Comparative roadmaps of reprogramming and oncogenic transformation identify Bcl11b and Atoh8 as broad regulators of cellular plasticity

Coordinated changes of cellular plasticity and identity are critical for pluripotent reprogramming and oncogenic transformation. However, the sequences of events that orchestrate these intermingled modifications have never been comparatively dissected. Here, we deconvolute the cellular trajectories of reprogramming (via Oct4/Sox2/Klf4/c-Myc) and transformation (via Ras/c-Myc) at the single-cell resolution and reveal how the two processes intersect before they bifurcate. This approach led us to identify the transcription factor Bcl11b as a broad-range regulator of cell fate changes, as well as a pertinent marker to capture early cellular intermediates that emerge simultaneously during reprogramming and transformation. Multiomics characterization of these intermediates unveiled a c-Myc/Atoh8/Sfrp1 regulatory axis that constrains reprogramming, transformation and transdifferentiation. Mechanistically, we found that Atoh8 restrains cellular plasticity, independent of cellular identity, by binding a specific enhancer network. This study provides insights into the partitioned control of cellular plasticity and identity for both regenerative and cancer biology.

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In particular, it would be essential to:  Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. activation of endogenous OSKM genes, suggesting Atoh8 might repress these genes. However, Atoh8 ChIP-seq showed that it predominantly binds enhancers. Does Atoh8 bind OSKM genes? Atoh8 presumably suppresses Wnt signaling by upregulating Wnt inhibitors. Is there any evidence of Atoh8 binding to these genes?" C) Improve the data regarding MEF identity as raised by Reviewer 3: "The authors report the finding that acquisition of cellular plasticity precedes loss of cellular identity as a major conceptual advance. This is based on the observation that RI4/TI4 cells did not downregulate MEF identity (Figure 3f). However, in Fig 1j the MEF identity score seems to decrease pretty quickly, especially along the transformation trajectory. As the RI4/TI4 intermediates are defined by loss of both Bcl11b and Thy1 which, according to Figure 2c, would place them towards the end of the trajectories where the MEF identity has been lost, can the authors add clarity where these group 4 intermediates might lie along the trajectory? Importantly, how MEF identity is defined and what makes up the MEF identity score should be clearly stated, even if it is derived from other publications." D) All other referee concerns pertaining to strengthening existing data, providing controls, methodological details, clarifications and textual changes as applicable should also be addressed. E) Finally please pay close attention to our guidelines on statistical and methodological reporting (listed below) as failure to do so may delay the reconsideration of the revised manuscript. In particular please provide: -a Supplementary Figure including unprocessed images of all gels/blots in the form of a multi-page pdf file. Please ensure that blots/gels are labeled and the sections presented in the figures are clearly indicated.
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Reviewer #1:
Remarks to the Author: In this manuscript, the authors reported a novel genetic pathway that is shared by the initial steps of the reprogramming and transformation. They generated a mouse model in which both reprogramming and transformation events can be induced: the reprogramming of somatic cells to pluripotent stem cells by tet-inducible Oct4/Sox2/Klf4/Myc and the transformation of somatic cells to cancer-like cells by tamoxifen-inducible K-Ras G12D in combination with viral transfection of Myc. Using this system, they found that the cell populations with high plasticity can be separated by the Bcl11-low/Thy1-low expressions at the initial steps of both cases. Then they found that Atoh8 is commonly down-regulated in these populations. Functional validation revealed that Atoh8 depletion enhances both transformation and reprogramming. The analysis of Atoh8 binding site by ChIP-seq combined with other data set deduced the Myc/Atoh8/Sfrp pathway driving the Wnt signal is functionally important as a roadblock of both reprogramming and transformation. The mechanistic overlap between reprogramming and transformation events at the molecular level is interest question to be addressed in cell biology. However, the wide-range variations of both events make it difficult to reveal the general answer. The reprogramming of somatic cells to induced pluripotent stem (iPS) cells has been well analyzed and it was shown that the process depends on the reprogramming methods and the types of somatic cells. Here the authors applied the canonical reprogramming model, reprogramming of MEF by Oct4/Sox2/Klf4/Myc, but they never addressed whether the finding is applicable for other reprogramming events with different starting cells and different reprogramming methods. For the transformation of MEF, the combination of K-Ras G12D and Myc was applied. The problem is the common use of Myc in both processes. As the result, they found the Myc/Atoh8/Sfrp pathway as a roadblock, but it may depend on the function of Myc in both reprogramming and transformation. What will be observed if the reprogramming and transformation events without Myc are analyzed? The role of Atoh8 in reprogramming is already reported by Divvela et al (Cells, 2019) as the authors referred. Surprisingly, the result shown in this previous report is completely opposite to the result shown in this manuscript. Divvela et al demonstrated that Atoh8/Math6 is up-regulated during reprogramming and Atoh8/Math6 KO MEFs show lower efficiency of reprogramming although they analyzed the same reprogramming event (MEF with Oct4/Sox2/Klf4/Myc). Since there are many data sets available for the transcriptomic change in the reprogramming process (Kaji, Hochedlinger etc), the authors should confirm the universality of their finding and address the reason of the controversy to the result by Divvela et al. Because of the points described above, it is difficult to find the general importance and interest in this manuscript that is required for the publication in Nature Cell Biology, I think.
Reviewer #2: Remarks to the Author: Huyghe and colleagues provide a comprehensive view of reprogramming and oncogenic transformation at single-cell resolution. They generate a novel mouse model, which helps them identify novel regulators of somatic cell identity and cellular plasticity. Mechanistically, the authors identified Blc11b as a new marker of fibroblast identity and Atoh8 as a novel modulator of the Wnt pathway and cellular plasticity. Overall, the idea is original, the findings are well presented, the 6 Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. datasets and transgenic models they generated are helpful for the scientific community, and the paper is of general interest. I have some suggestions and minor points to help improve the manuscript: 1. The authors uncovered that Bcl11b is highly expressed in MEFs and becomes downregulated during reprogramming and oncogenic transformation. This raises the intriguing possibility that Bcl11b controls somatic cell identity and acts as a barrier to cell fate change. It would be helpful if the authors could gather more mechanistic insights into the role of Bcl11b. Although they use it prevalently as a marker, Bcl11b is a transcription factor (mostly know as lymphoid and leukemia factor) and could have an active role in controlling MEF identity. Indeed, previous work has shown that depletion of Bcl11b causes loss of T cell identity (Li et al., Science 2010). I would suggest performing knockdown (KD) and overexpression (OE) experiments to determine whether it plays a role in cell fate changes. I expect that the KD accelerates cell fate transitions. In the presence of a phenotype, a Bcl11b ChIP experiment would also help gather some mechanistic insights. My suspect is that it could play a more substantial role than Atoh8.
2. Figure 2l, m is a bit puzzling. The reprogramming and transformation plots at day 5 resemble the plots at days 10-17 in Figure 2e. So, the reprogramming and transformation in Figure 2l,m seem massively accelerated. Moreover, based on the authors finding on Bcl11b-cells, I would expect that the population RI2 (Bcl11b-/Thy-1+) reprograms better than the population RI3 (Bcl11b+/Thy-1-). The author should explain these discrepancies.

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3. The cell cycle is only affected in "transfo" conditions but not in the "repro+transfo." The author should clarify this discrepancy and show the original FACS plots. 4. Figure 3f does not help and can be removed, as the populations RI4 and TI4 still have MEF identity. Moreover, the axis is starting at 3.5 while it should begin at 0.
Reviewer #3: Remarks to the Author: Review of Huyghe et al NCB 2021 This study interrogated the early stages of cellular reprogramming and cellular transformation, to identify the molecular changes that define and drive these processes, and to examine whether loss of cellular identity is coupled to gain of plasticity. The authors developed and utilized a mouse model that enabled inducible reprogramming or transformation of mouse embryonic fibroblasts (MEFs) ex vivo. Reprogramming of MEFs to induced pluripotent stem cells (iPSCs) was achieved by doxycyclineinducible expression of Oct4, Sox2, Klf4 and Myc (OSKM) whereas oncogenic transformation was achieved by tamoxifen-induced activation of oncogenic K-ras (sometimes with cMyc overexpression and trp53 knockdown). Established functional readouts were used to assess each model (teratoma formation and alkaline phosphatase positive colonies by reprogrammed iPSCs, and soft agar colony formation/anchorage independent growth and in vivo tumor formation for transformed MEFs). Multiple molecular modalities were used to characterize early time points of each process (single cell and bulk RNA-seq, ATAC-seq). The main findings include identification of Bcl11b as a novel marker of MEF cell identity and whose downregulation (together with the previously described marker Thy1) can be used to prospectively isolate cells prone to (or already undergoing) reprogramming/transformation; use of ATAC-seq data to identify FosL1 as having divergent roles in transformation (loss blocks) and reprogramming (loss enhances); purported uncoupling of loss of cell identity and gain of plasticity; identification of shared gene expression changes that occur during both reprogramming and transformation; and identification of the Atoh8 transcription factor as a nonspecific constraint to reprogramming and transformation through its presumed role in suppressing Wnt signaling. The evidence that Bcl11b is a faithful marker of transformation and reprogramming is well supported by the cell sorting experiments presented in Figure 2, although experimental perturbation would strengthen the functional relevance of this marker even further. Identification of Atoh8 as a relatively nonspecific constrainer of cellular plasticity is also well supported by the data, including examining Atoh8 knock-down in additional models of transformation and trans-differentiation. Overall, this study illustrates the power of examining dynamic cellular processes in a highly controlled, defined manner using time-resolved high-resolution molecular read-outs. The main conclusions of the study could be significantly strengthened by additional experiments, clarifying aspects of the experimental design, and analyzing the wealth of data in additional ways, as outlined below.
Major comments 1. The authors report the finding that acquisition of cellular plasticity precedes loss of cellular identity as a major conceptual advance. This is based on the observation that RI4/TI4 cells did not 8 Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. downregulate MEF identity ( Figure 3f). However, in Fig 1j the MEF identity score seems to decrease pretty quickly, especially along the transformation trajectory. As the RI4/TI4 intermediates are defined by loss of both Bcl11b and Thy1 which, according to Figure 2c, would place them towards the end of the trajectories where the MEF identity has been lost, can the authors add clarity where these group 4 intermediates might lie along the trajectory? Importantly, how MEF identity is defined and what makes up the MEF identity score should be clearly stated, even if it is derived from other publications. 2. Bcl11b downregulation was identified as a marker of transformation and reprogramming at very early time points and the sub-population sorting experiment in Figure 2 suggests that Bcl11b downregulation is a key event in both processes. Given that a) Bcl11b has not been explored in the context of MEF cell identity or transformation/reprogramming, b) Bcl11b is a well-known regulator of T cell differentiation with similar properties (constraining cell fate) and c) the claim of uncoupling between loss of cell identity and gain of plasticity is based on Bcl11b-defined populations, functional perturbation of Bcl11b would increase the significance of this finding and these claims. The experimental system appears sufficiently established to feasibly test the impact of Bcl11b knock-down (and possibly rescue) on transformation and reprogramming. 3. In this study, multiple factors were identified as correlating with (Thy1, Bcl11b) or directly impacting (FosL1, Atoh8, Srfp1/2) MEF cell reprogramming and transformation, however whether and how these factors functionally interact with each other was not discussed. Moreover, how these factors interact with the factors that actually drive each process (e.g. OSKM for reprogramming and oncogenic Kras for transformation) was not explored or discussed. Although this would not necessarily impact the main conclusions of the paper, some attempt at tying these diverse factors together within the context of these central cellular processes would improve the broader relevance to the field. Some of these points are easily explored with the data already generated. For example, Atoh8 knock-down hastened activation of endogenous OSKM genes, suggesting Atoh8 might repress these genes. However, Atoh8 ChIP-seq showed that it predominantly binds enhancers. Does Atoh8 bind OSKM genes? Atoh8 presumably suppresses Wnt signaling by upregulating Wnt inhibitors. Is there any evidence of Atoh8 binding to these genes?
Minor comments and suggestions 1. Experimental details are missing in the following places • How was the MEF identity score identified and applied to the data generated in this manuscript? • page 10 mentions a result showing expression change of Dmrtc2 and Pou3f1 but these genes are not shown in Extended Data Fig. 4i (and the gene Shisa8 is shown in ED 4i but not listed in the results section). • Page 11 mentions a "protein class analysis with pantherdb" was used to find master regulators of a transcriptional program, but unclear how this analysis was performed, and it is not mentioned in the Methods section (what genes were used as input? How were they compiled? How did this analysis lead to the subsequent focus on the genes highlighted in Cluster 2 of fig. 3k?) • Figure 6i: how long after Atoh8 knockdown were cells taken for RNA-seq? This is important for interpreting that the MEF identity score did not change. In many figures, the time point of the experiment was difficult to track down, so adding the timepoint to the figure itself or including in the figure legend each time would be helpful. 2. Based on the wide-spread use of the term 'cellular plasticity' it would be good to clearly define this term within the context of the read-outs presented in this study.
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3. Adding Bcl11B/Thy1 expression (high/low) to figure 2n would aid interpretation. 4. A suggestion is to analyze the joint Bcl11B/Thy1 expression in the single cell data from Figure 1 as support for the transition kinetics inferred in Figure 2 (how does the frequency of Bcl11b/Thy1 high/low cells change along each trajectory? What about the pattern in the bifurcation area, which is relatively under-discussed in this manuscript?) 5. An additional suggestion would be to identify genes that significantly correlate with Bcl11b expression during reprogramming and transformation to identify other genes putatively regulated by or regulating Bcl11b expression during these processes. Are they similar in the repro/transfo context? Particularly given the GO terms of the common set in Figure 3h, it is likely worthwhile to describe a putative Bcl11b controlled gene expression program in each context. 6. Unsupervised hierarchical clustering of the ATAC-seq data to identify clusters in an unsupervised manner is another way to identify potentially interesting clusters and see how it compares to the supervised classification of C1-C6 in Fig. 3c,d 7. Extended data fig. 4i should also show Dmrtc2 and Pou3f1 but these are missing. Shisa8 is shown but isn't listed in the main text (p.10) 8. Fig. 3a: add the % variance explained by each component 9. Why is p53 knock-down used in the experiments shown in Figure 4, but not in previous experiments of the same experimental system? 10. The significance of the gene expression results presented in Figure 4f-j is unclear; gene expression differences can be seen, but how they support the conclusion that "Atoh8 constrains cellular plasticity" is not clear. The functional read outs on the other hand are very convincing. 11. Also figure 4: the colony assay and western blots show that Atoh8 KD has an impact at early time points (d.3 and d.6) but analysis of gene expression was performed at day >30, so it's unclear if the time points directly impacted by Atoh8 are missed (e.g. the results in Figs4f,h,I,j are simply reflecting emergence of different cell states/types that are consequential to Atoh8 knock-down). 12. Figure 4j. References for Ube2c and Top2a as "promoting cancer cell invasion and migration"? 13. In the discussion, it is stated that the authors identified "the existence of a molecular program that commonly emerges during both processes" but this program remains obscure after reading the manuscript. Is this program all of the genes mentioned (Thy1, Bcl11b, Atoh8, Wnt regulators, etc.?). Is this program the "bifurcation area" shown in Figure 1k? 14. Page 6: "let us" should be "led us"" 15. Page 7: "reflect the ability" should be "reflects the ability" 16. Fig4 legend: "Cells were splited" should be "Cells were split" or "Cells were passaged at least 10 times"

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Author Rebuttal to Initial comments
Reviewers' Comments: Reviewer #1: Remarks to the Author: In this manuscript, the authors reported a novel genetic pathway that is shared by the initial steps of the reprogramming and transformation. They generated a mouse model in which both reprogramming and transformation events can be induced: the reprogramming of somatic cells to pluripotent stem cells by tetinducible Oct4/Sox2/Klf4/Myc and the transformation of somatic cells to cancer-like cells by tamoxifeninducible K-Ras G12D in combination with viral transfection of Myc. Using this system, they found that the cell populations with high plasticity can be separated by the Bcl11-low/Thy1-low expressions at the initial steps of both cases. Then they found that Atoh8 is commonly down-regulated in these populations. Functional validation revealed that Atoh8 depletion enhances both transformation and reprogramming. The analysis of Atoh8 binding site by ChIP-seq combined with other data set deduced the Myc/Atoh8/Sfrp pathway driving the Wnt signal is functionally important as a roadblock of both reprogramming and transformation.
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The mechanistic overlap between reprogramming and transformation events at the molecular level is interest question to be addressed in cell biology. However, the wide-range variations of both events make it difficult to reveal the general answer.
We thank Reviewer 1 (R1) for his/her interest for exploring "the mechanistic overlap between reprogramming and transformation…". We improved significantly the manuscript by addressing his/her constructive comments: The reprogramming of somatic cells to induced pluripotent stem (iPS) cells has been well analyzed and it was shown that the process depends on the reprogramming methods and the types of somatic cells. Here the authors applied the canonical reprogramming model, reprogramming of MEF by Oct4/Sox2/Klf4/Myc, but they never addressed whether the finding is applicable for other reprogramming events with different starting cells and different reprogramming methods. For the transformation of MEF, the combination of K-Ras G12D and Myc was applied. The problem is the common use of Myc in both processes. As the result, they found the Myc/Atoh8/Sfrp pathway as a roadblock, but it may depend on the function of Myc in both reprogramming and transformation. What will be observed if the reprogramming and transformation events without Myc are analyzed?
We thank R1 for his/her constructive comment. We conducted different approaches to assess the relevance of our findings in alternative somatic cell types and with other reprogramming/transformation/trandifferentiation methods.
1-We assessed first whether the emergence of Bcl11b low /Thy1 low (BLTL) cells constitutes a broad hallmark of reprogramming and transformation rather than an event specifically linked to the use of c-Myc and MEFs.
-We assessed whether BLTL cells emerged when alternative reprogramming and transformation methods excluding c-Myc were used. For reprogramming, we compared OSKM 1 with (i) OSK + Wnt inhibitor IWP2 and (ii) Sall4, Nanog, Esrrb and Lin28 (SNEL) 2 . For transformation, we compared K-Ras G12D /c-Myc with (i)K-Ras G12D +c-Myc+p53KD, (ii) H-Ras G12V +CyclinE+p53KD and (iii) H-Ras G12V +p53KD. Strikingly, we found that BLTL cells emerged in all of these conditions at different efficiencies. Please see Figure 3e and Extended Data Fig. 3e. In addition, we showed by FACS isolation followed by replating that BLTL cells generated with OSK+IWP2 were more efficient at forming pluripotent derivatives. Data are presented in Figure 3g and Extended Data Fig.  3e.
-We next derived mouse adult ear fibroblasts (MAEFs) to conduct reprogramming and transformation experiments because we found that they express both Bcl11b and Thy1. Of note, even Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
if MAEFs and MEFs are mesenchymal cells, their molecular and functional features are heavily different 3 . We found that BLTL cells emerged when Bcl11b-tdTomato knock-in MAEFs were induced for reprogramming (OSKM) and transformation (K-Ras G12D /c-Myc), as observed with MEFs. Moreover, FACS isolation demonstrated that these BLTL cells were prone to generate iPS colonies. These data are incorporated in Figure 3f, h and Extended Data Fig. 3f. 2-We attempted to evaluate Atoh8 function during reprogramming in other cell types but it was not expressed in MAEFs or in T lymphocytes (Extended Data Fig. 6b-c). However, during the review, we identified the Bcl11b TF as a regulator of cell fate in MEFs. In line with R1 request, we also evaluated Bcl11b function in reprogramming of T lymphocytes as an alternative cell type, as well as during MEFs to neuron transdifferentiation. Data are incorporated in an entirely new Figure 2, panels k-n. We thank R1 for this very important comment. We summarized below the four main controversies with the potential explanations for the discrepancies: Controversy 1: Atoh8 is not expressed in mouse adult ear fibroblasts (MAEF) (Divvela) -Atoh8 is expressed in mouse embryonic fibroblasts (MEF) (our study) Of high importance, Divvela et al. used mouse adult ear fibroblasts (MAEFs) for reprogramming (please see page 4 of the publication "To evaluate the expression of Math6 during somatic cell reprogramming, we prepared fibroblasts from the ears of adult Math6Flag-tag mice and subjected them to reprogramming using Oct4, Sox2, Klf4 and c-Myc…".). In contrast, we used mouse embryonic fibroblasts (MEFs). Therefore, the two studies work on completely different reprogramming events, and it might explain the difference of Atoh8 expression. To assess it, we compared Atoh8 expression in MAEFs, MEFs and iPS cells by Q-RTPCR and WB. We showed that Atoh8 is indeed expressed at very low levels in MAEFs, when compared with MEFs. These data are included in Extended Data Fig. 6b-c. The first discrepancy might therefore be explained by the use of different cells-of-origin in the two studies.
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As an additional note, Divvela et al. used a Math6-Flag knock-in MAEFs and assessed the expression of a fused Atoh8-flag protein. In contrast, we analyzed the expression of the endogenous native Atoh8 using a commercial antibody (that we validated in gain-and loss-of-function approaches). Therefore, we cannot exclude the possibility that the presence of the flag fused to Atoh8 affects its expression, localization and function.
Controversy 2: Atoh8 promotes MAEFs reprogramming (Divvela) -Atoh8 constrains MEFs reprogramming (our study) As stated before, the two studies used different cells-of-origin (MAEFs vs MEFs) to evaluate Atoh8 function. Therefore, we cannot rule out the possibility that Atoh8 functions in a cellular contextdependent manner, as found for the NuRD complex during reprogramming 4 . The discrepancy here might be explained by the different cells-of-origin in the two studies.
To reinforce our findings, we asked Jose Polo's lab to reproduce independently in his own lab the experiments of Atoh8 depletion in MEFs prior to reprogramming. As shown below in Reviewer Figure 1, using MEFs coming from 3 different embryos, an independent lab confirmed that Atoh8 constrains MEF reprogramming efficiency.
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Controversy 3: Atoh8 is induced during MAEFs reprogramming (Divvela) -Atoh8 is downregulated during MEFs reprogramming (our study)
A very important difference is that Divvela et al. analyzed Atoh8 expression at the whole population level while we focused on FACS-sorted subset of reprogramming cells. Just as a reminder, reprogramming events are quite rare in the culture and can be obscured by the non-reprogramming cells when analyzing whole population of cells. To assess whether the discrepancy comes from that difference, we interrogated 6 independent public RNA-seq datasets for Atoh8 expression, including data from total populations (Alex Meissner lab 5 , Andreas Nagy lab 6  In the 6 independent datasets, Atoh8 expression is completely undetectable in iPS cells, at the bulk but also single-cell levels (Extended Data Fig. 6a). In addition, we conducted WB and Q-RTPCR analyses on MEFs, MAEFs and iPS cells that confirmed that Atoh8 is not expressed in naïve pluripotent cells (Extended Data Fig. 6b-c). We hypothesize that the expression detected by Divvela et al. might come from (i) the feeder cells (MEFs) on which iPS cells are grown and/or (ii) by the genetic modification of the Atoh8 locus that disturbs its expression. -On the IF Figure 1C, Atoh8-Flag staining is exclusively cytoplasmic, which is not expected for a bHLH transcription factor, supporting the hypothesis that the modification of the protein might impact its function.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Because of the points described above, it is difficult to find the general importance and interest in this manuscript that is required for the publication in Nature Cell Biology, I think.
We thank R1 because her/his comments allowed us to demonstrate that our findings are not strictly limited to c-Myc and MEFs reprogramming but rather constitute a broad hallmark of reprogramming/transformation/transdifferentiation in various somatic cell types (MEFs, MAEFs, Tlymphocytes, Human dermal fibroblasts). We believe that the data generated to address them improved significantly the impact and interest of the work. We hope that she/he will agree.
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Reviewer #2:
Remarks to the Author: Huyghe and colleagues provide a comprehensive view of reprogramming and oncogenic transformation at single-cell resolution. They generate a novel mouse model, which helps them identify novel regulators of Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. somatic cell identity and cellular plasticity. Mechanistically, the authors identified Blc11b as a new marker of fibroblast identity and Atoh8 as a novel modulator of the Wnt pathway and cellular plasticity. Overall, the idea is original, the findings are well presented, the datasets and transgenic models they generated are helpful for the scientific community, and the paper is of general interest. I have some suggestions and minor points to help improve the manuscript: We first would like to thank R2 for her/his strong support of our work, especially for noticing the originality and general interest of our paper.
1. The authors uncovered that Bcl11b is highly expressed in MEFs and becomes downregulated during reprogramming and oncogenic transformation. This raises the intriguing possibility that Bcl11b controls somatic cell identity and acts as a barrier to cell fate change. It would be helpful if the authors could gather more mechanistic insights into the role of Bcl11b. Although they use it prevalently as a marker, Bcl11b is a transcription factor (mostly know as lymphoid and leukemia factor) and could have an active role in controlling MEF identity. Indeed, previous work has shown that depletion of Bcl11b causes loss of T cell identity (Li et al., Science 2010). I would suggest performing knockdown (KD) and overexpression (OE) experiments to determine whether it plays a role in cell fate changes. I expect that the KD accelerates cell fate transitions. In the presence of a phenotype, a Bcl11b ChIP experiment would also help gather some mechanistic insights. My suspect is that it could play a more substantial role than Atoh8.
We sincerely thank R2 for his/her pertinent comment. As suggested, we conducted a large number of approaches to dissect Bcl11b function during reprogramming, transformation and transdifferentiation that are now presented in an entirely new Figure 2: 1-We started by modulating Bcl11b expression prior to induce reprogramming or transformation. Bcl11b gain-and loss-of-function approaches in MEFs (using RNA interference but also Bcl11b conditional KO MEFs 11 ) led to demonstrate that Bcl11b depletion significantly increased reprogramming and transformation efficiencies, as suggested by R2 (please see Fig. 2e-i). Even if not requested, we next conducted teratoma formation assays to show that Bcl11b depletion has no major detrimental effect on the acquisition of in vivo multilineage differentiation potential (Fig. 2j). Therefore, we demonstrated that Bcl11b functionally acts as a gatekeeper of the somatic state.
2-We also assessed, as requested, whether Bcl11b accelerates cell state transitions. We showed unexpectedly that Bcl11b depletion does not accelerate the emergence of cells that activated the endogenous Oct4 reporter, in contrast to Atoh8. Data are presented below in Reviewer Figure 2 as we decided not to include them in the revised draft.
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3-As indicated by R2
, Bcl11b is highly expressed in T lymphocytes. Therefore, even if not requested, and in order to broaden the impact of our finding, we evaluated Bcl11b role in the reprogramming of T lymphocytes. Using OSKM Dox-inducible primary T cells, we demonstrated that Bcl11b constrains reprogramming of this alternative somatic cell type. Data are presented in Figure 2k-l. We also showed that Bcl11b constrains MEFs to neuron transdifferentiation triggered by Brn2, Ascl1 and Myt1l (Fig.  2m-n).
4-Next, in order to understand Bcl11b function, we combined RNA-seq and ChIP-seq analyses. We compared the transcriptomes of control, Bcl11b-overexpressing (OE) and Bcl11b-knockdown (KD) MEFs. In parallel, we optimized the Bcl11b ChIP-seq conditions by combining two commercial antibodies (Abcam, ab18465, Bethyl, A300-385A). We identified 7430 specific peaks of Bcl11b binding to the MEF genome (Fig. 2p). We found that Bcl11b modulation impacts 979 genes (adjusted p-value< 5.10-2; log2 FC >0.5 or <-0.5) with 122 genes (13%) presenting a Bcl11b binding site within 10kb of the TSS (Fig. 2s). Finally, the fact that several Bcl11b targets were related to the Mapk signalling pathway led us to demonstrate that Bcl11b directly regulates pErk1/2 levels in MEFs (Fig.  2t), providing a potential explanation of its mode of action during reprogramming 12 .
Overall, we invested important efforts to address the pertinent suggestion of R2. We believe that the results presented in Figure 2 improves significantly the impact of the manuscript by identifying Bcl11b as Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. a new broad-range gatekeeper of somatic states from reprogramming, transdifferentiation and transformation.
2. Figure 2l, m is a bit puzzling. The reprogramming and transformation plots at day 5 resemble the plots at days 10-17 in Figure 2e. So, the reprogramming and transformation in Figure 2l,m seem massively accelerated.
We thank R2 for his/her pertinent comment and we apologize for the lack of clarity that led to that misunderstanding. For Figure 2e (now Fig. 3b), we FACS-sorted Bcl11b high /Thy1 high (BHTH) cells prior to induce reprogramming/transformation (as indicated in the initial draft). Therefore, by starting with this subset of highly refractory cells, we significantly slowed down the reprogramming and transformation processes. Similar enrichment procedures are commonly used in the reprogramming field, for example for Thy1 by K. Hochedlinger lab 13 . In contrast, for the panels Fig. 2l and 2m (now Fig. 3l-m), we started the experiments with unsorted MEFs. This difference of experimental design explains that the process appeared somewhat accelerated in those panels. We sincerely apologize for this lack of accuracy, as we forgot to provide this crucial information. In the revised draft, we removed the initial misleading panels and indicated the difference of experimental design in the figure legends of Figure 3b and 3l-m on page 26.
We thank R2 for noticing this point that becomes even more important now that we showed that Bcl11b functionally restrains reprogramming and transformation (new Figure 2). As noticed by R2, when we FACS sorted cells based on the combined expression of Bcl11b and Thy1, the downregulation of Bcl11b in RI2 (BLTH) cells does not correlate with a significant improvement of reprogramming/transforming efficiency. To understand this apparent discrepancy, we hypothesized that, even if BLTH cells downregulated Bcl11b expression, they still harbor molecular differences with BLTL cells. To answer this question, we FACS sorted the 4 subpopulations at day5 of reprogramming and transformation and conducted Q-RTPCR analyses. We noticed that BLTH cells still harbor high levels of Atoh8 transcript (equivalent to refractory BHTH cells) when compared with BLTL cells during both reprogramming and transformation, which might explain their functional properties. The data are presented in the Reviewer Figure 3 below because we could not find a pertinent way to integrate them within the manuscript. Indeed, Atoh8 identification is presented in Figure 3 after the capture of the cellular intermediates in Figure 2.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Figure 2, the authors mainly focused on downregulated markers during the reprogramming and transformation processes. It would be great to discuss whether transient upregulated markers mark cell populations poised to change fate. This could indicate whether intermediates can be sorted during the process for further characterization in future studies.

In
We thank R2 for his/her pertinent comment. We now discussed the transient induction of some cell surface markers, such as CD14, CD53, CD72 and CD84, that might be used to refine the tracking of reprogramming/transforming intermediates (see discussion page 18). 4. The effect of Atoh8 on reprogramming and transdifferentiation is relatively minor, and the author could include some experiments to corroborate the data presented in Figure 5. For example, the authors could check whether Atoh8 KD specifically affects the population RI4 by flow cytometry.
We thank R2 for this interesting comment. We analyzed by FACS the dynamic emergence of RI4 (now called BLTL) cells during transformation and reprogramming in "Ctrl" and "Atoh8-KD" settings. We found that Atoh8 depletion significantly accelerates the appearance of RI4 cells during transformation but not reprogramming. For reprogramming, we already showed that Atoh8 depletion accelerates the arrival of Pou5f1-GFP+ cells. To reconcile this apparent discrepancy, we hypothesize that, even if the number of BLTL cells is not increased by Atoh8 depletion, the Atoh8-depleted BLTL cells might be more "advanced" in their reprogramming stage than control BLTL cells. However, addressing this hypothesis in detail will require additional approaches such as transcriptomic characterization that are, we believe, out of the scope of the study. The data are now presented in Figure 5c-d.
The authors should also include experiments to prove that Atoh8 OE impairs cell fate changes. Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. We performed the required experiments but found that Atoh8 OE does not significantly affect reprogramming or transformation efficiencies (see Reviewer Fig. 4 below). For reprogramming, we hypothesize that the massive epigenetic reorganization induced by OSKM in the early days of the process, especially on the Atoh8-bound regions (as demonstrated in Figure 7), is not sufficiently counteracted by the sole exogenous Atoh8 expression. Of note, in the context of the review, we observed a similar lack of effect with the exogenous expression of Bcl11b, in agreement with the hypothesis that the exogenous expression of these single TFs is not sufficient to counteract OSKM action.
Given its role in Wnt signaling regulation, the authors should test the effect of GSK3Bi (a known enhancer of reprogramming) together with Atoh8 KD (if Atoh8 depletion activates Wnt, the authors should not see an increase with GSK3Bi treatment).
We thank R2 for this comment. We employed different approaches to assess whether Atoh8 effect is strictly due to Wnt signalling modulation. We attempted to use GSK3Bi, as proposed by R2, but we observed a strong cellular toxicity on MEFs. Therefore, we showed first that Atoh8 depletion has no additive effect on reprogramming/transformation when combined with Wnt signalling activation via Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Sfrp1 KD (Fig. 7o). Moreover, we demonstrated that the effect of Atoh8 depletion was completely abrogated if Wnt signalling activation is blocked by recombinant Sfrp1 during reprogramming/transformation. These data are now incorporated in Fig. 7p-q. Finally, even if not requested, we also demonstrated the existence of a feedback loop linking Atoh8 and Wnt signalling, with Wnt3a treatment that activates Atoh8 expression in MEFs (Fig. 7r).
Minor points: 1. The authors should show the actual numbers throughout the Figures and not foldchange/percentage/fold ratio (for example, figure 2F, 4C).
We attempted to modify the figures but it was very difficult to present the actual numbers because of the important variability observed between experiments and MEF batches, as reported by other labs 14,15 . We maintained the ratio but we can easily provide the detailed numbers of each experiment as raw data if requested by R2. Figure S1g, no error bars are presented for two conditions. The authors should include the missing data.

In
Data have been included accordingly in the Extended Data Figure 1e. 3. The cell cycle is only affected in "transfo" conditions but not in the "repro+transfo." The author should clarify this discrepancy and show the original FACS plots.
We thank R2 for this observation. We now included the FACS plots in Extended Data Fig. 1f. We also commented on this result by enlarging the protective effect of OSKM on DNA damage and cell cycle features in the main text page 5. Figure 3f does not help and can be removed, as the populations RI4 and TI4 still have MEF identity. Moreover, the axis is starting at 3.5 while it should begin at 0.

4.
As requested, we removed Fig. 3f from the main figures. Figure S1i, S2d, S2m, 5m, 5q, 6j, the authors present statistical differences and mention "n=3" in the Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. figure legends; however, only two samples per condition are shown. The authors should include the missing data points.

In
We modified the figure legends and/or figures accordingly.

Reviewer #3:
Remarks to the Author:

Review of Huyghe et al NCB 2021
This study interrogated the early stages of cellular reprogramming and cellular transformation, to identify the molecular changes that define and drive these processes, and to examine whether loss of cellular identity is coupled to gain of plasticity. The authors developed and utilized a mouse model that enabled inducible reprogramming or transformation of mouse embryonic fibroblasts (MEFs) ex vivo. Reprogramming of MEFs to induced pluripotent stem cells (iPSCs) was achieved by doxycyclineinducible expression of Oct4, Sox2, Klf4 and Myc (OSKM) whereas oncogenic transformation was achieved by tamoxifen-induced activation of oncogenic K-ras (sometimes with cMyc overexpression and trp53 knockdown). Established functional readouts were used to assess each model (teratoma formation and alkaline phosphatase positive colonies by reprogrammed iPSCs, and soft agar colony Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. formation/anchorage independent growth and in vivo tumor formation for transformed MEFs). Multiple molecular modalities were used to characterize early time points of each process (single cell and bulk RNA-seq, ATAC-seq). The main findings include identification of Bcl11b as a novel marker of MEF cell identity and whose downregulation (together with the previously described marker Thy1) can be used to prospectively isolate cells prone to (or already undergoing) reprogramming/transformation; use of ATACseq data to identify FosL1 as having divergent roles in transformation (loss blocks) and reprogramming (loss enhances); purported uncoupling of loss of cell identity and gain of plasticity; identification of shared gene expression changes that occur during both reprogramming and transformation; and identification of the Atoh8 transcription factor as a nonspecific constraint to reprogramming and transformation through its presumed role in suppressing Wnt signaling.
The evidence that Bcl11b is a faithful marker of transformation and reprogramming is well supported by the cell sorting experiments presented in Figure 2, although experimental perturbation would strengthen the functional relevance of this marker even further. Identification of Atoh8 as a relatively nonspecific constrainer of cellular plasticity is also well supported by the data, including examining Atoh8 knockdown in additional models of transformation and trans-differentiation. Overall, this study illustrates the power of examining dynamic cellular processes in a highly controlled, defined manner using timeresolved high-resolution molecular read-outs. The main conclusions of the study could be significantly strengthened by additional experiments, clarifying aspects of the experimental design, and analyzing the wealth of data in additional ways, as outlined below.
We sincerely thank R3 for his/her strong support of the work and for her/his constructive comments.
Major comments 1. The authors report the finding that acquisition of cellular plasticity precedes loss of cellular identity as a major conceptual advance. This is based on the observation that RI4/TI4 cells did not downregulate MEF identity (Figure 3f). However, in Fig 1j the MEF identity score seems to decrease pretty quickly, especially along the transformation trajectory. As the RI4/TI4 intermediates are defined by loss of both Bcl11b and Thy1 which, according to Figure 2c, would place them towards the end of the trajectories where the MEF identity has been lost, can the authors add clarity where these group 4 intermediates might lie along the trajectory? Importantly, how MEF identity is defined and what makes up the MEF identity score should be clearly stated, even if it is derived from other publications.
We sincerely apologize for this lack of accuracy. In the initial draft, the MEF identity score was defined by a list of 298 MEF genes defined by Schiebinger et al., 2019 7 . The color code that we used in Fig. 1j was confusing and gave the feeling that the identity was decreasing pretty quickly, as noticed by R3. We apologize for this inaccuracy and proceeded as follow to correct: Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1-With the help of our bioinformatics department, we modified the color code to render it more accurate and to show that the MEF identity score is decreasing far more slowly than initially presented and is still maintained in some cells after 10 days of reprogramming or transformation (please see new Fig. 1k). 2-We also added subsequent representations (Pseudo-time analysis in Fig. 1l and Violin plots encompassing the final iPS and cancer cells samples in Extended Data Fig. 1n) to reinforce the idea that the MEF identity is not rapidly and homogeneously lost during both processes. 3-Moreover, in order to strengthen this important result, we reproduced the analyses with a second independent MEF identity score (defined in Nefzger et al., 2017 6 ) that consists of 395 genes. Similar results were obtained with the two independent MEF scores and integrated in Extended Data Fig. 1l-m but also throughout the manuscript (Extended Data Fig. 2m and 7l).
As requested by R3, we also attempted to position the RI4 and TI4 (now called BLTL) cells on their respective single cell trajectories. The direct comparison of sc-RNA-seq data with bulk-RNA-seq is hindered by the difference in resolution between the two techniques. We circumvented this issue in 2 ways: 1-We first located the cells that downregulated both Bcl11b and Thy1 on the single cell trajectories and observed that they appeared at day5 of both processes, especially in the bifurcation area that we identified (see Diffusion map in Fig. 3a and Violin plots in Extended Data Fig. 3b). 2-We used a trick by finding differentially expressed genes between each intermediate (RI4-BLTL and TI4-BLTL) and untreated MEFs in bulk-RNA-seq, and converting them into a "signature score". These scores were next calculated for each individual cell and projected on the sc-RNA-seq diffusion map. For reprogramming and to a lesser extent transformation, we noticed the emergence of a reduced number of cells harboring a high activity of the BLTL score in the bifurcation area, suggesting that these cells might correspond to the bulk intermediates (please see Fig. 4g).
2. Bcl11b downregulation was identified as a marker of transformation and reprogramming at very early time points and the sub-population sorting experiment in Figure 2 suggests that Bcl11b downregulation is a key event in both processes. Given that a) Bcl11b has not been explored in the context of MEF cell identity or transformation/reprogramming, b) Bcl11b is a well-known regulator of T cell differentiation with similar properties (constraining cell fate) and c) the claim of uncoupling between loss of cell identity and gain of plasticity is based on Bcl11b-defined populations, functional perturbation of Bcl11b would increase the significance of this finding and these claims. The experimental system appears sufficiently established to feasibly test the impact of Bcl11b knock-down (and possibly rescue) on transformation and reprogramming.
We thank R3 for this inspiring comment, redundant with R2. As suggested, we conducted a large number of approaches to dissect Bcl11b function during reprogramming, transformation and transdifferentiation that are now presented in a fully new Figure 2: Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1-We started by modulating Bcl11b expression prior to induce reprogramming or transformation. Bcl11b gain-and loss-of-function approaches in MEFs (using RNA interference but also Bcl11b conditional KO MEFs 11 ) led to demonstrate that Bcl11b depletion significantly increased reprogramming and transformation efficiencies (please see Fig. 2e-i). Even if not requested by R3, we next conducted teratoma formation assays to show that Bcl11b depletion has no major detrimental effect on the acquisition of in vivo multilineage differentiation potential (Fig. 2j). Therefore, we demonstrated that Bcl11b functionally acts as a gatekeeper of the somatic state.
2-Bcl11b is highly expressed in T lymphocytes. Therefore, even if not requested, and in order to broaden the impact of our finding, we evaluated Bcl11b role in the reprogramming of T lymphocytes. Using OSKM Dox-inducible primary T cells, we demonstrated that Bcl11b constrains reprogramming of this alternative somatic cell type. Data are presented in Figure 2k-l. We also showed that Bcl11b also constrains MEFs to neuron transdifferentiation triggered by Brn2, Ascl1 and Myt1l (Fig. 2m-n).
3-Next, in order to understand Bcl11b function, we combined RNA-seq and ChIP-seq analyses. We compared the transcriptomes of control, Bcl11b-overexpressing (OE) and Bcl11b-knockdown (KD) MEFs. In parallel, we optimized the Bcl11b ChIP-seq conditions by combining two commercial antibodies (Abcam, ab18465, Bethyl, A300-385A). We identified 7430 specific peaks of Bcl11b binding to the MEF genome (Fig. 2p). We found that Bcl11b modulation impacts 979 genes (adjusted p-value< 5.10-2; log2 FC >0.5 or <-0.5) with 122 genes (13%) presenting a Bcl11b binding site within 10kb of the TSS (Fig. 2s). Finally, the fact that several Bcl11b targets were related to the Mapk signalling pathway led us to demonstrate that Bcl11b directly regulates pErk1/2 levels in MEFs (Fig.  2t), providing a potential explanation of its mode of action during reprogramming 12 .
Overall, we invested massive efforts to address the pertinent suggestion of R3 that led to improve significantly the impact of the manuscript by identifying Bcl11b as a new broad-range gatekeeper of somatic states from reprogramming, transdifferentiation and transformation.
3. In this study, multiple factors were identified as correlating with (Thy1, Bcl11b) or directly impacting (FosL1, Atoh8, Srfp1/2) MEF cell reprogramming and transformation, however whether and how these factors functionally interact with each other was not discussed. Moreover, how these factors interact with the factors that actually drive each process (e.g. OSKM for reprogramming and oncogenic Kras for transformation) was not explored or discussed. Although this would not necessarily impact the main conclusions of the paper, some attempt at tying these diverse factors together within the context of these central cellular processes would improve the broader relevance to the field.
We than R3 for this pertinent comment. To address this point, we conducted different approaches: Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1-We focused in particular on Atoh8 and Bcl11b and, by comparing the ChIP-seq profiles, we revealed the existence of a co-binding on 533 peaks on the MEF genome (see Fig. 7e). We also noticed an overlap of 173 genes that are commonly deregulated by Bcl11b and Atoh8 KD.
2-We individually knockdown the expression of Bcl11b, Atoh8, FosL1 and Sfrp1 in MEFs and assessed the effect on the expression of the other factors. This approach showed that most of the factors do not regulate each other, at least in MEFs, except Sfrp1 that appear to repress FosL1 expression. Deeper investigations will be required but we believe that they are out of the scope of the present study. These data are presented in Extended Data Fig. 7s. 3-We assessed whether the expression of Oct4 (reprogramming) and K-Ras G12D (transformation) was impacted by Bcl11b, Atoh8, FosL1 and Sfrp1 KD. No significant impact was observed, ruling out the possibility that the phenotypes observed were due to a direct action of the candidates on the factors that drive the processes. These data are presented in Extended Data Fig. 7r. 4-Finally, we significantly extended the exploration of the interplay between WNT signalling and Atoh8. We showed in particular that Atoh8 effect is abrogated by recombinant Sfrp1 (see Fig. 7p-q) and we also revealed the existence of a feedback loop by which WNT signalling promotes Atoh8 expression. These data are presented in Fig. 7r. The interplays between the actors are now presented in the model Fig. 7s.
Some of these points are easily explored with the data already generated. For example, Atoh8 knockdown hastened activation of endogenous OSKM genes, suggesting Atoh8 might repress these genes. However, Atoh8 ChIP-seq showed that it predominantly binds enhancers. Does Atoh8 bind OSKM genes? Atoh8, presumably suppresses Wnt signaling by upregulating Wnt inhibitors. Is there any evidence of Atoh8 binding to these genes?
We thank R3 for this suggestion. We interrogated the ChIP-seq datasets and no direct binding of Atoh8 was detected on OSKM, Ras and Wnt-associated genes. We believe that the fact that Atoh8 hinders the activation of the endogenous Oct4 gene might be due to indirect effects, but additional approaches, that appear to us out of the scope of the present study, will be required.
Minor comments and suggestions 1. Experimental details are missing in the following places • How was the MEF identity score identified and applied to the data generated in this manuscript?
We apologize for this inaccuracy. These informations can now be found in the main text page 6 but also in the methods section on page 41.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
• page 10 mentions a result showing expression change of Dmrtc2 and Pou3f1 but these genes are not shown in Extended Data Fig. 4i (and the gene Shisa8 is shown in ED 4i but not listed in the results section).
We apologize for this inaccuracy and thank R3 for noticing it. We corrected the sentence accordingly on page 12.
• Page 11 mentions a "protein class analysis with pantherdb" was used to find master regulators of a transcriptional program, but unclear how this analysis was performed, and it is not mentioned in the Methods section (what genes were used as input? How were they compiled? How did this analysis lead to the subsequent focus on the genes highlighted in Cluster 2 of fig. 3k?) We corrected the revised draft on page 12 by removing the sentence.
• Figure 6i: how long after Atoh8 knockdown were cells taken for RNA-seq? This is important for interpreting that the MEF identity score did not change. In many figures, the time point of the experiment was difficult to track down, so adding the timepoint to the figure itself or including in the figure legend each time would be helpful.
We apologize for this inaccuracy and added time points in a large number of panels throughout the figures and/or in the figure legends. The consequences of Atoh8 depletion were analyzed 5 days after the infection with the lentiviral particles inducing the KD. Even if we cannot rule out that this timing was too short to induce changes of MEF identity, it was sufficient to induce severe transcriptomic changes, suggesting that at least part of the GRN controlled by Atoh8 is impacted at this timing. However, because we believe that this comment is important, we modified the text accordingly claiming that "at this time point, Atoh8 depletion has no significant impact on MEF identity…" on page 16.
2. Based on the wide-spread use of the term 'cellular plasticity' it would be good to clearly define this term within the context of the read-outs presented in this study.
We now included a definition of the term cell plasticity and cited relevant literature in the Introduction page 3.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
We fully agree with R3 that the previous nomenclature was difficult to track. In order to gain clarity, we modified the nomenclature for the reprogramming and transforming intermediates throughout the manuscript. The nomenclature now includes the Bcl11b/Thy1 expression levels with initials (BHTH for Bcl11b high /Thy1 high ) in the new figure 3n. 4. A suggestion is to analyze the joint Bcl11B/Thy1 expression in the single cell data from Figure 1 as support for the transition kinetics inferred in Figure 2 (how does the frequency of Bcl11b/Thy1 high/low cells change along each trajectory? What about the pattern in the bifurcation area, which is relatively under-discussed in this manuscript?) We thank R3 for this pertinent suggestion. We analyzed the joint expression of Bcl11b and Thy1 in the sc-RNA-seq data to visualize the transition inferred in Fig. 2. As shown in Fig. 3a and Extended Data Fig.  3b, we visualized and quantified the emergence of Bcl11b low /Thy1 low cells along each trajectory. We also discussed in more detail the existence and significance of this area in the discussion page 18.
5. An additional suggestion would be to identify genes that significantly correlate with Bcl11b expression during reprogramming and transformation to identify other genes putatively regulated by or regulating Bcl11b expression during these processes. Are they similar in the repro/transfo context? Particularly given the GO terms of the common set in Figure 3h, it is likely worthwhile to describe a putative Bcl11b controlled gene expression program in each context.
As presented in the new Figure 2, we deciphered the GRN controlled by Bcl11b by conducting RNA-seq in gain-and loss-of-function settings with ChIP-seq analyses. For this specific question, we compared the genes responding to Bcl11b KD in MEFs with the genes that significantly correlate with Bcl11b during reprogramming and transformation. This analysis led to reveal a significant overlap, in agreement with the fact that these intermediates lost Bcl11b expression. These data are presented in Figure 4f. 6. Unsupervised hierarchical clustering of the ATAC-seq data to identify clusters in an unsupervised manner is another way to identify potentially interesting clusters and see how it compares to the supervised classification of C1-C6 in Fig. 3c,d We included a heatmap visualizing relative peak intensities in the data (see Extended Data Fig. 4b) for differential loci. It allows indeed to visualize the high-dimensional structure of the data as well as our cluster group assignments. However, in this case, the data appear a bit too complex to do a single hierarchical clustering to identify peak groups, potentially explaining why the clusters don't just show up as neat blocks due to the high dimensionality.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. fig. 4i should also show Dmrtc2 and Pou3f1 but these are missing. Shisa8 is shown but isn't listed in the main text (p.10)

Extended data
We apologize for this inaccuracy and thank R3 for noticing it. We corrected the sentence accordingly on page 12. 8. Fig. 3a: add the % variance explained by each component We corrected this part and added the % variance in figure 4a. 9. Why is p53 knock-down used in the experiments shown in Figure 4, but not in previous experiments of the same experimental system?
We included p53 KD to accelerate the transformation process in several experiments but we obtained similar results with or without p53. 10. The significance of the gene expression results presented in Figure 4f-j is unclear; gene expression differences can be seen, but how they support the conclusion that "Atoh8 constrains cellular plasticity" is not clear. The functional read outs on the other hand are very convincing.
We agree with R3. We modified the main text in order to focus more specifically on the adhesion properties of the cells that are modulated by Atoh8, especially via Cdh1. We also modified the title of the paragraph accordingly for "Atoh8 regulates the acquisition of malignant features" page 13.
11. Also figure 4: the colony assay and western blots show that Atoh8 KD has an impact at early time points (d.3 and d.6) but analysis of gene expression was performed at day >30, so it's unclear if the time points directly impacted by Atoh8 are missed (e.g. the results in Figs4f,h,I,j are simply reflecting emergence of different cell states/types that are consequential to Atoh8 knock-down).
We understand R3 comment and separated the 2 biological questions in the revised draft page 13. We focused first on the "early" effects of Atoh8 depletion on the efficiency and pace of oncogenic transformation. In a second paragraph, we asked a different question, namely whether "Atoh8 depletion leads to the emergence of different cell states". We also removed the kinetic assays showing the early induction of cdh1 because they were potentially confusing for the overall message.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Decision Letter, first revision:
7th April 2022 Dear Fabrice, Thank you for submitting your revised manuscript "The comparative roadmaps of reprogramming and transformation unveiled that cellular plasticity is broadly controlled by Bcl11b and Atoh8" (NCB-L46074A). It has now been seen by the original referees and their comments are below. The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Cell Biology, pending minor revisions to satisfy the referees' final requests and to comply with our editorial and formatting guidelines.
Please, note that we wish you to address ALL the remaining referee points, EXCEPT for point 2 of referee 1, asking you to address the commonality of the initial events of reprogramming and transformation (which currently mostly relies on the gene expression profiles and the roles of few regulators) that -although interesting and potentially insightful-was not raised in the initial round of review and we do not consider necessary for publication of your article at Nature Cell Biology.
If the current version of your manuscript is in a PDF format, please email us a copy of the file in an editable format (Microsoft Word or LaTex)--we can not proceed with PDFs at this stage.
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Best regards,
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Final Decision Letter:
Dear Fabrice, I am pleased to inform you that your manuscript, "Comparative roadmaps of reprogramming and oncogenic transformation identify Bcl11b and Atoh8 as broad regulators of cellular plasticity.", has now been accepted for publication in Nature Cell Biology. Congratulations to you and your team! Thank you for sending us the final manuscript files to be processed for print and online production, and for returning the manuscript checklists and other forms. Your manuscript will now be passed to our production team who will be in contact with you if there are any questions with the production quality of supplied figures and text.
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