Molecular mechanisms of promoter silencing by transcriptional readthrough

Transcriptional interference is defined as the in cis suppression of one transcriptional process by another. While most mammalian genes are well insulated from such interference through proper transcriptional termination and chromatin boundary elements, disruption of these safeguards can lead to durable changes in gene activity and, in some cases, heritable epigenetic silencing. Here, we developed a tractable experimental system to investigate the mechanistic consequences of antisense transcriptional readthrough. Using mouse mesenchymal progenitor cells and CRISPR-Cas9, we modeled the epi-cblC phenotype through targeted deletion of the Prdx1 intron 5 splice acceptor site, resulting in transcriptional readthrough across the Mmachc locus. This led to aberrant deposition of H3K36me3, the establishment of DNA methylation, and stable, mitotically heritable silencing of Mmachc. Through systematic perturbations of transcription, and the H3K36 and DNA methylation machinery, we deconstruct the epigenetic and transcriptional mechanisms that contribute to readthrough-mediated silencing. We demonstrate that antisense transcription is the primary driver of repression, while H3K36me3 contributes an intrinsic silencing component that is further required for the establishment of DNA methylation, which in turn confers long term stability. Together, these findings define a hierarchical silencing cascade in which transcriptional readthrough is converted into stable epigenetic memory across cell divisions, and establish transcription as an instructive regulatory process that is capable of reshaping local chromatin environments and gene expression.