Molecular mechanisms of promoter silencing by transcriptional readthrough

Transcriptional interference is the in cis suppression of one transcriptional process by another. To define the mechanisms by which transcriptional readthrough is converted into stable gene silencing, we develop a mouse cell line model of cobalamin C deficiency associated with MMACHC silencing (epi-cblC) by deleting the Prdx1 intron 5 splice acceptor, thereby inducing antisense transcription across Mmachc. Readthrough results in ectopic H3K36me3 deposition, de novo DNA methylation, and stable silencing of Mmachc. Through systematic perturbations of transcription and the H3K36 and DNA methylation machinery, we demonstrate that antisense transcription is the primary driver of repression, whereas H3K36me3 contributes directly to promoter silencing and is further required for the establishment of DNA methylation, which in turn confers long-term stability. Together, these findings define a hierarchical silencing cascade by which transcriptional readthrough establishes stable gene repression and demonstrate that transcription itself can direct local chromatin remodeling and changes in gene expression.