A test of the pioneer factor hypothesis using ectopic liver gene activation [ATACseq]

The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. We tested the PFH by expressing the endodermal PF FoxA1 and nonPF Hnf4a in K562 lymphoblast cells. While co-expression of FoxA1 and Hnf4a activated a burst of endoderm-specific gene expression, we found no evidence for functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and “pioneered” for each other, although FoxA1 required fewer copies of its motif to bind at inaccessible sites. A subset of targets required both TFs, but the mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we propose an alternative to the PFH where “pioneer activity” depends not on the existence of discrete TF subclasses, but on TF binding affinity and genomic context. Overall design: To measure accessibility, we treated clonally derived K562 cell lines that were previously transduced with lentiviral vectors carrying doxycycline-inducible FoxA1, Hnf4a, or both FoxA1-Hnf4a with +/- 0.5µg/ml doxycycline for 24 hours in duplicate. We input 2e5 cells/sample into the OMNI-ATAC protocol (Corces et al. 2017) and isolate 5e4 nuclei/sample for tagmentation and library preparation. We size selected the libraries with AmpureXP beads and then sequenced them libraries on an Illumina NextSeq 500 using 2x75 paired-end reads.