DNA methylation directs a time-dependent repression of transcription initiation.

The regulation of DNA methylation is required for differential expression of imprinted genes during vertebrate development. Earlier studies that monitored the activity of the Herpes simplex virus (HSV) thymidine kinase (tk) gene after injection into rodent cells have suggested that assembly of chromatin influences the methylation-dependent repression of gene activity. Here, we examine the mechanism of methylation-dependent HSV tk gene regulation by direct determination of nucleoprotein organization during the establishment of a transcriptionally silenced state after microinjection of templates with defined methylation states into Xenopus oocyte nuclei. The transcriptional silencing conferred by a methylated DNA segment was not immediate, as methylated templates were initially assembled into active transcription complexes. The eventual loss of DNase I hypersenitive sites and inhibition of transcription at the HSV tk promoter only occurred after several hours. Flanking methylated vector DNA silenced the adjacent unmethylated HSV tk promoter, indicative of a dominant transmissible repression originating from a center of methylation. The resulting repressive nucleoprotein structure silenced transcription in the presence of activators that are able to overcome repression of transcription by nucleosomes. Silencing of transcription by DNA methylation is achieved at the level of transcription initiation and involves the removal of transcriptional machinery from active templates. This transcriptional repression can occur by indirect mechanisms involving the time-dependent assembly of repressive nucleoprotein complexes, which are able to inhibit transcription more effectively than nucleosomes alone.