Overall changes in chromatin sensitivity to DNase I during differentiation.
The DNase I sensitivity of total chromatin was studied in fixed cells and nuclei isolated from proliferating and terminally differentiated cells, by measuring the incorporation of labelled nucleotides into DNase-sensitive sites, and electrophoresis of DNA isolated from DNase-treated nuclei. The unfixed nuclei were sensitive to digestion at around 10 micrograms/ml, the fixed cells at 30 ng/ml DNase I concentration. Proliferating Rauscher leukemia cells were more digestible than normal spleen cells. The DNase I sensitivity of the human HL60 leukemia line decreased upon DMSO-induced differentiation but still exceeded the digestibility of nuclei from normal human peripheral blood. A novel flow-cytometric technique was developed to study DNase sensitivity at the cell level. It confirmed the relative resistance of differentiated cells to DNase I and ruled out the possibility that this could be due to an altered distribution of cell cycle phases. The overall DNase I sensitivity of chromatin was compared with the sensitivity of the c-myc gene and the myc-associated hypersensitive sites. The latter sites were detected at 1 microgram/ml DNase I in HL60 nuclei. They disappeared partially upon DMSO-induced differentiation. At 10 micrograms/ml, myc was degraded in both growing and differentiating HL60, but not in HPB cells. These data suggest that a progressive condensation of the chromatin occurs during terminal differentiation which gradually involves specific genes that need to be inactivated.
Szabó
G
G
Damjanovich
S
S
Sümegi
J
J
Klein
G
G
D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002843:Chromatin / Q000648:ultrastructure*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D007942:Leukemia, Experimental / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D014176:Protein Biosynthesis; D013154:Spleen / Q000166:cytology*
D002843:Chromatin; D003850:Deoxyribonuclease I