A quantitative metric of pioneer activity reveals that HNF4A has stronger in vivo pioneer activity than FOXA1

We and others have suggested that pioneer activity–a transcription factor’s (TF’s) ability to bind and open inaccessible loci–is not a qualitative trait limited to a select class of pioneer TFs. We hypothesize that most TFs display pioneering activity that depends on the TF concentration and the motif content at their target loci. Here we present a quantitative measure of pioneer activity that captures the relative difference in a TF’s ability to bind accessible versus inaccessible DNA. The metric is based on experiments that use CUT&Tag to measure binding of doxycycline (dox) inducible TFs. For each location across the genome we determine a “dox50,” the concentration of dox required for a TF to reach half-maximal occupancy. We propose that the ratio of a TF’s average dox50 between ATAC-seq labeled inaccessible and accessible binding sites, its Δdox50, is a measure of its pioneer activity. We measured Δdox50’s for the endodermal TFs FOXA1 and HNF4A and show that HNF4A has a smaller Δdox50 than FOXA1, suggesting that HNF4A has stronger pioneer activity than FOXA1. We further show that FOXA1 binding sites with more copies of its motif have a lower Δdox50, suggesting that strong motif content may compensate for weak pioneer activity. Our results suggest that Δdox50s, or other similar measures that assess the difference in TF affinity for inaccessible and accessible DNA, are reasonable measures of pioneer activity. We performed these experiments with clonal K562 lines that express either doxycycline-inducible FOXA1 or HNF4A. We split cells into two replicate flasks, treated each replicate with either 0ug/ml (uninduced), 0.005ug/ml, 0.05ug/ml, 0.1ug/ml, 0.25ug/ml, 0.5ug/ml, or 5ug/ml doxycycline for 24 hours, and then used the CUTANA Direct-to-PCR CUT&Tag protocol from Epicypher to measure binding. We input 1e5 cells from each replicate into either a plus- or minus-primary antibody and then used species-matched secondary antibodies. Due to extremely low background in both the uninduced and minus antibody samples and based on recommendation from the lab that developed the technology (Kaya-Okur 2020), we only sequenced the induced and plus-antibody samples. We size selected the libraries with AmpureXP beads and sequenced them on an Illumina NextSeq 500 using 2x150 paired-end reads.