Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by PU.1

Abstract

PU.1 is a dominant but transient regulator in early T-cell precursors and a potent transcriptional controller of developmentally important pro-T cell genes. Before T-lineage commitment, open chromatin is frequently occupied by PU.1, and many PU.1 sites lose accessibility when PU.1 is later downregulated. Pioneering activity of PU.1 was tested in in this developmentally dynamic context, by quantitating the relationships between PU.1 occupancy and site quality and accessibility as PU.1 levels naturally declined in pro-T cell development, and by using stage-specific gain and loss of function perturbations to relate binding to effects on target genes. PU.1 could bind closed genomic sites, but rapidly opened many of them, despite the absence of its frequent collaborators, C/EBP factors. The dynamic properties of PU.1 engagements implied that PU.1 binding affinity and concentration determine its occupancy choices, but with quantitative tradeoffs for occupancy between site sequence quality and stage-dependent site accessibility in chromatin. At non-promoter sites PU.1 binding criteria were more stringent than at promoters, and PU.1 was also much more effective as a transcriptional regulator at non-promoter sites where local chromatin accessibility depended on the presence of PU.1. Runx motifs and Runx1 binding were often linked to PU.1 at open sites, but highly expressed PU.1 could bind its sites without Runx1. Notably, closed chromatin presented a qualitative barrier to occupancy by the PU.1 DNA binding domain alone. Thus, effective pioneering at closed chromatin sites also depends on requirements beyond site recognition served by non-DNA binding domains of PU.1.