Pioneer factors: nature or nurture?

Figures & data

Figure 1. Some evidence suggests that PFs are a unique subset of TFs. (a) The apparent binding affinity of PFs for target sites on nucleosomes (K_D,nuc) is similar to that of free DNA (K_D,DNA). (b) some PFs have a stronger binding affinity for nucleosomal sites due to a dissociation rate compensation mechanism, where the nucleosomal off-rate (k_off,nuc) is lower than the off-rate from free DNA (k_off,DNA). (c) many PFs possess unique structural features in their DBDs that can better accommodate nucleosome substrates. (d) PFs are able to target internal nucleosomal motifs, like those at the dyad, while non-PFs primarily target motifs on free DNA or at the nucleosome entry-exit site. (e) PFs can target nucleosomal sites irrespective of the underlying chromatin landscape, whereas the binding of non-PFs tends to be inhibited at unmodified and repressive (red) sites but permitted at activating (green) sites. (f) PFs are the first TFs to bind to cis-regulatory elements that initiate gene regulatory events. Non-PFs tend to bind more downstream. (g) PFs can remain bound to chromatin throughout mitosis, whereas non-PFs dissociate from mitotic chromosomes.

Figure 2. Some evidence suggests that PFs are not special and all/many TFs can function as PFs. (a) TF concentration correlates positively with nucleosomal targeting and opening. (b) Some PFs do not exhibit the dissociation rate compensation and have a higher nucleosomal off-rate (k_off,nuc) than the off-rate from free DNA (k_off,DNA). (c) many PFs have a stronger preference for binding to free DNA or at the nucleosome entry-exit site than to internal nucleosomal motifs. (d) Some evidence suggests that PFs are able to target unmarked and activating (green) sites but unable to target repressive (red) sites. (e) Some PFs are not retained on mitotic chromatin, whilst some TFs not characterized as PFs have been shown to remain bound to mitotic chromatin. (f) Some PFs rely on co-binding with other TFs (green or purple) to direct context-specific binding. (g) Some PFs rely on ATP-dependent chromatin remodelers for binding, despite the definition of PFs as being the most upstream factors in a gene regulatory pathway.

Table 1. Standard assay for PF classification.

Method	In vitro	In vivo	Interpretation	Note
Measure the apparent KD of a TF on free DNA and the same DNA reconstituted onto a nucleosome.	X		PFs tend to have comparable binding affinities toward naked DNA and nucleosomes, while non-PFs tend to have much lower affinities toward nucleosome in comparison to naked DNA	This KD measurement may depend on the number, strength, location, and orientation of the binding motifs, as well as the nucleosome positioning element used. If this property uniformly applies to all PFs, and if high affinity toward nucleosome is required for pioneering activities in vivo, require further testing.
Measure the binding / dissociation rates of a TF for free DNA and the same DNA reconstituted onto a nucleosome.	X		Some PFs have slower dissociation rate on nucleosome in comparison with that on naked DNA, while some canonical TFs show the opposite trend	Same complications as above
Structural studies (crystallography or Cryo-EM) on TF/nucleosome complex.	X		PFs are thought to have structural features that are compatible with nucleosomes, including resemblance to linker histone, special DBD structure, partial motif recognition, and interactions with histones	Structures of TF/nucleosome complex also depend on the nucleosome and motif configurations. More structures of PFs vs non-PFs need to be compared to extract the systematic differences between these two classes of factors.
Measure the binding of a TF in vivo using ChIP-seq or CUT&RUN and calculate the fraction of its occupied motifs		X	PF binding is less affected by the presence of nucleosomes and therefore more likely to bind a larger fraction of their consensus motifs than non-PFs	This criterion is likely to work better with PFs that can bind independently without sequence-specific co-factors. PFs with binding partner can only occupy a small fraction of their recognition sites. Potential sensitivity to chromatin states (like heterochromatin) can further restrict the binding sites.
Measure chromatin accessibility or nucleosome occupancy change in the presence of absence of a TF through induction or differentiation		X	Chromatin accessibility increase (most commonly, ATAC-seq peak increase) in the presence of the TF is often used as a indication for its pioneering activity.	Non-PFs that bind to preexisting linkers or NDRs may also further extend the open regions and cause ATAC signal increase. This scenario needs to be differentiated from the true PFs that invade into nucleosomes.
Measure chromatin accessibility or nucleosome occupancy change over integrated synthetic oligonucleotides (ISO) ± TF motifs embedded inside a well-positioned nucleosome		X	Nucleosome depletion or chromatin accessibility increase in the presence of the TF motif indicates the pioneering activity of the TF.	This assay can provide more unambiguous evidence for nucleosome invasion. It can be used as a high throughput method to screen motifs that lead to nucleosome displacement. The factors that associate with the motifs may require further validation.

Figure 3. Pioneer binding and activity is determined by a combination of intrinsic protein characteristics and context-dependent factors. A TF will bind to and potentially open a nucleosomal site given sufficient “pioneering” features. Pioneering features can be protein-intrinsic (having a low K_D,nuc and k_off,nuc, targeting internal nucleosomal motifs, possessing a nucleosome-compatible DBD, engaging in physical histone contacts, binding to unmarked/repressive chromatin, and recruiting chromatin remodelers) or context-specific (high TF concentration and availability of cofactors).