p300 is an obligate integrator of combinatorial transcription factor inputs

Transcription coactivators are proteins or protein complexes that mediate transcription factor (TF) function. However, they lack DNA-binding capacity, prompting the question of how they engage target loci. Three non-exclusive hypotheses have been posited: coactivators are recruited by complexing with TFs, by binding histones through epigenetic reader domains, or by partitioning into condensates through their extensive intrinsically disordered regions. Using p300 as a prototypical coactivator, we systematically mutated its annotated domains and show by single-molecule tracking in live U2OS cells that coactivator-chromatin binding depends entirely on combinatorial binding of multiple TF-interaction domains. Furthermore, we demonstrate that acetyltransferase activity opposes p300-chromatin association and that the N-terminal TF-interaction domains regulate that activity. Single TF-interaction domains are insufficient for chromatin binding and regulation of catalytic activity, implying a principle that we speculate could broadly apply to eukaryotic gene regulation: a TF must act in coordination with other TFs to recruit coactivator activity. [Display omitted] •The structured Core domain of p300 is dispensable for chromatin engagement•p300-chromatin binding requires multiple TF-interaction domains•p300 KAT activity opposes chromatin binding•p300 KAT activity may be regulated by its N-terminal TF-interaction domains Ferrie et al. demonstrate through live-cell single-molecule imaging that the coactivator p300, a central player in eukaryotic gene regulation, carries out its function of integrating multiple transcription factor (TF) inputs at the level of chromatin binding. Their results support a TF- rather than histone-mediated model of coactivator recruitment.