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

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. To measure binding, 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 then split each uninduced and induced samples and treated with +/- primary antibody followed by a species-specific secondary antibody. Then, we isolated 1e5 cells/sample and followed the CUTANA Direct-to-PCR CUT&Tag protocol from Epicypher. Due to extremely low background in both the uninduced and negative antibody samples and based on recommendation from the lab that developed the technology (Kaya-Okur 2020), we only sequenced +doxycycline +antibody samples. We size selected the libraries with AmpureXP beads and sequenced them on an Illumina NextSeq 500 using 2x150 paired-end reads.