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 factors (TFs) 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 TFs 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 TFs average dox50 between ATAC-seq labeled inaccessible and accessible binding sites, its {Delta}dox50, is a measure of its pioneer activity. We measured {Delta}dox50s for the endodermal TFs FOXA1 and HNF4A and show that HNF4A has a smaller {Delta}dox50 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 {Delta}dox50, suggesting that strong motif content may compensate for weak pioneer activity. The quantitative analysis of binding suggests different modes of binding for FOXA1, including an anti-cooperative mode of binding at certain accessible loci. Our results suggest that {Delta}dox50s, or other similar measures that assess the difference in TF affinity for inaccessible and accessible DNA, are reasonable measures of pioneer activity.