OTX2 controls chromatin accessibility to direct somatic versus germline differentiation

  1. Elisa Barbieri
  2. Ian Chambers

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Abstract

The choice between somatic and germline fates is essential for species survival. This choice occurs in embryonic epiblast cells, as these cells are competent for both somatic and germline differentiation. The transcription factor OTX2 regulates the choice between somatic and germline fates, as Otx2 -null epiblast-like cells (EpiLCs) form primordial germ cell-like cells (PGCLCs) with enhanced efficiency. However, the mechanisms by which OTX2 achieves this function are not fully characterized. Here we show that OTX2 controls chromatin accessibility at specific chromatin loci to modulate gene transcription and enable somatic differentiation. By performing CUT&RUN for OTX2 and ATAC-seq in wild-type and Otx2 -null embryonic stem cells and EpiLCs, we identified regions where OTX2 binding opens chromatin, either alone or in cooperation with a ZIC family protein. Enforced OTX2 expression maintains accessibility at these regions and, in addition, induces opening of ∼4,000 somatic-associated regions in cells differentiating in the presence of PGC-inducing cytokines. Once cells have acquired germline identity, these additional somatic-associated regions can no longer respond to OTX2 and remain closed. Our results indicate that OTX2 works in cells with dual competence for both somatic and germline differentiation to increase accessibility of somatic regulatory regions and induce the somatic fate at the expense of the germline.

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    Reply to the reviewers

    1. General Statements [optional]

    *We thank the reviewers for finding the manuscript enjoyable and well-written, with experiments that were performed well, show solid results and provide useful data for the community. The reviewers have provided meaningful feedback to improve this study. We have addressed the comments point-by-point below. The main text will also be further modified to incorporate new analysis where it has not yet been done. *

    2. Description of the planned revisions

    Reviewer 1:

    Summary OTX2 is a pivotal transcription factor that regulates the fate choice between somatic and primordial germ cell (PGC) lineages in early mouse development. In the current study, the authors use in vitro stem cell models to demonstrate that OTX2 mediates this developmental fate decision through controlling chromatin accessibility, whereby OTX2 helps to activate putative enhancers that are associated with somatic fate. By extension, those somatic-associated regulatory regions therefore become inaccessible in cells adopting PGC identity in which Otx2 is downregulated.

    Comments I enjoyed reading this manuscript. The experiments have been carried out well and for the most part the results provide convincing evidence to support the claims and conclusions in the manuscript. I particularly liked the experiments using the inducible Otx2 transgene to examine the acute changes in chromatin accessibility following restoration of OTX2.

    I include some suggestions below to the authors for additional analyses that I feel would further strengthen their study.

    I also felt that the authors focus almost exclusively on the subset of OTX2-bound sites that lose accessibility in the absence of OTX2. But, as they show in several figure panels, these sites tend to be the minority and that most OTX2-occupied sites do not lose accessibility in Otx2-null cells (actually, more sites tend to gain accessibility). I encourage the authors to modify the text and some of the analyses to give a better balance to their study. We are pleased that this reviewer enjoyed our manuscript. As suggested by the reviewer, we included analyses on the regions that are bound by OTX2 but do not show an increase in accessibility (see section 3 reviewer 1 point 6). The text will be expanded to include the new data and to include the description of the subset of OTX2 sites that do not show accessibility changes in the absence of OTX2. We have responded to other points they raised as detailed in the sections below

    Figure 1: The authors write: "...OTX2 binds mostly to putative enhancers." Whether these distal sites are enhancers is not sufficiently evidenced in the manuscript, but it is important information to collect to support their model of OTX2 function. The authors should strengthen their analysis by examining whether OTX2 peaks are enriched at previously defined enhancer regions.

    We plan to compare OTX2 bound regions with defined lists of enhancers identified in ESCs grown in Serum/LIF (e.g. Whyte et al 2013) and, if available, in 2i/LIF and EpiLCs. We will also analyse publicly available datasets for H3K4me1 (enhancer marker) and H3K27ac (marker of active regulatory regions) at the regions bound by OTX2 in ESCs and EpiLCs.

    Figure 2: I'm still puzzled why the authors did not examine flow-sorted WT+cyto cells?

    *We agree with the reviewer that it would be interesting to examine flow-sorted WT +cyto PGCLCs. Unfortunately, the expression of CD61 and SSEA1 only becomes visible from day 4 of PGCLC differentiation. Therefore, we were not able to isolate PGCLC at day 2 from WT cells differentiated in the presence of cytokines. We then used OTX2-/- cells at day 2 to model PGCLCs. This is based on the assumption that because day 6 Otx2-/- PGCLCs are transcriptionally similar to sorted day 6 WT cells (Zhang, Zhang et al Nature 2018), the same will be true at day 2. We will modify the text in the final version of this manuscript to clarify this point that has also been raised by reviewers 2 and 3. *

    Figure 3: I would be tempted to put Figure S3A and S3B into Figure 3. It would be better to show all 1246 DARs together, either ordered by OTX2 CT&RUN signal, or presented in two pre-defined groups (OTX2-bound vs unbound). I also suggest that the author show OTX2 signals and ATAC-seq signals for the 3028 DARs that gain accessibility in Otx2-null EpiLCs (this could be added to a supplemental figure).

    Although the analysis has been carried out and the figures have been amended, the main text will be modified in a future updated version of the manuscript to incorporate these results.

    Figure 3: What is special about the 8% of OTX2-bound site that lose accessibility, versus the 92% of sites that do not?

    *The 8% of the OTX2-bound regions that lose accessibility in the absence of OTX2 appear to be more sensitive to the loss of OTX2. One possible explanation is that the accessibility of the rest of OTX2 bound regions relies on other TFs, such as OCT4, that are expressed in EpiLCs. We will modify the main text to discuss this interesting point raised by the reviewer. *

    Figure 6F: If the 4221 sites are split into those bound by OTX2 versus those that are not (related to Figure 6C) then is there a difference? i.e. are the OTX2-bound sites opening up?

    We separated the 4,221 sites in OTX2 bound and unbound. The result is reported below:

    *Although there is a slight increase in accessibility in the OTX2 bound subset, the average accessibility reaches less than ¼ of the accessibility of these regions when OTX2 is present from day 0 to day 4, while the OTX2 unbound regions do not show an increase in accessibility. Although we can not rule out that a longer treatment with tamoxifen may lead to higher accessibility in the OTX2 bound subset, the dynamics are extremely slower compared to the EpiLC regions where accessibility reaches 50% of the d0-d4 sample in just 1 hour of tamoxifen treatment. *

    Is there any evidence that OTX2 binds and compacts PGCLC enhancers in somatic cells? I appreciate this is different to the main thrust of the authors' model, but being able to show that OTX2 does not compact these sites lends further support to their preferred model of OTX2 opening sites of somatic lineages.

    *Comparing the ATAC-seq in PGCLCs with ESCs and EpiLCs, we identify a subset of regions that are open in PGCLC only (PGCLC-specific accessible regions, see below). These regions do not show binding of OTX2 in WT EpiLCs or the d0-d2 Tam sample, suggesting that OTX2 does not bind and compact PGCLC-specific enhancers. *

    PGCLC-specific regions showing high accessibility only in PGCLCs.

    • OTX2 CUT&RUN signal in WT EpiLC, OTX2-ERT2 PGCLCs in presence or absence of Tamoxifen, showing that OTX2 does not bind PGCLC-specific regions even when it is overexpressed in GK15 medium.*

    *These analyses will be incorporated in the manuscript. *

    Discussion: Have prior studies established a connection between OTX2 and chromatin remodellers that can open chromatin? Or, if not, then perhaps this could be proposed as a line of future research.

    We thank the reviewer for suggesting to amplify the discussion on the possible connection between OTX2 and chromatin remodellers. Although there is no evidence in the literature of a direct interaction between OTX2 and chromatin remodellers, this can not be excluded. The connection might also be indirect: OTX2 is known to interact with OCT4, which in turn interacts and recruits to chromatin the catalytic subunit of the SWI/SNF complex, BRG1. This point will be discussed in a modified version of the manuscript.

    Reviewer 2:

    Barbieri and Chambers explore the role of OTX2 on mouse pluripotency and differentiation. To do so, they examine how the chromatin accessibility and OTX2 binding landscape changes across pluripotency, the exit of pluripotency towards formative and primed states, and through to PGCLC/somatic differentiation. The work mostly represents a resource for the community, with possible implications for our understanding of how OTX2 might mediate the germline-soma switch of fates. While the findings of the work are modest, the results seem solid and the manuscript is clear and well-written.

    *We are pleased that this reviewer found our results solid and the manuscript clear. *

    I have some comments as indicated below:

    1. The comparison between Otx2-/- cells in the presence of PGCLC cytokines compared to WT cells in the absence of cytokines seems like it is missing controls to me. I assume the authors wanted to enable homogeneous populations to facilitate their bulk sequencing methods, but it seems to me like they are comparing apples with oranges. It would have been better to have the reciprocal situations (Otx2-/- cells in basal differentiation medium, and WT cells in PGCLC cytokines) with a sorting strategy to better unpick the differences between the presence and absence of Otx2 in the 2 protocols. Having said that, the authors are careful not to draw many comparisons between those populations so I don't think this omission affects their current claims. They should however clarify whether the flow cytometry (Supp Fig2) was used for sorting cells or if all cells were taken for bulk sequencing. *We agree with the reviewer that it would be of interest to compare the PGCLC and somatic population derived from the OTX2-/- cells in GK15 without cytokines with the same populations derived from WT cells differentiated in the presence of cytokines. Our work aims to identify what happens at the stages of PGCLC differentiation when cells are still competent for both germline and somatic differentiation. Previous work from the lab showed that this dual competence is lost after day 2, therefore we focus our attention on this time of differentiation. Unfortunately, the two surface markers characteristics of PGCs (CD61 and SSEA1) are not expressed at day2 and, therefore we are not able to sort PGCLCs derived from OTX2-/- cells in GK15 without cytokines or WT cells differentiated in the presence of cytokines. As recognised by this reviewer, we aimed to obtain two homogenous populations that can model PGCLCs and somatic cells. This is based on data obtained at day 6 when Otx2-/- PGCLCs show a similar transcriptome to sorted day 6 WT cells (Zhang, Zhang et al Nature 2018) and the assumption that the same will be true at day 2. We will clarify that the supplementary Figure 2 is not a sorting strategy. As this point has been raised by reviewers 1 and 2 as well, we will modify the text to clarify the choice and the assumption behind using OTX2-/- cells in the presence of cytokines and WT cells in the absence of cytokines to model PGCLCs and somatic cells respectively. *

    Throughout the text, the authors subject cells (WT / Otx2-/- /Otx2ER ) to different protocols to look at accessibility and Otx2 binding, but with no mention of the cell fate differences that occur in these different conditions. For instance, it is unclear to me to which fate the WT cells without PGCLC cytokines go - I presume this is neural but perhaps this is a mixed fate, given that they are in GK15 rather than N2B27. Likewise, the OTX2ER experiments may promote a mixed population between PGCLC/somatic fates, and this is never described. Ideally transcriptomic data would be collected, but failing that, qPCR data should be obtained to examine this more closely.

    *We are planning to generate RT-qPCR data for germ layer markers (ectoderm, endoderm and mesoderm) in WT cells in GK15 without cytokines at day 2, as well as OTX2-ERT2 cells with and without Tamoxifen at day 2 (noTam, d0-d2) and day 4 (no Tam, d0-d4). *

    The authors also state that "OTX2 facilitates Fgf5 transcription' (page10) but provide no transcriptional data to substantiate this claim. Again RT-qPCR would help make this point.

    *We will analyse the level of Fgf5 by RT-qPCR in OTX2-ERT2 EpiLCs treated for 1 hour and 6 hours with Tamoxifen to show the effect of OTX2 on Fgf5 transcription. *

    It is unclear to me what the 'increase[d] accessibility' (eg abstract final sentence, Figure 3E) really means at the cellular level. Does this indicate that more cells have this site open, and does this have implications for the heterogeneity of cell fates observed? Since the authors are concerned with fate decisions, this seems like an important consideration that should at least be discussed.

    The possibility that the increased accessibility is due to higher heterogeneity in the population is interesting and it will be included in the discussion in a revised version of the manuscript.

    Reviewer 3:

    In this manuscript, the authors perform OTX2 CUT&RUN and ATAC-seq in Otx2-null and WT ESCs, EpiLCs and PGCLCs to understand whether the role of OTX2 in restricting mouse germline entry that they previously described (Zhang Nature 2018) mechanistically depends on chromatin remodeling. They identify differentially accessible regions (DARs) between Otx2-null and WT cells at different stages of differentiation and show that many of these are OTX2 bound in WT. They then show using cells expressing OTX2-ER^T2 in Otx2-null Epiblast cells that when OTX2 is moved into the nucleus, the regions that were differentially closed in Otx2-null open within an hour, suggesting chromatin accessibility is directly controlled by OTX2 (rather than indirect effects involving transcription and translation which one would expect to take longer). The scope is narrow, but this is nice work and useful data for the mouse PGC field. However, there are a few places where the data could be strengthened, and the writing is a little confusing in places, for example by stating as fact in early sections what is not proven until later.

    We thank the reviewer for finding our work nice and useful for the mouse PGC field, and for the useful comments to improve the manuscript. We have included new analysis and modified the text as suggested to improve the writing, avoiding early statements that were not fully proven until later in the manuscript. We have responded to other points they raised as detailed below and in the next section.

    1. "we compared Otx2-/- cells cultured in the presence of PGC-promoting cytokines with wild-type cells cultured in the absence of PGC-promoting cytokines. Under these conditions Otx2-/- cells produce an essentially pure (>90%) CD61+/SSEA1+ population that we refer to as PGCLCs, while wild-type cells yield a cell population from which PGCLCs are absent"

    This is not a controlled comparison since one cannot separate the day 2 effect of cytokines from that of the Otx2 knockout. The manuscript would be strengthened if the authors include WT somatic and PGCLCs from the +cytokine conditions, which could be easily sorted out as shown in Supp. Fig. 2. Ideally they would also include Otx2-null somatic cells, although Supp. Fig. 2 shows those are rare under the conditions considered.

    *This work aimed to analyse early stages of EpiLC to PGCLC differentiation when cells are still competent for both somatic and germline differentiation. This stage has been described previously to be at day 2 of differentiation in GK15 + cytokines (PGCLC differentiation medium, Zhang, Zhang et al, Nature 2018). Unfortunately, CD61 and SSEA1 are not expressed at day 2 of PGCLC differentiation, and they start to be expressed on the cell surface by day 4. Consequently, it is impossible to sort cells at day 2 using the CD61+/SSEA1+ strategy. To overcome this problem, we used WT cells grown in GK15 without cytokines to model a population of somatic cells and OTX2-/- cells grown in GK15+ cytokines to model a homogeneous population of PGCLCs. As explained in a similar point raised by reviewers 2 and 3, we assumed that, as OTX2-/- cells grown in the presence of cytokines are transcriptionally similar to sorted WT cells at day 6 (Zhang, Zhang et al, Nature 2018), OTX2-/- cells at day 2 are similar to their WT counterpart at day 2. The main text will be modified to clarify that we are using homogeneous populations to model both PGCLC and somatic cells and that Figure S2 does not show a sorting strategy. *

    1. "In ESCs, OTX2 binds We are planning to perform a statistical analysis to ascertain that the small number of DARs bound by OTX2 are or are not bound by chance by OTX2.
    1. It would be good if the discussion was broadened to include both human and other transcription factors that are involved. How much of these conclusions could one expect to carry over to human or other mammals? There is some work from the Surani lab considering OTX2 in human. One could even look at published ATAC or OTX2 chip-seq data in hPSCs and potentially learn something interesting. Furthermore, there are studies on other transcription factors modulating chromatin accessibility in the decision between germline and somatic cells, for example PRDM1, PRDM14 (refs in e.g. Tang et al Nat Rev Gen 2016) or TFAP2A (at least in human (Chen et al Cell Rep 2019)). Do these factors affect the same genes? Is a coherent picture emerging of their respective roles in germline entry?

    *As suggested by the reviewer, we will discuss the role of OTX2 in human PGCLC formation and include studies on PGC-specific transcription factors concerning changes in chromatin accessibility in germline and somatic cells. This will be included in a revised version of the manuscript. *

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer 1:

    1. Figure 1: The authors report in the methods that they performed OTX2 CUT&RUN in biological duplicates. It would strengthen their results if they showed in Figure S1 some representative data from each replicate separately to show the consistency. As suggested by the reviewer to show consistency between replicates, two representative tracks of the two CUT&RUN replicates at the Tet2 (ESCs) and Fgf5 (EpiLCs) loci have been included in Figure S1A. The corresponding tracks of the average bigwig files are reported in Figure 1E. The main text (page 5) and the figure legends have been amended to incorporate the new panels.

    Figure 2: I think it would be helpful to remind the reader here that Otx2 is normally downregulated in PGCs, and that Otx2 expression is maintained (at least initially) in somatic cells. This would help explain the logic behind the choice of samples that were profiled.

    *We modified the text with the following sentence, as suggested by the reviewer, emphasising the level of OTX2 in early somatic vs early PGCLCs: “Otx2 expression is rapidly downregulated in the EpiLC to PGCLC transition while its expression is maintained longer in cells entering the somatic lineage [8]” (page 7). *

    Figure 2D: I appreciate that the highlighted region at the Tet2 locus is a DAR, but from the genome tracks it looks as though the region still has high accessibility. Are there any other examples to exemplify a more obvious DAR? Additionally, since twice as many DARs gain accessibility in Otx2-null ESCs compared to lose accessibility, why not show examples of these as well? The same is true of EpiLCs. (Or alternatively, provide a good explanation for why not to show these other categories)

    We substituted the Tet2 DAR with a more clear example of ESC DAR located in the Hes1 locus that shows low accessibility in Otx2-/- ESCs versus WT ESCs. Examples of ESC DARs and EpiLC DARs that show higher accessibility in Otx2-/- vs WT cells have been added as new panels 2E (DAR in Pebp4 locus) and 2G (DAR in Tdh locus). We also simplified the panels showing only ATAC-seq tracks in WT and OTX2-/- cells, either ESCs (2D-E) or EpiLCs (G-H). Text and figure legends have been modified to accommodate the changes made in Figure 2.

    Figure 3: I would be tempted to put Figure S3A and S3B into Figure 3. It would be better to show all 1246 DARs together, either ordered by OTX2 CT&RUN signal, or presented in two pre-defined groups (OTX2-bound vs unbound). I also suggest that the author show OTX2 signals and ATAC-seq signals for the 3028 DARs that gain accessibility in Otx2-null EpiLCs (this could be added to a supplemental figure).

    Figures S3A and S3B have been moved to the main figure. Figure S3A is now part of Figure 3C, where all the 1,246 DARs are shown together, separated into two groups (OTX2-bound and -unbound). Figure S3B is now part of Figure 3F. A new heatmap showing the OTX2 and ATAC-seq signals for the 3028 regions that gain accessibility in Otx2-/- EpiLCs has been added as new Figure S3B. Only 28 out of the 3,028 regions overlap an OTX2 peak as shown in the new Figure S3A. These regions appear to be already open in ESCs (Figure S3C) and they do not fully close when OTX2 is absent. This can be explained by either a) the lack of expression of an OTX2 target gene that represses these regions or b) the continuous expression of a gene that is usually repressed by OTX2 in the transition to EpiLCs. In both cases, OTX2 does not directly repress these regions. Figure legends have been amended to incorporate the new panels. The main text will be modified to incorporate these results.

    Figure 6: Do the PGCLCs with OTX2 expression have chromatin accessibility profiles similar to somatic cells? Consider adding WT somatic cell data to Figure 6A, which could be an interesting comparison with the Tam d0-d2 samples.

    *The heatmap showing the ATAC-seq signal at the additional OTX2-induced regions in somatic cells has been added to Figure 6A. The data show that the regions induced by OTX2 are not open in somatic cells generated in GK15. One possible explanation is the overexpression of OTX2 induces the opening of neural-associated regions, but neural differentiation is not fully supported in GK15 medium (see reviewer 2, point 3). As suggested by reviewer 2, we will perform RT-qPCR of germ layer markers to analyse the identity of somatic cells grown in GK15 (without cytokines) and somatic cells induced by OTX2 overexpression. *

    Reviewer 2:

    The authors focus solely on the activating role of Otx2 in their data, but given the substantial proportion of DARs that decrease following Otx2 depletion, I presume it is possible that it also has a repressive effect? Either way, this should be discussed.

    *As also suggested by reviewer 1 (point 6), we analysed the accessibility level and the OTX2 signal at the 3,028 regions that gain accessibility in Otx2-/- EpiLCs (new Figure S3A-C). These regions show high accessibility in ESCs suggesting that these are ESC regions that do not close properly in the transition to EpiLCs in the absence of OTX2. OTX2 CUT&RUN show a low to absent signal at these regions, with just 28 regions overlapping EpiLCs DARs that show higher accessibility in Otx2-/- cells, suggesting that OTX2 does not have a direct suppressive effect on them. *

    The authors state that d2 PGCLCs "show an intermediate position between ESCs and EpiLCs" based on the PCA location. They should be careful to qualify that this is only in the first 2 principal components, because it may well be the case (and is likely) that in other components the PGCLC population is far removed from the pluripotent states.

    • The text has been updated as follows: d2 PGCLCs “show an intermediate position between ESCs and EpiLCs on both PC1 and PC2”.*

    Reviewer2 Minor Suggestions:

    Presumably the regions bound by OTX2 in Tet2, Mycn and Fgf5 (Fig1E) are called enhancers because these are known from existing literature. It would be helpful to cite the relevant references to this in the text for those unfamiliar with these. References (Whyte et al, Cell, 2018 – Tet2 and Mycn, Buecker et al, Cell Stem Cell, 2013, Thomas et al, Mol Cell 2021 – Fgf5) have been added to the text and the figure legends.

    On page 13, the authors say "To determine whether OTX2 expression is essential to maintain chromatin accessibility in somatic cells..." but this does not seem to be what they test because they are using PGCLC medium. Perhaps I misunderstood, but this could be clarified.

    *Expression of OTX2 during the first 2 days of PGCLC differentiation leads to a block of germline differentiation as previously shown in Zhang, Zhang et al, Nature 2018. After 2 days of tamoxifen treatment, cells have acquired somatic fate and cells will undergo somatic differentiation even after tamoxifen is withdrawn after day 2. Nevertheless, we agree with the reviewer that the sentence is of difficult interpretation and we modified the sentence as shown below and as reported in the updated manuscript: “To determine whether OTX2 expression is essential to maintain chromatin accessibility in cells differentiating in the presence of PGC-inducing cytokines after day 2” (page 12). *

    On page 14 the authors claim, "These results indicate that...the partner proteins that OTX2 act alongside differ...". While this may be the case, their results do not substantiate this, it is just speculation. Should be toned down.

    The text has been modified as follows: "These results suggest that...the partner proteins that OTX2 act alongside differ..."

    Page 18, PGCLC differentiation method sections needs to be described as such (ie. Add "For PGCLC differentiation..." before the second paragraph)

    *The text “For PGCLC differentiation” has been added at the beginning of the PGCLC differentiation method section. *

    It would be helpful to indicate time on the protocol schematics (eg Fig4A, 5A, 5D etc) as I had to keep checking the methods to find out how long the full differentiation time-course was.

    *Indication of time has been added to Figures 1, 2, 4, 5 and 6. *

    Since the authors compare between the Tam d0-d2 treatments assessed at d2 versus d4 (Figure5B vs 5E) it would be helpful to make the colourbars the same scale, for both ATAC and Cut&Run datasets.

    *The heatmap in Figure 5B has been modified. The colourbars of Figure 5B and 5E are now using the same scale. *

    Reviewer 3:

    1. As a minor point related to this, the second sentence is confusing since it kind of sounds like Otx2-/- and WT cells are compared under the same conditions unless one carefully reads the previous sentence.

    The text has been modified to clarify the different medium conditions for WT and OTX2-/- cells, as follows: “In the presence of PGC-inducing cytokines, Otx2-/- cells produce an essentially pure (>90%) CD61+/SSEA1+ population that we refer to as PGCLCs, while wild-type cells differentiated in GK15 medium without cytokines yield a cell population from which PGCLCs are absent” (page 7).

    1. "This suggests that OTX2 acts as a pioneer TF to regulate the accessibility of enhancers E1, E2 and E3."

    This is from the text corresponding to Fig. 2. That data actually only shows that Otx2-null cells have DARs, so somehow OTX2 affects chromatin accessibility but it could be indirect by controlling transcription of genes that modify chromatin accessibility. It is not until figure 4 that the data suggests that OTX2 directly affects accessibility, perhaps as a pioneer TF.

    The authors continue to make many statements about the direct action of OTX2 before the data supporting this is shown, on which I got hung up as a reader. I suggest the authors edit the manuscript to improve this. E.g. "OTX2 may directly control accessibility at these sites (Figure 3E)." and the fact that in 3E and other figure, it says "DARs increased by OTX2 binding" which at that point is not proven, so would better say "Otx2-null vs WT DARs" or something like that.

    The sentence "This suggests that OTX2 acts as a pioneer TF to regulate..” has been removed from the text (page 9). The sentence “OTX2 may directly control accessibility at these sites” has been modified with “*suggesting that the presence of OTX2 affects accessibility at these sites” (page 9). The sentence “ Together, these results suggest that OTX2 is required to open these chromatin regions” has been modified to “Together, these results suggest that OTX2 is required for the accessibility of these chromatin regions”. *

    The subset of DARs that increase in WT EpiLC and are bound by OTX2 that was called “DARs increased by OTX2 binding” has been renamed as “DARs higher in WT with OTX2 binding”. For consistency, the subset of DARs showing increased accessibility in WT EpiLCs that are not bound by OTX2 are now called “DARs higher in WT without OTX2 binding” (Figure 3, Figure 4, main text and figure legends). We will further revise the manuscript to avoid statements or hypotheses that are not yet supported by data throughout the text.

    Reviewer 3 – minor comments:

    1. "Comparing wild-type and Otx2-/- ESCs identified 375 differentially accessible regions (DARs) with increased accessibility in wild-type cells, and 743 regions with higher accessibility in Otx2-/- ESCs (Figures 2C). An example of ESC DARs where accessibility is increased in cells expressing OTX2 is the intragenic enhancer of Tet2. Tet2 is expressed at high levels in ESCs but at low levels in EpiLCs."

    The authors compare Otx2-null and WT ESCs then proceed to give an example comparing ESCs to EpiLCs, instead of Otx2-null vs WT ESCs, which is confusing.

    Furthermore, here and in other places the authors describe ESCs as not expressing OTX2. However, they also show CUT&RUN data for OTX2 in ESCs etc, clearly indicating that it is expressed, just lower (otherwise how could one get anything?).

    *We originally chose Tet2 enhancer as an example of the 375 ESC DAR with higher accessibility in WT vs Otx2-/- ESCs as it shows a slightly decreased level of accessibility and OTX2 binding in ESCs. Therefore, the sentence “where accessibility is increased in cells expressing OTX2” refers to WT cells (expressing OTX2) when compared to Otx2-/- cells (OTX2-null). The text has been changed to describe the new panel. The rest of the main text will be checked and modified where appropriate to avoid possible misinterpretations. *

    *We also appreciate that the change in accessibility is not clearly visible in the original Figure 2, as also pointed out by Reviewer 1 (point 6). In the updated Figure 2, we show a region in the Hes1 locus as an example of the 375 ESC DARs. Moreover, we simplified the panels showing ATAC-seq tracks of WT and OTX2-/- ESC (Fig. 2D-E) or EpiLCs (Fig. G-H). *

    1. "In contrast, in EpiLCs, OTX2 binds almost 40% (446 out of 1,246) of the DARs that are more accessible in wild-type than in Otx2-/- cells (Figure 3B-C). Notably, these regions are mainly located distal to genes (91%, Figure 3D), despite the increased fraction of promoter regions bound by OTX2 in EpiLCs (Figure S1A)."

    Are the authors rounding percentages with 2 significant digits, as suggested by the "91%"? If so, 446/1245 ~ 36%, not 40%.

    *The text has been modified from “OTX2 binds almost 40%” to “OTX2 binds 36%”. *

    1. The results in Figure 4 are nice and the real meat of the paper. One suggestion: It would be helpful is Fig. 4B were split up between the 446 and 800 genes instead of showing all 1246, and if the WT control was shown in the same figure as well.

    *Panels with the 446 and 800 regions have been added to Figure 4 instead of the panels with all 1246 regions. WT control has been inserted in Figure 4. The main text and the figure legends have been updated accordingly. *

    1. "Enforced OTX2 expression opens additional somatic regulatory regions" - it would be clearer to say "OTX2 overexpression opens additional somatic regulatory regions", since this is really about DARs between EpiLCs that already express OTX2 and those forced to express higher than WT endogenous levels by the OTX2-ER system?

    We thank the reviewer for their suggestion. The text has been modified (page 12)

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, the authors perform OTX2 CUT&RUN and ATAC-seq in Otx2-null and WT ESCs, EpiLCs and PGCLCs to understand whether the role of OTX2 in restricting mouse germline entry that they previously described (Zhang Nature 2018) mechanistically depends on chromatin remodeling. They identify differentially accessible regions (DARs) between Otx2-null and WT cells at different stages of differentiation and show that many of these are OTX2 bound in WT. They then show using cells expressing OTX2-ER^T2 in Otx2-null Epiblast cells that when OTX2 is moved into the nucleus, the regions that were differentially closed in Otx2-null open within an hour, suggesting chromatin accessibility is directly controlled by OTX2 (rather than indirect effects involving transcription and translation which one would expect to take longer). The scope is narrow, but this is nice work and useful data for the mouse PGC field. However, there are a few places where the data could be strengthened, and the writing is a little confusing in places, for example by stating as fact in early sections what is not proven until later.

    Major Comments:

    1. "we compared Otx2-/- cells cultured in the presence of PGC-promoting cytokines with wild-type cells cultured in the absence of PGC-promoting cytokines. Under these conditions Otx2-/- cells produce an essentially pure (>90%) CD61+/SSEA1+ population that we refer to as PGCLCs, while wild-type cells yield a cell population from which PGCLCs are absent"

    This is not a controlled comparison since one cannot separate the day 2 effect of cytokines from that of the Otx2 knockout. The manuscript would be strengthened if the authors include WT somatic and PGCLCs from the +cytokine conditions, which could be easily sorted out as shown in Supp. Fig. 2. Ideally they would also include Otx2-null somatic cells, although Supp. Fig. 2 shows those are rare under the conditions considered.

    As a minor point related to this, the second sentence is confusing since it kind of sounds like Otx2-/- and WT cells are compared under the same conditions unless one carefully reads the previous sentence.

    1. "This suggests that OTX2 acts as a pioneer TF to regulate the accessibility of enhancers E1, E2 and E3."

    This is from the text corresponding to Fig. 2. That data actually only shows that Otx2-null cells have DARs, so somehow OTX2 affects chromatin accessibility but it could be indirect by controlling transcription of genes that modify chromatin accessibility. It is not until figure 4 that the data suggests that OTX2 directly affects accessibility, perhaps as a pioneer TF.

    The authors continue to make many statements about the direct action of OTX2 before the data supporting this is shown, on which I got hung up as a reader. I suggest the authors edit the manuscript to improve this. E.g. "OTX2 may directly control accessibility at these sites (Figure 3E)." and the fact that in 3E and other figure, it says "DARs increased by OTX2 binding" which at that point is not proven, so would better say "Otx2-null vs WT DARs" or something like that.

    1. "In ESCs, OTX2 binds <10% (30 out of 375) of DARs that are more accessible in wild-type cells than in Otx2-/- cells (Figure 3A), suggesting that accessibility of ESC DARs is directly due to OTX2 in a small subset of DARs."

    When a small number of DARs are OTX2 bound, it does not necessarily suggest that that small set is directly affected by OTX2. It could just mean no DARs are controlled directly by OTX2 and then some are bound by chance by OTX2. Some appropriate statistical null hypotheses about the occurrence of OTX2 motifs might help to see if 10% is more than chance.

    1. It would be good if the discussion was broadened to include both human and other transcription factors that are involved. How much of these conclusions could one expect to carry over to human or other mammals? There is some work from the Surani lab considering OTX2 in human. One could even look at published ATAC or OTX2 chip-seq data in hPSCs and potentially learn something interesting. Furthermore, there are studies on other transcription factors modulating chromatin accessibility in the decision between germline and somatic cells, for example PRDM1, PRDM14 (refs in e.g. Tang et al Nat Rev Gen 2016) or TFAP2A (at least in human (Chen et al Cell Rep 2019)). Do these factors affect the same genes? Is a coherent picture emerging of their respective roles in germline entry?

    Minor comments:

    1. "Comparing wild-type and Otx2-/- ESCs identified 375 differentially accessible regions (DARs) with increased accessibility in wild-type cells, and 743 regions with higher accessibility in Otx2-/- ESCs (Figures 2C). An example of ESC DARs where accessibility is increased in cells expressing OTX2 is the intragenic enhancer of Tet2. Tet2 is expressed at high levels in ESCs but at low levels in EpiLCs."

    The authors compare Otx2-null and WT ESCs then proceed to give an example comparing ESCs to EpiLCs, instead of Otx2-null vs WT ESCs, which is confusing. Furthermore, here and in other places the authors describe ESCs as not expressing OTX2. However, they also show CUT&RUN data for OTX2 in ESCs etc, clearly indicating that it is expressed, just lower (otherwise how could one get anything?).

    1. "In contrast, in EpiLCs, OTX2 binds almost 40% (446 out of 1,246) of the DARs that are more accessible in wild-type than in Otx2-/- cells (Figure 3B-C). Notably, these regions are mainly located distal to genes (91%, Figure 3D), despite the increased fraction of promoter regions bound by OTX2 in EpiLCs (Figure S1A)."

    Are the authors rounding percentages with 2 significant digits, as suggested by the "91%"? If so, 446/1245 ~ 36%, not 40%.

    1. The results in Figure 4 are nice and the real meat of the paper. One suggestion: It would be helpful is Fig. 4B were split up between the 446 and 800 genes instead of showing all 1246, and if the WT control was shown in the same figure as well.
    2. "Enforced OTX2 expression opens additional somatic regulatory regions" - it would be clearer to say "OTX2 overexpression opens additional somatic regulatory regions", since this is really about DARs between EpiLCs that already express OTX2 and those forced to express higher than WT endogenous levels by the OTX2-ER system?

    Significance

    Also see summary. Understanding what restricts cells to germline vs somatic lineages is an important question. By providing functional data showing that OTX2 directly controls chromatin accessibility, the authors add an important layer of understanding to their previous finding that OTX2 plays a key role in preventing mouse germline entry. The use of their previously established OTX2-null cells expressing OTX2-ER to rapidly induce nuclear OTX2 in a mutant background or the most part makes their experiments elegant and convincing. In focusing on the role of one gene in one event in one species, it is specialized and narrow in scope and will mostly be of interest to experts in the field, but there is nothing wrong with that.

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    Referee #2

    Evidence, reproducibility and clarity

    Barbieri and Chambers explore the role of OTX2 on mouse pluripotency and differentiation. To do so, they examine how the chromatin accessibility and OTX2 binding landscape changes across pluripotency, the exit of pluripotency towards formative and primed states, and through to PGCLC/somatic differentiation. The work mostly represents a resource for the community, with possible implications for our understanding of how OTX2 might mediate the germline-soma switch of fates. While the findings of the work are modest, the results seem solid and the manuscript is clear and well-written. I have some comments as indicated below:

    • The comparison between Otx2-/- cells in the presence of PGCLC cytokines compared to WT cells in the absence of cytokines seems like it is missing controls to me. I assume the authors wanted to enable homogeneous populations to facilitate their bulk sequencing methods, but it seems to me like they are comparing apples with oranges. It would have been better to have the reciprocal situations (Otx2-/- cells in basal differentiation medium, and WT cells in PGCLC cytokines) with a sorting strategy to better unpick the differences between the presence and absence of Otx2 in the 2 protocols. Having said that, the authors are careful not to draw many comparisons between those populations so I don't think this omission affects their current claims. They should however clarify whether the flow cytometry (Supp Fig2) was used for sorting cells or if all cells were taken for bulk sequencing.
    • The authors focus solely on the activating role of Otx2 in their data, but given the substantial proportion of DARs that decrease following Otx2 depletion, I presume it is possible that it also has a repressive effect? Either way, this should be discussed.
    • Throughout the text, the authors subject cells (WT / Otx2-/- /Otx2ER ) to different protocols to look at accessibility and Otx2 binding, but with no mention of the cell fate differences that occur in these different conditions. For instance, it is unclear to me to which fate the WT cells without PGCLC cytokines go - I presume this is neural but perhaps this is a mixed fate, given that they are in GK15 rather than N2B27. Likewise, the OTX2ER experiments may promote a mixed population between PGCLC/somatic fates, and this is never described. Ideally transcriptomic data would be collected, but failing that, qPCR data should be obtained to examine this more closely.
    • The authors also state that "OTX2 facilitates Fgf5 transcription' (page10) but provide no transcriptional data to substantiate this claim. Again RT-qPCR would help make this point.
    • The authors state that d2 PGCLCs "show an intermediate position between ESCs and EpiLCs" based on the PCA location. They should be careful to qualify that this is only in the first 2 principal components, because it may well be the case (and is likely) that in other components the PGCLC population is far removed from the pluripotent states.
    • It is unclear to me what the 'increase[d] accessibility' (eg abstract final sentence, Figure 3E) really means at the cellular level. Does this indicate that more cells have this site open, and does this have implications for the heterogeneity of cell fates observed? Since the authors are concerned with fate decisions, this seems like an important consideration that should at least be discussed.

    Minor Suggestions:

    • Presumably the regions bound by OTX2 in Tet2, Mycn and Fgf5 (Fig1E) are called enhancers because these are known from existing literature. It would be helpful to cite the relevant references to this in the text for those unfamiliar with these.
    • On page 13, the authors say "To determine whether OTX2 expression is essential to maintain chromatin accessibility in somatic cells..." but this does not seem to be what they test because they are using PGCLC medium. Perhaps I misunderstood, but this could be clarified.
    • On page 14 the authors claim, "These results indicate that...the partner proteins that OTX2 act alongside differ...". While this may be the case, their results do not substantiate this, it is just speculation. Should be toned down.
    • Page 18, PGCLC differentiation method sections needs to be described as such (ie. Add "For PGCLC differentiation..." before the second paragraph)
    • It would be helpful to indicate time on the protocol schematics (eg Fig4A, 5A, 5D etc) as I had to keep checking the methods to find out how long the full differentiation time-course was.
    • Since the authors compare between the Tam d0-d2 treatments assessed at d2 versus d4 (Figure5B vs 5E) it would be helpful to make the colourbars the same scale, for both ATAC and Cut&Run datasets.

    Significance

    The study examines the binding of OTX2 and subsequent chromatin accessibility in pluripotent, primed and differentiated (PGCLC/Somatic) cell states, including through Otx2-/- cell lines and temporally-controlled exogenous expression of Otx2. As such, it represents a valuable resource into the potential direct targets of Otx2 and their change in accessibility state across cell types. The work is likely to be of interest to those working on understanding the exit of pluripotency, gene regulatory networks, and chromatin remodelling. My expertise is in cell fate decisions, pluripotency regulation and PGC(LC) differentiation.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    OTX2 is a pivotal transcription factor that regulates the fate choice between somatic and primordial germ cell (PGC) lineages in early mouse development. In the current study, the authors use in vitro stem cell models to demonstrate that OTX2 mediates this developmental fate decision through controlling chromatin accessibility, whereby OTX2 helps to activate putative enhancers that are associated with somatic fate. By extension, those somatic-associated regulatory regions therefore become inaccessible in cells adopting PGC identity in which Otx2 is downregulated.

    Comments

    I enjoyed reading this manuscript. The experiments have been carried out well and for the most part the results provide convincing evidence to support the claims and conclusions in the manuscript. I particularly liked the experiments using the inducible Otx2 transgene to examine the acute changes in chromatin accessibility following restoration of OTX2.

    I include some suggestions below to the authors for additional analyses that I feel would further strengthen their study.

    I also felt that the authors focus almost exclusively on the subset of OTX2-bound sites that lose accessibility in the absence of OTX2. But, as they show in several figure panels, these sites tend to be the minority and that most OTX2-occupied sites do not lose accessibility in Otx2-null cells (actually, more sites tend to gain accessibility). I encourage the authors to modify the text and some of the analyses to give a better balance to their study.

    1. Figure 1: The authors report in the methods that they performed OTX2 CUT&RUN in biological duplicates. It would strengthen their results if they showed in Figure S1 some representative data from each replicate separately to show the consistency.
    2. Figure 1: The authors write: "...OTX2 binds mostly to putative enhancers." Whether these distal sites are enhancers is not sufficiently evidenced in the manuscript, but it is important information to collect to support their model of OTX2 function. The authors should strengthen their analysis by examining whether OTX2 peaks are enriched at previously defined enhancer regions.
    3. Figure 2: I think it would be helpful to remind the reader here that Otx2 is normally downregulated in PGCs, and that Otx2 expression is maintained (at least initially) in somatic cells. This would help explain the logic behind the choice of samples that were profiled. That said, I'm still puzzled why the authors did not examine flow-sorted WT+cyto cells?
    4. Figure 2D: I appreciate that the highlighted region at the Tet2 locus is a DAR, but from the genome tracks it looks as though the region still has high accessibility. Are there any other examples to exemplify a more obvious DAR? Additionally, since twice as many DARs gain accessibility in Otx2-null ESCs compared to lose accessibility, why not show examples of these as well? The same is true of EpiLCs. (Or alternatively, provide a good explanation for why not to show these other categories)
    5. Figure 2: the authors write: "This suggests that OTX2 acts as a pioneer TF...". However, at this point in the manuscript, there is no evidence to support that OTX2 might have pioneer activity. I think this claim would be better suited to later in the manuscript, or in the discussion, following the finding that reintroduction of OTX2 can induce chromatin accessibility at previously closed sites.
    6. Figure 3: I would be tempted to put Figure S3A and S3B into Figure 3. It would be better to show all 1246 DARs together, either ordered by OTX2 CT&RUN signal, or presented in two pre-defined groups (OTX2-bound vs unbound). I also suggest that the author show OTX2 signals and ATAC-seq signals for the 3028 DARs that gain accessibility in Otx2-null EpiLCs (this could be added to a supplemental figure).
    7. Figure 3: What is special about the 8% of OTX2-bound site that lose accessibility, versus the 92% of sites that do not?
    8. Figure 6: Do the PGCLCs with OTX2 expression have chromatin accessibility profiles similar to somatic cells? Consider adding WT somatic cell data to Figure 6A, which could be an interesting comparison with the Tam d0-d2 samples.
    9. Figure 6F: If the 4221 sites are split into those bound by OTX2 versus those that are not (related to Figure 6C) then is there a difference? i.e. are the OTX2-bound sites opening up?
    10. Is there any evidence that OTX2 binds and compacts PGCLC enhancers in somatic cells? I appreciate this is different to the main thrust of the authors' model, but being able to show that OTX2 does not compact these sites lends further support to their preferred model of OTX2 opening sites of somatic lineages.
    11. Discussion: Have prior studies established a connection between OTX2 and chromatin remodellers that can open chromatin? Or, if not, then perhaps this could be proposed as a line of future research.

    Significance

    Strengths

    The results presented provide a careful dissection of the role of OTX2 in controlling chromatin accessibility in different stages of pluripotent to somatic and PGC fates. The authors do a good job of revealing the stage-specific differences in OTX2 occupancy and chromatin accessibility as well as the different responses following the acute reintroduction of OTX2.

    Limitations

    I felt that the authors could present/discuss a bit more on alternative possibilities and models, as it would help the reader to better understand why they favour one model over other ones, and presenting these other possibilities could also provide more support for their preferred model.

    Whether OTX2 is binding to putative enhancers is inferred but could be evidenced more strongly, as that is important for their model.

    Advance

    This study provides key information to understand the mechanisms of OTX2 function in cell fate choice. Similar functions have been shown in other contexts for other transcription factors, but this is a nicely done study and adds to our understanding of how transcription factors function in early development to direct cell-fate decisions.

    Expertise

    My field of expertise lies in the gene regulatory control of early developmental decisions.

  5. Version published to 10.1101/2025.02.18.638827 on bioRxiv