Molecular mechanisms of promoter silencing by transcriptional readthrough [RNA-seq]

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.