SELECTIVE HYPERMETHYLATION OF TRANSCRIBED NUCLEOSOMAL DNA BY SODIUM-BUTYRATE

Previous studies have shown that treatment of cultured fibroblasts wit h millimolar concentrations of sodium butyrate results in increased me thylation of cytosine residues in DNA. In this study, active nucleosom es were fractionated from the inactive ones by organomercurial agarose column chromatography. DNA in each fraction was hydrolyzed to its con stituent bases and subjected to HPLC analysis in order to determine th e 5-methylcytosine content. In control cells, the active nucleosomal D NA was hypomethylated (0.97 +/- 0.27% 5-methylcytosine) when compared with the inactive DNA fraction (1.61 +/- 0.15%). This result was not u nexpected since DNA hypermethylation is generally associated with gene inactivation. Treatment of cells with sodium butyrate, however, resul ted in increased methylation of the active nucleosomal DNA such that i t was comparable to that of the inactive fraction of control cells (1. 73 +/- 0.02% 5-methylcytosine). A much smaller increase in B-methylcyt osine content was detected in the inactive DNA fraction of sodium buty rate-treated cells (from 1.61 to 1.89%). Removal of the sodium butyrat e followed by a chase in butyrate-free medium for up to 120 h failed t o reverse the butyrate-induced hypermethylation. Reversal was achieved only after continuous culture in butyrate-free medium for 10 days. (C ) 1994 Academic Press, Inc.