A test of the pioneer factor hypothesis using ectopic liver gene activation
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Evaluation Summary:
How transcription factors access their DNA binding motifs in chromatin and cooperate with other transcription factors in DNA binding remains a contentious question. It is clear that some transcription factors ("pioneer transcription factors") play a dominant role in opening chromatin during development and reprogramming, but it has also been clear that the ability of transcription factors to do so lies on a spectrum, that pioneer transcription factors may mutually interact with other transcription factors in their pioneering activity and that their mode of binding is still poorly understood. This manuscript's presentation attempts to refute an overly simplified pioneer factor hypothesis. Overall, this is an important topic and the authors use a good experimental approach, but the analyses are limited and the interpretation too simplified.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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.
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Evaluation Summary:
How transcription factors access their DNA binding motifs in chromatin and cooperate with other transcription factors in DNA binding remains a contentious question. It is clear that some transcription factors ("pioneer transcription factors") play a dominant role in opening chromatin during development and reprogramming, but it has also been clear that the ability of transcription factors to do so lies on a spectrum, that pioneer transcription factors may mutually interact with other transcription factors in their pioneering activity and that their mode of binding is still poorly understood. This manuscript's presentation attempts to refute an overly simplified pioneer factor hypothesis. Overall, this is an important topic and the authors use a good experimental approach, but the analyses are limited and the …
Evaluation Summary:
How transcription factors access their DNA binding motifs in chromatin and cooperate with other transcription factors in DNA binding remains a contentious question. It is clear that some transcription factors ("pioneer transcription factors") play a dominant role in opening chromatin during development and reprogramming, but it has also been clear that the ability of transcription factors to do so lies on a spectrum, that pioneer transcription factors may mutually interact with other transcription factors in their pioneering activity and that their mode of binding is still poorly understood. This manuscript's presentation attempts to refute an overly simplified pioneer factor hypothesis. Overall, this is an important topic and the authors use a good experimental approach, but the analyses are limited and the interpretation too simplified.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The title of the manuscript is overly broad and does not reflect the specific experiments presented in this manuscript. Specifically, this manuscript narrowly evaluates the ability of the two factors FOXA1 and HNF4A to either independently or cooperatively activate inaccessible regulatory DNA elements in the K562 human cancer cell line when overexpressed at likely non-physiological levels. Given the narrow scope of the experiments presented in this manuscript, it is disingenuous to state that this manuscript is actually "A Test of the Pioneer Factor Hypothesis", which implies a much broader scope.
Overall, their data supports the notion that in K562 cells, overexpressed FOXA1 acts as a pioneering factor, and overexpressed HNF4A can similarly act as a pioneering factor. The authors rightly state that the …
Reviewer #1 (Public Review):
The title of the manuscript is overly broad and does not reflect the specific experiments presented in this manuscript. Specifically, this manuscript narrowly evaluates the ability of the two factors FOXA1 and HNF4A to either independently or cooperatively activate inaccessible regulatory DNA elements in the K562 human cancer cell line when overexpressed at likely non-physiological levels. Given the narrow scope of the experiments presented in this manuscript, it is disingenuous to state that this manuscript is actually "A Test of the Pioneer Factor Hypothesis", which implies a much broader scope.
Overall, their data supports the notion that in K562 cells, overexpressed FOXA1 acts as a pioneering factor, and overexpressed HNF4A can similarly act as a pioneering factor. The authors rightly state that the HNF4A results are surprising given its prior status as a non-pioneering factor. However, it may be the case that HNF4A acts as a pioneering factor in K562 cells when overexpressed, but may not have pioneering activity in other tissues or samples, or when expressed to a lesser degree. Similarly, it is possible that HNF4A is cooperatively interacting with other TFs that are endogenously expressed in K562 cells. The authors need to discuss the possible limitations of their experimental setup.
The authors need to attempt to reconcile their findings with prior studies showing HNF4A as a non-Pioneering factor. What is potentially causing these divergent conclusions?
Similarly, please clarify what specific data is used to draw the conclusion that "... the mode of action of these targets did not conform to the sequential activity predicted by the PFH." If anything, Figure 4 presents data that a substantial proportion of both FOXA1 and HNF4A elements require cooperative occupancy of both of these factors to be opened.
Pg. 16, line 313-5. "Pioneer activity may best be summarized then by the free energy balance between TFs, nucleosomes and DNA ... rather than as a property of specific classes of TFs." As discussed above, this conclusion seems overly broad given the data presented only applied to two factors in a single cell line.
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Reviewer #2 (Public Review):
How transcription factors access their DNA binding motifs in chromatin and cooperate with other transcription factors in DNA binding remains a contentious question. It is clear that some transcription factors ("pioneer transcription factors") play a dominant role in opening chromatin during development and reprogramming. But it has also been clear that the ability of such transcription factors to do so lies on a spectrum, that pioneer transcription factors may mutually interact with other transcription factors in their pioneering activity (e.g. Swinstead et al. 2016) and that their mode of binding is still poorly understood (see recent crystal structures).
This study clearly illustrates our lack of understanding and thus provides impetus for an important discussion. I particularly like that the study uses de …
Reviewer #2 (Public Review):
How transcription factors access their DNA binding motifs in chromatin and cooperate with other transcription factors in DNA binding remains a contentious question. It is clear that some transcription factors ("pioneer transcription factors") play a dominant role in opening chromatin during development and reprogramming. But it has also been clear that the ability of such transcription factors to do so lies on a spectrum, that pioneer transcription factors may mutually interact with other transcription factors in their pioneering activity (e.g. Swinstead et al. 2016) and that their mode of binding is still poorly understood (see recent crystal structures).
This study clearly illustrates our lack of understanding and thus provides impetus for an important discussion. I particularly like that the study uses de novo chromatin accessibility in vivo as a readout for pioneering activity. There has been much effort into measuring the ability to bind nucleosomes in vitro or their behavior in imaging assays in vivo, but ultimately the more relevant assay for understanding enhancer function is the assay the authors performed, and this is not done often enough. Having said that, the manuscript's presentation relies on refuting an overly simplified pioneer factor hypothesis. Furthermore, it is doing so by only highlighting the inconsistencies of a single transcription factor, Hnf4a, without providing new evidence for a more consistent hypothesis. There is insufficient genome-wide evidence to support the suggestion that Foxa1 and Hnf4a's pioneering activity at individual sequences is linked to affinity or sequence context. Overall, this is an important topic and a good experimental approach, but the limited analysis and overly simplified interpretation limits the scope of the discussion.
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