DNA METHYLATION DIRECTS A TIME-DEPENDENT REPRESSION OF TRANSCRIPTION INITIATION

Background: The regulation of DNA methylation is required for differen tial expression of imprinted genes during vertebrate development, Earl ier studies that monitored the activity of the Herpes simplex virus (H SV) thymidine kinase (tk) gene after injection into rodent cells have suggested that assembly of chromatin influences the methylation-depend ent repression of gene activity, Here, we examine the mechanism of met hylation-dependent HSV tk gene regulation by direct determination of n ucleoprotein organization during the establishment of a transcriptiona lly silenced state after microinjection of templates with defined meth ylation states into Xenopus oocyte nuclei. Results: The transcriptiona l silencing conferred by a methylated DNA segment was not immediate, a s methylated templates were initially assembled into active transcript ion complexes. The eventual loss of DNase I hypersensitive sites and i nhibition of transcription at the HSV rk promoter only occurred after several hours. Flanking methylated vector DNA silenced the adjacent un methylated HSV tk promoter, indicative of a dominant transmissible rep ression originating from a center of methylation. The resulting repres sive nucleoprotein structure silenced transcription in the presence of activators that are able to overcome repression of transcription by n ucleosomes. Conclusions: 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 tran scriptional repression can occur by indirect mechanisms involving the time-dependent assembly of repressive nucleoprotein complexes, which a re able to inhibit transcription more effectively than nucleosomes alo ne.