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In previous work, three clonal cell lines with extremely low DNA methylation levels were derived by multiple consecutive treatments of C3H 10T1/2 C18 (10T1/2) cells with 5-aza-2'-deoxycytidine (5-aza-CdR). In this study we examined the methylation status of genes in these three methyl-deficient clones to assess the specificity of the induced hypomethylation. Complete demethylation of virtually all 5'-CCGG-3' sites was observed in four genes examined, but some sites common to all three clones were persistently methylated even after further exhaustive 5-aza-CdR treatment. Thus, there is a subset of methylation sites within these cells which can never be stably demethylated. The extensive demethylation was not always associated with changes in the level of RNA expression of the genes examined but was strongly correlated with an altered chromatin structure of the unexpressed alpha 1-globin gene and the muscle determination gene MyoD1. These results provide a direct correlation between hypomethylation and the induction of a transcriptionally competent chromatin state.

Gene structure and transcription in mouse cells with extensively demethylated DNA.