Different Patterns of Chromatin Opening By Hematopoietic Pioneer Factors

Pioneer transcription factors engage nucleosomal DNA in chromatin to initiate gene regulatory events that ultimately control cell fate. Pioneer factors can elicit local exposure of a nucleosome within chromatin and ultimately recruit co-regulators and remodelers to yield open chromatin sites seen in vivo. Yet how different pioneer transcription factors initially expose a targeted nucleosome in compacted chromatin structures is unclear. We used nucleosome arrays in vitro with a central nucleosome that can be targeted by the hematopoietic Ets factor PU.1 and the bZIP factors C/EBPα and C/EBPβ. Each class of factor can elicit targeted nucleosome exposure on linker histone-compacted arrays, but with different hypersensitivity patterns, as discerned from long-read nanopore sequencing. The DNA binding domains (DBDs) of PU.1 and C/EBPα are sufficient for mononucleosome binding but are much less efficient than the full-length proteins, which can function cooperatively, in opening compacted chromatin. Thus, pioneer factors use their DBD to bind nucleosomes and other domains to elicit distinct patterns of target nucleosome exposure within compacted chromatin in vitro. Finally, nucleosome exposure by PU.1 enables further chromatin remodeling by a SWI/SNF complex. Together our data give a mechanistic view of how transcription factors cooperate to disrupt chromatin structures to initiate DNA accessibility to additional regulatory factors.