rapidly, consistent with a more rapid rise in IL-2 mRNA levels. Using a number of pharmacological inhibitors we show that remodeling is dependent on the presence of specific transcription factors, but not on the modification of histones. The development of this novel chromatin accessibility assay based on real-time PCR has allowed rapid, sensitive, and quantitative measurements on the IL-2 gene following cellular activation in both T cell lines and primary cells.
Rao
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S
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia.
Procko
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E
Shannon
M F
MF
D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D003199:Computer Systems; D000242:Cyclic AMP / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D018664:Interleukin-12 / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology*
D002843:Chromatin; D018664:Interleukin-12; D016572:Cyclosporine; D000242:Cyclic AMP