Effects of DNA binding proteins on DNA methylation in vitro

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Abstract
The inheritance of DNA methylation patterns may play an important role in the stability of the differentiated state. The inhibitory effects of DNA binding proteins on DNA methylation in vitro were studied. Mouse L1210 cells grown in the presence of 5-azacytidine acquire hemimethylated sites in their DNA. Purified hemimethylated DNA accepted methyl groups from S-adenosyl-L-methionine in the presence of a crude maintenance methylase more readily than purified DNA isolated from cells not exposed to 5-azacytidine. Chromatin fractions isolated from cells grown in the presence or absence of 5-azacytidine were poor substrates for the maintenance methylase irrespective of the number of hemimethylated sites present in the DNA. Inhibition of DNA methylation was shown to be associated primarily with chromatin proteins bound to DNA, and trypsinization of nuclei increased their methyl accepting abilities. Methyl acceptance was increased by salt extraction of chromosomal proteins. Association of histones with DNA may play a role in the modulation of methylation patterns.