Simultaneous induction of epigenetic variants

Epigenetic changes at one, two, or three loci were induced with 5-azacytidine in CHO-K1 cells, using markers (proline dependence, asparagine dependence, and thymidine kinase deficiency) which respond by high-frequency reversion to wild-type states. The observed incidence of dual or triple revertants was compared with the frequency expected by chance as the product of single frequencies measured separately for the markers involved. Values observed for dual reversions consistently exceeded levels predicted by this purely stochastic model, and for triple reversion the divergence was 1000-fold. Coordinate responses could account for this disparity, if minority cell types with higher reactivity to 5-azacytidine exist in target cell populations. To test this hypothesis, single colonies were isolated under nonselective conditions from a mass culture of CHO-K1 cells that had been treated with 5-azacytidine. These clones showed wide and reproducible differences in competence for reversion from proline dependence to independence. Our data thus suggest that simultaneity of epigenetic changes depends on random events which are modulated by a mosaic of inductive potentials within individual cells of the reacting system.