Oct4 differentially regulates chromatin opening and enhancer transcription in pluripotent stem cells
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Evaluation Summary:
The manuscript by Xiong et al provides high resolution kinetic information on transcriptional events and enhancer activity after loss of Oct4. The authors conclude that Oct4 mainly acts as an activator whereas Sox2 maintains chromatin accessibility. These results are of interest for stem cell biologists and developmental biologists.
(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. Reviewer #3 agreed to share their name with the authors.
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Abstract
The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2.
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Author Response:
Reviewer #3 (Public Review):
In this work by Le Xiong et al., the authors focus on the role of Oct4 in activating transcription at target enhancers and genes and its ability to regulate chromatin accessibility. To do so, they used a previously established Tet-off Oct4 system to deplete Oct4 levels gradually over a period of 15 hours. They performed TT-seq and ATAC-seq experiments over this time frame with a time resolution of 3 hours. They found that eRNA transcription rapidly decreases in response to a decrease in Oct4 levels. Among the enhancers decreasing eRNA synthesis in response to a decrease in Oct4 levels, about half of them displayed a decrease in accessibility and the other half does not. They found that chromatin accessibility changes at loci that do decrease their accessibility in response to Oct4 …
Author Response:
Reviewer #3 (Public Review):
In this work by Le Xiong et al., the authors focus on the role of Oct4 in activating transcription at target enhancers and genes and its ability to regulate chromatin accessibility. To do so, they used a previously established Tet-off Oct4 system to deplete Oct4 levels gradually over a period of 15 hours. They performed TT-seq and ATAC-seq experiments over this time frame with a time resolution of 3 hours. They found that eRNA transcription rapidly decreases in response to a decrease in Oct4 levels. Among the enhancers decreasing eRNA synthesis in response to a decrease in Oct4 levels, about half of them displayed a decrease in accessibility and the other half does not. They found that chromatin accessibility changes at loci that do decrease their accessibility in response to Oct4 knockdown are delayed as compared to changes in transcriptional activity. They also find that Sox2 occupancy is maintained or decreased at loci that do not change or decrease their accessibility in response to Oct4 knockdown, respectively. From these results, they conclude that Oct4 regulates transcriptional activity but is not critical for regulation of chromatin accessibility.
The major strengths of the paper is the high quality of the experiments that assess chromatin state and acute transcriptional changes using state of the art methods. The fine kinetics of transcriptional/chromatin accessibility changes upon Oct4 removal, and the detailed dissection of how different genomic loci are temporally affected by these changes is a very valuable resource to the field of transcription at large.
The main weakness of this paper is that the central conclusions are not convincingly supported by the data, as explained below.
- Upon removal of Oct4, the authors found that some regions bound by Oct4 decrease in accessibility and some do not. However, the fact that some Oct4-bound regions do not require Oct4 to maintain their accessibility does not imply that Oct4 does not play a central role in regulating chromatin accessibility at other regions. Also note that regions bound by Oct4 but differentially dependent on Oct4 for their accessibility were described before using the same cell line (King and Klose, eLife 2017, Friman et al., eLife 2019).
We agree with the reviewer. Note, although regions bound by Oct4 but differentially dependent on Oct4 for their accessibility were described before using the same cell line (King and Klose, eLife 2017, Friman et al., eLife 2019), by combining TT-seq, ATAC-seq and examining earlier time points, we demonstrated that down-regulation of eRNA and target gene synthesis occurred earlier than a decrease in chromatin accessibility for Oct4-occupied enhancers.
- Upon removal of Oct4, the authors found that regions maintaining their accessibility maintain Sox2 binding, while regions losing accessibility lose Sox2 binding. The authors use these findings (also already described before in the refs cited above) in support of a model where Sox2 transiently maintains accessibility in the absence of Oct4. The authors do not explain why Sox2 has a differential ability to maintain its binding in these two classes of regions. No Sox2 loss of function experiments were attempted to substantiate this statement. Friman et al., eLife 2019 defined regions that depend on Oct4, Sox2 or both of them for maintenance of their accessibility using the same Oct4 Tet-off cell line, as well as a Sox2 Tet-off cell line. Le Xiong et al. did not compare this dataset to theirs nor discuss it. Importantly, upon rapid Sox2 depletion, Friman et al. showed that more than half of Oct4 binding sites retained their accessibility. Thus, functional analysis has shown that Oct4 can maintain accessibility at a large fraction of its targets in the absence of Sox2. Taken together, their data together with previous literature converge on a different model, i.e. Oct4 controls chromatin accessibility at a (large) subset of regions it binds, and thereby regulates Sox2 binding. This explains why upon Oct4 knockdown, Sox2 binding decreases at regions that lose accessibility. In contrast, at regions bound by Oct4 but independent of Oct4 for their accessibility, Sox2 binding is maintained because chromatin accessibility does not change.
We thank the reviewer for the comment. We have performed Oct4 recovery experiments which agree with the proposed alternative model. In light of the added recovery experiments and reviewer comments, we reinterpreted some of our results, clarified the role of Sox2, and modified the discussion section of the manuscript accordingly.
- King et al., eLife 2017 have shown that Oct4 directly recruits the Brg1 subunit from the BAF complex, which colocalizes strongly with Oct4-bound regions in ES cells. This strongly suggests a direct role for Oct4 in the regulation of chromatin accessibility.
We agree.
- Friman et al., eLife 2019, performed rapid depletion of Oct4 using an Auxin-inducible system, and they observed a loss of accessibility at a large number of Oct4-bound regions that is quasi-synchronized with Oct4 loss. This also argues that Oct4 directly regulates chromatin accessibility.
We agree.
- Using the Tet-off Oct4 cell line, the authors observed a delayed loss of chromatin accessibility as compared to changes in transcriptional activity. From this observation, they conclude that Oct4 is a not crucial for regulating chromatin accessibility at these loci. However, this inference can only be true if there is an identical concentration-dependent activity of Oct4 in transcriptional activation and pioneer activity. Importantly, there is no reason to assume that this is the case. Transcriptional activity changes in response to changes in Oct4 levels might be very sensitive to slight decreases in Oct4 levels. Chromatin accessibility as observed by ATAC-seq might only start to decrease once Oct4 levels go below a certain threshold. In fact, it was reported (Strebinger et al., Molecular Systems Biology 2019) that cells with low endogenous Oct4 levels do not show changes in chromatin accessibility at pluripotency enhancers. This suggests that chromatin accessibility is relatively resilient to mild changes in Oct4 concentrations, which is what occurs after 3 hours of dox treatment in the present study.
We agree.
- The conclusions on the minor role of Oct4 in regulating chromatin accessibility are also weakened by the absence of Oct4 recovery experiments (i.e. dox treatment for 15-24 hours, and dox removal to re-express Oct4). In fact, Auxin-inducible degradation followed by recovery of Oct4 levels as well as recovery of Oct4 levels after mitotic degradation have shown to allow partial recovery in chromatin accessibility at a large number of Oct4-bound regions (Friman et al., eLife 2019). This also suggests a direct role for Oct4 in opening chromatin.
We thank the reviewer for the suggestion. We have performed Oct4 recovery experiments.
In summary, the data described in this paper are definitely very valuable. Their results allow to quantitatively describe the differential timing/sensitivity of transcriptional changes vs accessibility changes upon Oct4 knockdown, which is clearly new and insightful to understand the interplay between different mechanisms by which transcription factors regulate gene expression. A re-interpretation of this data could thus make this manuscript even more interesting.
We thank the reviewer for the critical review and the insightful suggestions that helped us to improve the manuscript.
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Evaluation Summary:
The manuscript by Xiong et al provides high resolution kinetic information on transcriptional events and enhancer activity after loss of Oct4. The authors conclude that Oct4 mainly acts as an activator whereas Sox2 maintains chromatin accessibility. These results are of interest for stem cell biologists and developmental biologists.
(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. Reviewer #3 agreed to share their name with the authors.
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Reviewer #1 (Public Review):
While the work is of great interest, some of the key conclusions are not supported by the data as the manuscript stands, due to the progressive loss (rather than dissaperance) of Oct4 activity, the possible differences among regions that require or not Oct4 binding and the role of Sox2, which requires experimental support.
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Reviewer #2 (Public Review):
In this manuscript, the authors attempt to identify the essential role of Oct4 in mouse ESCs. They utilized existing Tet-OFF Oct4 ESCs and looked for changes in gene expression, enhancer RNA expression and enhancer chromatin accessibility in a timecourse while depleting Oct4. This led to the discovery that many enhancer elements bound by Oct4 showed downregulation of enhancer RNAs following loss of Oct4. This typically occurred before reduction of gene expression and before or in the absence of loss of chromatin accessibility. They further showed that Sox2 remained bound at high levels for some time at enhancers that proceeded to be inactivated, while enhancers at which Oct4 was not required for activity retained Sox2 binding throughout the timecourse.
The in-depth analysis of nascent enhancer RNA expression …Reviewer #2 (Public Review):
In this manuscript, the authors attempt to identify the essential role of Oct4 in mouse ESCs. They utilized existing Tet-OFF Oct4 ESCs and looked for changes in gene expression, enhancer RNA expression and enhancer chromatin accessibility in a timecourse while depleting Oct4. This led to the discovery that many enhancer elements bound by Oct4 showed downregulation of enhancer RNAs following loss of Oct4. This typically occurred before reduction of gene expression and before or in the absence of loss of chromatin accessibility. They further showed that Sox2 remained bound at high levels for some time at enhancers that proceeded to be inactivated, while enhancers at which Oct4 was not required for activity retained Sox2 binding throughout the timecourse.
The in-depth analysis of nascent enhancer RNA expression and chomatin accessibility changes during a timecourse following loss of Oct4 are interesting and provide insights into the order of events that occur during enhancer decommissioning. However, there are several key limitations to this study with regard to identifying the primary function of Oct4 in the control of pluripotency, and as such several of the key conclusions may be somewhat overstated. -
Reviewer #3 (Public Review):
In this work by Le Xiong et al., the authors focus on the role of Oct4 in activating transcription at target enhancers and genes and its ability to regulate chromatin accessibility. To do so, they used a previously established Tet-off Oct4 system to deplete Oct4 levels gradually over a period of 15 hours. They performed TT-seq and ATAC-seq experiments over this time frame with a time resolution of 3 hours. They found that eRNA transcription rapidly decreases in response to a decrease in Oct4 levels. Among the enhancers decreasing eRNA synthesis in response to a decrease in Oct4 levels, about half of them displayed a decrease in accessibility and the other half does not. They found that chromatin accessibility changes at loci that do decrease their accessibility in response to Oct4 knockdown are delayed as …
Reviewer #3 (Public Review):
In this work by Le Xiong et al., the authors focus on the role of Oct4 in activating transcription at target enhancers and genes and its ability to regulate chromatin accessibility. To do so, they used a previously established Tet-off Oct4 system to deplete Oct4 levels gradually over a period of 15 hours. They performed TT-seq and ATAC-seq experiments over this time frame with a time resolution of 3 hours. They found that eRNA transcription rapidly decreases in response to a decrease in Oct4 levels. Among the enhancers decreasing eRNA synthesis in response to a decrease in Oct4 levels, about half of them displayed a decrease in accessibility and the other half does not. They found that chromatin accessibility changes at loci that do decrease their accessibility in response to Oct4 knockdown are delayed as compared to changes in transcriptional activity. They also find that Sox2 occupancy is maintained or decreased at loci that do not change or decrease their accessibility in response to Oct4 knockdown, respectively. From these results, they conclude that Oct4 regulates transcriptional activity but is not critical for regulation of chromatin accessibility.
The major strengths of the paper is the high quality of the experiments that assess chromatin state and acute transcriptional changes using state of the art methods.
The fine kinetics of transcriptional/chromatin accessibility changes upon Oct4 removal, and the detailed dissection of how different genomic loci are temporally affected by these changes is a very valuable resource to the field of transcription at large.The main weakness of this paper is that the central conclusions are not convincingly supported by the data, as explained below.
1. Upon removal of Oct4, the authors found that some regions bound by Oct4 decrease in accessibility and some do not. However, the fact that some Oct4-bound regions do not require Oct4 to maintain their accessibility does not imply that Oct4 does not play a central role in regulating chromatin accessibility at other regions.
Also note that regions bound by Oct4 but differentially dependent on Oct4 for their accessibility were described before using the same cell line (King and Klose, eLife 2017, Friman et al., eLife 2019).2. Upon removal of Oct4, the authors found that regions maintaining their accessibility maintain Sox2 binding, while regions losing accessibility lose Sox2 binding. The authors use these findings (also already described before in the refs cited above) in support of a model where Sox2 transiently maintains accessibility in the absence of Oct4. The authors do not explain why Sox2 has a differential ability to maintain its binding in these two classes of regions. No Sox2 loss of function experiments were attempted to substantiate this statement.
Friman et al., eLife 2019 defined regions that depend on Oct4, Sox2 or both of them for maintenance of their accessibility using the same Oct4 Tet-off cell line, as well as a Sox2 Tet-off cell line. Le Xiong et al. did not compare this dataset to theirs nor discuss it. Importantly, upon rapid Sox2 depletion, Friman et al. showed that more than half of Oct4 binding sites retained their accessibility. Thus, functional analysis has shown that Oct4 can maintain accessibility at a large fraction of its targets in the absence of Sox2.
Taken together, their data together with previous literature converge on a different model, i.e. Oct4 controls chromatin accessibility at a (large) subset of regions it binds, and thereby regulates Sox2 binding. This explains why upon Oct4 knockdown, Sox2 binding decreases at regions that lose accessibility. In contrast, at regions bound by Oct4 but independent of Oct4 for their accessibility, Sox2 binding is maintained because chromatin accessibility does not change.3. King et al., eLife 2017 have shown that Oct4 directly recruits the Brg1 subunit from the BAF complex, which colocalizes strongly with Oct4-bound regions in ES cells. This strongly suggests a direct role for Oct4 in the regulation of chromatin accessibility.
4. Friman et al., eLife 2019, performed rapid depletion of Oct4 using an Auxin-inducible system, and they observed a loss of accessibility at a large number of Oct4-bound regions that is quasi-synchronized with Oct4 loss. This also argues that Oct4 directly regulates chromatin accessibility.
5. Using the Tet-off Oct4 cell line, the authors observed a delayed loss of chromatin accessibility as compared to changes in transcriptional activity. From this observation, they conclude that Oct4 is a not crucial for regulating chromatin accessibility at these loci.
However, this inference can only be true if there is an identical concentration-dependent activity of Oct4 in transcriptional activation and pioneer activity. Importantly, there is no reason to assume that this is the case. Transcriptional activity changes in response to changes in Oct4 levels might be very sensitive to slight decreases in Oct4 levels. Chromatin accessibility as observed by ATAC-seq might only start to decrease once Oct4 levels go below a certain threshold. In fact, it was reported (Strebinger et al., Molecular Systems Biology 2019) that cells with low endogenous Oct4 levels do not show changes in chromatin accessibility at pluripotency enhancers. This suggests that chromatin accessibility is relatively resilient to mild changes in Oct4 concentrations, which is what occurs after 3 hours of dox treatment in the present study.6. The conclusions on the minor role of Oct4 in regulating chromatin accessibility are also weakened by the absence of Oct4 recovery experiments (i.e. dox treatment for 15-24 hours, and dox removal to re-express Oct4). In fact, Auxin-inducible degradation followed by recovery of Oct4 levels as well as recovery of Oct4 levels after mitotic degradation have shown to allow partial recovery in chromatin accessibility at a large number of Oct4-bound regions (Friman et al., eLife 2019). This also suggests a direct role for Oct4 in opening chromatin.
In summary, the data described in this paper are definitely very valuable. Their results allow to quantitatively describe the differential timing/sensitivity of transcriptional changes vs accessibility changes upon Oct4 knockdown, which is clearly new and insightful to understand the interplay between different mechanisms by which transcription factors regulate gene expression. A re-interpretation of this data could thus make this manuscript even more interesting.
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