A pioneer factor locally opens compacted chromatin to enable targeted ATP-dependent nucleosome remodeling

Summary: Pioneer transcription factors engage nucleosomal DNA in chromatin to initiate gene regulatory events that control cell fate 1 . To determine how different pioneer transcription factors initiate the formation of a locally accessible environment within silent, compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome that can be targeted by the hematopoietic ETS factor PU.1 and bZIP factors C/EBPa, and C/EBPß. Each class of factor can expose target nucleosomes on linker histone-compacted arrays, but with different hypersensitivity patterns, as discerned by long-read sequencing. The DNA binding domain of PU.1 is sufficient for mononucleosome binding but requires an additional intrinsically disordered domain to bind and open compacted chromatin. The canonical mammalian SWI/SNF (BAF) complex, cBAF, was unable to act upon two forms of locally open chromatin, in the presence of linker histone, unless cBAF was enabled by the acidic- and glutamine-enriched transactivation domain of PU.1. However, cBAF complexes potentiate the nucleosome binding DBD of PU.1 to weakly open chromatin in the absence of the PU.1 unstructured domain. Together our findings provide a mechanism for how pioneer factors initially target chromatin structures to provide specificity for action by nucleosome remodelers that further open local domains. Overall design: MNase-seq performed on Cx3cr1 nucleosome arrays in the presence or absence of several transcription factors (CEBPa, CEBPb, PU.1, as well as combinations CEBPa+PU.1 or CEBPb+PU.1). Control conditions include those arrays without transcription factors or nucleosomes (free DNA), without transcription factors but with nucleosomes and linker histone H1 (NoTF), and without transcription factors or linker histone but with nucleosomes (array). Each condition was sequenced with two replicates. Additional samples are provided to examine the role of chromatin remodeler cBAF: an additional free DNA, array, and NoTF control, and two conditions incubated with PU.1 in the presence or absence of BAF. All conditions were sequenced with two replicates.