Hemimethylation of DNA prevents chromatin expression

The activity of hemimethylated herpes simplex virus thymidine kinase DNA and chromatin was analyzed by microinjection and thymidine incorporation into the DNA of thymidine kinase-negative Rat2 cells. Hemimethylated DNA was obtained by in vitro replication of single-stranded M13 DNA constructs and of chromatin produced by in vitro reconstitution of the DNA with purified chicken histone octamers. We found that methylation of either the coding or the noncoding DNA strand was sufficient to block expression of the hemimethylated chromatin. In contrast, the hemimethylated DNA was as active as the unmethylated control DNA after microinjection until chromatin formation occurred in the recipient cells. Microinjection of chromatin hemimethylated by bacterial Hae III methyltransferase excluded the possibility that inactivation was caused by symmetrical methylation of the injected molecules.