Time dependence of X gene reactivation induced by 5-azacytidine: possible progressive restructuring of chromatin.

According to the theoretical mechanism of DNA demethylation by 5-azacytidine, the complete demethylation of one site will require two cell divisions. If reexpression is directly related to demethylation, a maximal reexpression is expected after two cell divisions. In a hamster X human hybrid cell line containing an inactive human X chromosome treated by 5-azacytidine, we show that HPRT reactivation frequency is increased more than 10-fold when cells are allowed to divide 14 times before the selection for the HPRT reactivants.

Inactive X chromosome gene reactivation induced by 5-azacytidine, is not correlated with the modification of the pattern of replication.

The replication pattern of a human inactive X chromosome reactivated for one to four genes by 5-azacytidine has been studied by the BrdU-33258-Hoechst-Giemsa technique in four subclones of a somatic hamster-human hybrid. In one of them the pattern was clearly modified. In the three others the changes were not significant.