Solid phase transitions as a solution to the genome folding paradox

Ultra-long-range genomic contacts, which are key components of neuronal genome architecture^1-3, constitute a biochemical enigma. This is because regulatory DNA elements make selective and stable contacts with DNA sequences located hundreds of kilobases away, instead of interacting with proximal sequences occupied by the exact same transcription factors^1,4. This is exemplified in olfactory sensory neurons (OSNs), in which only a fraction of LHX2-, EBF1- and LDB1-bound sites interact with each other, converging into highly selective multi-chromosomal enhancer hubs⁵. To obtain biochemical insight into this process, here we assembled olfactory receptor (OR) enhancer hubs in vitro with recombinant proteins and enhancer DNA. Cell-free reconstitution of enhancer hubs revealed that OR enhancers form nucleoprotein condensates with unusual, solid-like characteristics. Assembly of these solid condensates is orchestrated by specific DNA motifs enriched in OR enhancers, which are likely to confer distinct homotypic properties on their resident LHX2-EBF1-LDB1 complexes. Single-molecule tracking and pulse-chase experiments in vivo confirmed that LHX2 and EBF1 assemble OR-transcription-competent condensates with solid properties in OSN nuclei, under physiological concentrations of protein. Thus, homophilic nucleoprotein interactions that are influenced by DNA sequence generate new types of biomolecular condensate, which might provide a generalizable explanation for the stability and specificity of long-range genomic contacts across cell types.