Mechanism Of PCG Redirecting Brd4 Phase Separation

As an epigenetic reader,Brd4 contain two N-terminal bromodomains(BD1 and BD2)which bind to acetyllysine of histone H3 and H4 and recruit other transcription factors to regulate transcription involved in cell proliferation and cell cycle.In addition,Brd4 is recognized as a major drug target owing to its implicated functions in oncogenesis and inflammation.Currently,the developed BET inhibitor(BETi)block the activity of Brd4 by competitively binding to the BD domain of Brd4 and disrupt the protein-protein interactions between Brd4 and acetyllysine.This type of BETi is not selective among BET family proteins.Recent studies have shown that Brd4 and MED1 are involved in the formation of phase separation of super enhancer(SE).Which further explains the transcription flexiblility mechanism of SE from a spatial perspective.Phase separation mediated by cooperative interactions between multivalent molecules in cells.Cells organize many of their biochemical reactions in the phase separated non-membrane structures.And the abnormally high density of transcription factors,co-factors,chromatin regulators,RNA pol Ⅱ and non-coding RNA are found to form phase separation at SE,and regulate the transcription of cell type-specific genes.SE driven genes are more sensitive to BETi due to the high density of Brd4 occupancy at SE.BETi has also been shown to disrupt phase separation through targeting Brd4 at SEs.Different from the currently developed BETi,we found a compound Procyanidin C-13,3’,3"-tri-O-gallate(PCG)targets the proline-rich sequence of Brd4,and works on the phase separation mechanism of Brd4.The compound PCG is a polyphenolic compound composed of a procyanidin trimer and three gallic substituents.PCG directly binds to Brd4 and form the SDS insoluble state.Further we find that PCG redirects Brd4 by proline rich regions through various Brd4 truncation.However prolin-rich sequences of Brd4 locat at Brd4 IDR which drive formation of Brd4 phase separation.Although proline rich regions don’t affect Brd4 phase separation in vivo and vitro,PCG transforms Brd4 phase separation into aggregation throuth these regions which renders Brd4 inactive towards transcription.And PCG effectively inhibits the enrichment of Brd4 at SEs.Therefore disruption of phase separation is an effective strategy in Brd4 suppression.