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

Long-range competition among 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 transcripts resulting in more competition. The inserted active promoter and its associated transcriptional unit function as an insulator, rendering competition position dependent. Competition is counteracted by the HUSH-mediated silencing of the inserted promoters. Together, our work uncovers the rules governing promoter competition, highlights its impact on tuning gene expression levels and genome evolution, and suggests that transcriptional units producing transcripts of sufficient level and length can mediate insulation independently of CTCF and cohesin.