Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments

Abstract

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H 2O 2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H 2O 2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H 2O 2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50–200 kb fragments. Chromatin digestion could be elicited with H 2O 2 concentrations as low as 50 μM. After the removal of H 2O 2, most >400 kb fragments were religated within 2 h; however, digestion into 50–200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H 2O 2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.