Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function [CUT&RUN]

While chromatin presents a barrier to the binding of many transcription factors, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type specific binding and activity. The mechanisms governing pioneer-factor occupancy and the relationship between chromatin occupancy and opening remain unclear. We studied three Drosophila transcription factors with distinct DNA-binding domains and biological functions: Zelda, Grainy head, and Twist. We demonstrated that the level of chromatin occupancy is a key determinant of activity. Multiple factors regulate occupancy, including motif content, local chromatin, and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. Our results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features. Overall design: CUT&RUN on cells ectopically expressing Drosophila transcription factors.