Selective phase separation of transcription factors is driven by orthogonal molecular grammar

Protein production is critically dependent on gene transcription rates, which are regulated by RNA polymerase and a large collection of transcription factors (TFs). Previous studies identified the formation of super enhancer regions where increased transcriptional activity is observed. This has been linked to phase separation, in which the differential condensation behaviour of separate TF families has been hypothesised to cause the selectivity in gene expression. The underlying molecular forces that are responsible for this selectivity, however, are unknown. Here, we conduct phase separation studies on six TFs (FUS, EWS, TAF15, SP1, SP2, and HNF1A) from three different TF families by carrying out residue-scale coarse-grained molecular dynamics simulations. Our exploration of ternary TF phase diagrams revealed four dominant sticker motifs and two orthogonal driving forces, consisting of hydrophobic (aromatic, aliphatic) interactions and electrostatic/cation-{pi} interactions. The contribution of these driving forces to the homotypic and heterotypic intermolecular strengths dictate the resultant condensate morphology. These results point to sequence-dependent orthogonal grammar as a generic mechanism responsible for selective transcriptional condensation in gene expression. Interestingly, our results also show how RNA polymerase is able to overcome this orthogonality to coalesce with TF condensate providing a framework in which co-condensation is the primary nuclear organisational principle that controls selective partitioning of RNAP to targeted genes for transcription.