A test of the pioneer factor hypothesis using ectopic liver gene activation.

Jeffrey L Hansen,Barak A Cohen,Kaiser J Loell

doi:10.7554/elife.73358

Jeffrey L Hansen, Barak A Cohen + Show 1 more

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https://doi.org/10.7554/elife.73358

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Abstract

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 (non-PFs) that activate batteries of silent genes. The PFH predicts that ectopic gene activation requires the sequential activity of qualitatively different TFs. We tested the PFH by expressing the endodermal PF FOXA1 and non-PF 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 a 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 for binding. A subset of targets required both TFs, but the predominant mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results, we hypothesize an alternative to the PFH where 'pioneer activity' depends not on categorically different TFs but rather on the affinity of interaction between TF and DNA.

Highlights

Transcription factors (TFs) face steric hindrance when instances of their motifs are occluded by nucleosomes(Kornberg 1974; Kaplan et al 2009)
When we examine all of the liver genes only activated by FOXA1-HNF4A coexpression, we find that in contradiction with the Pioneer Factor Hypothesis (PFH), there are roughly equal numbers of FP, HP, and Cooperatively Bound” (CB) sites (Figure 4C)
When we examined the bp of sequence centered upon genome-wide co-bound sites, we found that there was higher total motif content at inaccessible binding sites as compared to random (Figure 5D) and that FOXA1 and HNF4A motif content was higher at FP or HP sites, respectively, than CB sites (Figure 5E)

Summary

Introduction

Transcription factors (TFs) face steric hindrance when instances of their motifs are occluded by nucleosomes(Kornberg 1974; Kaplan et al 2009). The conversion from embryonic fibroblasts to induced endoderm progenitors offers one clear example(Sekiya and Suzuki 2011; Morris et al 2014) This reprogramming cocktail combines the canonical PF FOXA1(Lisa Ann Cirillo et al 2002) and nonPF HNF4A(Karagianni et al 2020). There are chromatin states that are prohibitive to PF binding(Mayran et al 2018; Zaret and Mango 2016) and, in at least two cases, FOXA1 requires help from other TFs to bind at its sites(Donaghey et al 2018; Swinstead et al 2016) These examples suggest that PFs are not always sufficient to open inaccessible chromatin. In contrast to the predictions of the PFH, we found that both FOXA1 and HNF4A could independently bind to inaccessible instances of their motifs, induce chromatin accessibility, and activate endoderm-specific gene expression. We suggest that our findings present an alternative to the PFH that eliminates the categorical distinction between PFs and nonPFs and instead posits that the energy required to pioneer occluded sites (“pioneer activity”) comes from the affinity of interaction between TFs and DNA

Results

Discussion

Materials and Methods