S. Ymer et Da. Jans, IN-VIVO CHROMATIN STRUCTURE OF THE MURINE INTERLEUKIN-5 GENE REGION -A NEW INTACT CELL SYSTEM, BioTechniques, 20(5), 1996, pp. 834
The study of chromatin involvement in the regulation of gene expressio
n has traditionally required the isolation of nuclei. However, cell fr
actionation techniques are subject to redistribution of proteins durin
g the isolation procedure, which prevents rigorous physiologically rel
evant analysis. To eliminate the need to isolate nuclei and to analyze
chromatin structures in vivo in response to agents regulating murine
interleukin-5 (IL-5) gene activation, we have established a novel lyso
lecithin permeabilized intact cell system for suspension cell types, i
n this case T cells. Nuclear integrity of permeabilized cells is demon
strated by nuclear transport assays using confocal laser scanning micr
oscopy. Results are identical in unstimulated and stimulated T cells,
indicating that the chromatin structure after activation is not the re
sult of gross alterations in nuclear protein transport properties. Pot
ential new IL-5 gene regulatory regions are identified by DNase I hype
rsensitivity mapping. Our lysolecithin permeabilized intact cell syste
m is amenable to physiologically relevant analysis of responses to sig
naling pathways at the level of chromatin, nuclear protein translocati
on and possibly other cellular functions in a variety of suspension an
d adherent cell types.