hnRNPK condensates facilitate enhancer-promoter looping and RNA polymerase II recruitment.
Enhancer RNAs interact with promoter-derived RNAs to dictate enhancer-promoter looping, but the RNA-binding protein that mediates this process has remained unidentified. Here we identify hnRNPK as a general structural regulator that preferentially binds to nascent RNAs transcribed from enhancer and promoter regions, promoting enhancer-promoter looping and transcriptional activation. We further show that hnRNPK forms phase-separated, cavity-containing condensates that encapsulate RNA polymerase II (Pol II) via its RPB3 subunit, facilitating chromatin looping and potentially enabling recruitment of Pol II from enhancers to promoters through protein dimerization. Notably, a mutation associated with Au-Kline syndrome in hnRNPK (c.953+1dupG) alters its condensates from a liquid-like to a gel-like state, leading to developmental defects in knock-in mice. Fibroblasts derived from these mutants display reduced enhancer-promoter looping and decreased Pol II recruitment at promoters of key developmental genes. These findings suggest that hnRNPK is a structural regulator of enhancer-promoter communication and highlight the importance of RNA-RNA interactions mediated by RNA-binding proteins in transcriptional regulation.