Promoter strength and position govern promoter competition via transcript-dependent insulation

Competition between promoters within a shared regulatory landscape has been implicated in development and disease, but the determinants of promoter competition remain unclear. Here, we introduce diverse promoters into defined genomic sites within the Sox2 locus and measure how these insertions attenuate endogenous Sox2 expression. We find that the level of reduction in endogenous Sox2 transcription is correlated with the strength of the inserted promoter. Transcription from the inserted promoter is required for competition, with longer transcript resulting in more competition. The inserted active promoter and its associated transcriptional unit function as an insulator, rendering competition position-dependent. Finally, we observe HUSH-mediated silencing of the inserted promoters, which counteracts competition. Together, our work uncovers the rules governing promoter competition, highlights its impact on tuning gene expression levels and genome evolution, and suggests that transcripts of sufficient level and length can mediate insulation independently of CTCF and cohesin.