Highlights • AGO1 enrichment on chromatin and transcriptional enhancers is mediated by eRNAs • AGO1 depletion alters global transcriptional output including eRNA transcripts • Enhancer-associated AGO1 contributes to the maintenance of 3D chromatin organization Summary The impact of mammalian RNA interference components, particularly, Argonaute proteins, on chromatin organization is unexplored. Recent reports indicate that AGO1 association with chromatin appears to influence gene expression.