The SynMuvA lin-15A licenses natural transdifferentiation by antagonizing identity safeguarding mechanisms

  1. Sarah Becker
  2. Marie-Charlotte Morin
  3. Julien Lambert
  4. Shashi Kumar Suman
  5. Francesco Carelli
  6. Alex Appert
  7. Stéphane Roth
  8. Sarah Hoff-Yoessle
  9. Jessica Medina-Sanchez
  10. Manuela Portoso
  11. Stéphanie Le Gras
  12. Julie Ahringer
  13. Sophie Jarriault

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Abstract

The mechanisms that restrict or enable latent cellular plasticity have attracted growing interest over the past decade, with important implications for cancer and regenerative therapies. However, the diversity of both pro- and anti-plasticity mechanisms remains incompletely understood. Here, we identify the THAP domain gene lin-15A as a novel factor involved in the natural rectal-to-neuronal Y-to-PDA transdifferentiation in Caenorhabditis elegans . We found that, unlike previously described essential factors, lin-15A is not a Driver of transdifferentiation. Instead, it antagonizes several chromatin-modifying complexes known to safeguard differentiated cell identities. We also show that lin-15A is not a core plasticity factor per se but acts as one specifically in the Y cell context. Together, our findings support a model in which diverse molecular players coordinate controlled cell identity conversions: plasticity factors function as Drivers, while others like lin-15A which we propose to term Licensers attenuate identity safeguarding mechanisms, thereby facilitating transdifferentiation.

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    Reply to the reviewers

    We thank the reviewers for their insightful comments.Please find below a point-by-point response.

    • As the authors acknowledge in the section at the end of the discussion (Limitations of this study) it is not established that LIN-15A has a cell-autonomous function in Y-to-PDA transdifferentiation. Given that LIN-15A has a cell non-autonomous function in vulval development (Herman and Hedgecock, 1990) it is possible that its function here could also be. The authors have used an egl-5 promoter to rescue lin-15A through expression in rectal cells; however, all these cells are in a neighborhood. The lack of a promoter that is specfic for Y has impeded answering this question (a standard genetic mosaic analysis would be problematic because of the incomplete penetrance of the mutation). Although this issue is addressed in the section at the end of the Discussion, I think most readers would like to see this acknowledged earlier in the presentation, perhaps after describing the egl-5 rescue experiment.

    We thank the reviewer for this comment and agree that our data do not formally demonstrate a Y-cell autonomous role for LIN-15A during Y-to-PDA transdifferentiation, as we discussed in the manuscript. As suggested, we have modified the Results section immediately after the egl-5 rescue experiment to explicitly acknowledge this limitation early-on (see p8, l168-170) and retained the discussion in the "Limitations of the study" section.

    • The experiment shown in Figure S1B is unconvincing. To show that they are detecting a LIN-15A-LIN-56 heterodimer, the authors need to show that antibody tags to both proteins detect the band. Mass spectrometry or biochemical purifications would also be helpful. As it is, they show the protein(s) detected depend genetically on lin-56 and lin-15A. It was also unclear what the other bands were in the mutant backgrounds

    We agree that the experiment shown in Figure S1B does not provide sufficient evidence to conclusively demonstrate the existence of a LIN-15A-LIN-56 heterodimer. While the detected species depend genetically on both lin-15A and lin-56, we agree that additional controls, such as detection through reciprocal tagging, biochemical purification, or mass spectrometry, would be required to firmly establish the molecular nature of the complex and to interpret the additional bands observed in the mutant backgrounds. As this experiment is not essential to the conclusions of the manuscript, we have removed Figure S1B and the associated statements from the revised version.

    • Lines 207-212. The authors are making an argument that LIN-15A and LIN-56 function as "Licensers" not "Drivers" because they are not strictly required but appear to facilitate the process. Could it be that LIN-15A and LIN-56 function as "Drivers" but that in their absence the fidelity of the process is compromise? There are many ways that genetic redundancy can be manifested at biochemical levels and the concern is that there are other interpretations of the data. In this regard, the authors should consider rewriting the Abstract to focus on the genetic results underpinning the work. The current version or the Abstract focuses on an interpretation of the data, not the data itself.

    We thank the reviewer for this thoughtful comment. We agree that the Driver/Licenser terminology represents an interpretation of the genetic data and that additional activities acting alongside LIN-15A may exist: lin-15A alleles used in this study correspond to null alleles - that is total loss of lin-15A activity - and approximately half of the animals still successfully undergo Y-to-PDA transdifferentiation in these null mutants. Thus, lin-15A activity is either not strictly required to facilitate the initiation of the process (e.g., threshold model). Or this may point to other factors (than lin-56) able to somewhat compensate for lin-15A absence and that remain to be identified. In line with this interpretation, while we retrieved several alleles for some of the genes identified in our forward genetic screen (in which lin-15A was identified), that screen may not have been saturated. Note that both these hypotheses are compatible with a role for LIN-15A as a licenser of the initiation of the process.

    Importantly, our distinction between "Drivers" and "Licensers" is not solely based on the incomplete penetrance of lin-15A and lin-56 null mutants. First, the distinction reflects the different biological roles inferred from our genetic analyses. The previously characterized factors CEH-6, SOX-2, SEM-4, EGL-27, EGL-5 and HLH-16 are conserved plasticity factors that promote the initiation of transdifferentiation. Their loss results in a complete, or near-complete, failure of Y-to-PDA initiation, and they act within a common plasticity-promoting network. By contrast, LIN-15A and LIN-56 define a genetically distinct pathway. They are neither upstream nor downstream of the Driver cassette, and display additive interactions with partial Driver mutants. Second, loss of LIN-15A does not affect the fidelity or outcome of transdifferentiation. In all defective animals examined, the Y cell retains its normal position, morphology and rectal markers, indicating a failure to initiate the process rather than the production of an aberrant cell type. Third, the fact that a core Driver set is involved in different transdifferentiation events (ie Y-to-PDA and K-to-DVB) but not lin-15A or lin-56 further argues against LIN-15A acting as a Driver. And finally, and most importantly, lin-15A and lin-56 antagonize SynMuvB chromatin regulators known to safeguard differentiated cell identities, while the Drivers do not. In fact, the transdifferentiation process is mostly restored in some lin-15A; SynMuvB double null mutants, suggesting that LIN-15A main function is to block these genes activities. We therefore favor a model in which the Drivers cassette triggers transdifferentiation, whereas LIN-15A and LIN-56 facilitate the process by alleviating inhibitory constraints imposed by identity-safeguarding mechanisms. We have reformulated this in the manuscript in order to make it clearer and also clarified how we define Drivers and Licencers activitities (see p10, l211-217; p13, l283-289 and p14 l312-315). We have further reformulated the abstract to integrate the reviewer's comments.

    Minor Points

    __ 1. Line 279. The authors state that "LIN-15A becomes dispensable when member of the SynMuvB factors are absent." This statement is not completely accurate as the suppression is incomplete.__

    • Addressed, the statement has been reworded in the revised version (see p13, l285-286)

    2. Line 294. The number in Tagble S1 is 58.8% not 65%

    • Addressed, thank you for spotting this, Table S1 was correct, and the typo in the Results section was corrected (see p15, l319).

    3__. Lines 300-301. I couldn't find the data for lin-40. __

    __- __The data can be found in Fig. 3Bii (which we have more clearly indicated in the text) and SI table 1.

    __. Line 363. Should be "represses cell cycle genes." __

    - Addressed

    __5. Line 862. AJM-1 is not a tight junction component. AJM-1 is best described as a component of apical junctions. __

    __- __Absolutely ! Addressed

    Reviewer 2

    • The conclusions derived from the presented data are generally comprehensible but should be phrased more carefully to grant full legitimacy. The reason is that the central mechanistic claim that LIN-15A licenses Td by antagonizing most of the SynMuvBs chromatin factors, including DREAM, rests on whole-animal ChIP-seq that cannot resolve the Y cell. The authors acknowledge that "it was not technically feasible to purify sufficient Y cells for analysis" and therefore use synchronized unstarved L1 whole-animal lysates. This is certainly legitimate, but demands more tact when using such a conclusion as the headline claim.

    We thank the reviewer for this important comment and agree that the mechanistic conclusions drawn from the ChIP-seq data should be presented more cautiously. As noted by the referee and in the manuscript, it was not technically feasible to isolate sufficient Y cells for chromatin profiling and therefore all ChIP-seq experiments were performed on synchronized whole-animal L1 populations. We agree that these experiments cannot directly establish the mechanism operating in the Y cell. Rather, our genetic analyses demonstrate that LIN-15A antagonizes identity-safeguarding SynMuvB factors during Y-to-PDA transdifferentiation. The ChIP-seq data provide an additional and independent line of evidence suggesting that this antagonism may involve modulation of DREAM chromatin occupancy. We have rephrased to state this more clearly. We thus have revised the Abstract (see p2, l7), Introduction (p6, L110-112), Results (see p18-19, l405-420) and Discussion (see p23-24, l523-542 and p25 l566-575) to more clearly separate the conclusions supported by the genetic analyses from the mechanistic interpretation suggested by the ChIP-seq data. We further clarify that the relevance of this mechanism to the Y cell remains a hypothesis consistent with, but not directly demonstrated by, the available data.

    • Also, in the context of the ChIP-Seq experiments, it is understandable that it could not be conducted in a cell-specific manner, but two duplicates in some ChIP-Seq experiments (as stated in the material and methods) is below standard.

    We thank the reviewer for this comment and agree that two biological replicates represent the lower end of what is generally desirable for ChIP-seq analyses. To clarify, more biological samples were initially generated than are represented in the final analysis. In total, five independent biological preparations were performed for each genotype. However, the experimental design imposed substantial technical constraints. Because the experiments required tightly synchronized fed L1 populations (ie, not using a starvation step), standard synchronization procedures could not be used and animals instead had to be collected through successive hatch pulses, resulting in considerably lower yields. Combined with the mutant backgrounds analyzed, this led to variable ChIP-seq quality across preparations. To ensure robustness, we restricted the final analyses to datasets that passed all predefined quality-control criteria. As a result, some conditions were ultimately represented by only two high-quality biological replicates. We agree that this limitation should be made more explicit and have added this information in the Materials and Methods section (p35 l774-779). Despite the reduced number of replicates retained for some conditions, the genome-wide binding patterns observed for LIN-15B and LIN-35 in wild-type animals closely recapitulated those reported previously by the Ahringer laboratory (Gal et al., 2022; SI table 2), supporting the overall robustness and biological validity of the datasets used in this study. More generally, we have also tempered the interpretation of the ChIP-seq experiments throughout the manuscript. We view these data as supportive evidence consistent with a chromatin-level mechanism, rather than as definitive mechanistic proof, and have revised the text to reflect this more clearly.

    • Regarding the genetic interactions with met-2: as MET-2 works in concert with other SET domain proteins, such as SET-25, and also HPL-2, is there a possibility they may be implicated?

    We also considered the possibility that the interaction observed with MET-2 could reflect a broader involvement of the H3K9 methylation machinery, given the well-established functional relationships between MET-2 and other SET domain proteins. To address this possibility, we tested whether SET-25 and SET-32 losses suppressed the lin-15A phenotype. In contrast to met-2 loss-of-function, neither set-25 nor set-32 mutations modified the transdifferentiation defects observed in lin-15A mutants. These observations suggest that the interaction is not a general property of all MET-2-associated SET domain proteins and may instead reflect a more specific role for MET-2 in this context, although we have not tested triple mutant combinations, such as met-2; set-25; lin-15A or met-2; set-32; lin-15A, and therefore cannot exclude additional contributions from these factors. However, based on the available genetic evidence, our data support a model in which the phenotype is more closely linked to the SynMuvB-centered identity-safeguarding machinery than to the canonical MET-2/SET pathways. We now mention these negative results p14, l290-295 and in the discussion (p22, l510-511) of the revised manuscript. HPL-2 itself was tested alongside the other SynMuvBs, as previously reported to be a SynMuvB (Fig. 4Ci). Loss of HPL-2 had the same effect than loss of the other SynMuvBs. Together these data further suggest that the canonical SynMuvB machinery is at play, including MET-2, but not a generic requirement for all H3K9 methyltransferases, and instead points toward a more specific role of MET-2 within the SynMuvB.

    • The fact that Y-to-PDA in males (which involves a cell division) shows the same lin-15A dependence as in hermaphrodites is informative and a bit underplayed. Since this argues against a cell-cycle-coupled mechanism (an important aspect of the reprogramming field) for LIN-15A, it is worth elaborating on this in the discussion.

    We thank the reviewer for this insightful comment and agree that this result deserves further discussion. One of our initial hypotheses was indeed that LIN-15A might be specifically required in transdifferentiation events that occur without a cell division. Cell division and DNA replication have long been proposed to facilitate cellular reprogramming by promoting the dilution or resetting of identity-safeguarding mechanisms. In this context, it was conceivable that LIN-15A and LIN-56 might compensate for the absence of such a process during hermaphrodite Y-to-PDA transdifferentiation. However, our data do not support this model. We found that LIN-15A and LIN-56 are similarly required for Y-to-PDA transdifferentiation in males, despite the fact that this event occurs through a cell division. Conversely, neither factor is required for the K-to-DVB transdifferentiation, which also occurs in the rectum at a similar developmental stage and likewise involves a cell division. Together, these observations argue that the requirement for LIN-15A is not determined by the presence or absence of cell division. Rather, they suggest that the Licensers activity is context-dependent and linked to specific cellular identities. We agree that this point also strengthens the notion that Licensers are distinct from Driver factors, which function in both Y-to-PDA and K-to-DVB transdifferentiation. We have therefore modified the discussion (see p20 l441-443 and l455-480).

    Minor: __ - in the legend of Figure 1 and other places, it should be "Fisher's exact" instead of "Fisher exact"

    • line 31; exhibits instead of exhibit
    • line 85: results instead of result
    • line 228: involvement instead of involvment
    • line 293: "of missing" in loss of lin-36 had no effect while loss ... lin-53 further
    • lines 297 - 299: check sentence; reads not correct
    • line 395: "with an increase"
    • line 484: "with regard"__
    • All points were all addressed in the revised version.

    Reviewer 3

    Based on the observation that LIN-15A does not affect SynMuvB expression in Y (figure S4), the authors conclude that antagonism of the SynMuvBs by LIN-15A is not likely mediated by a negative control of their expression, but rather by impacting their activity. However, as suggested by the authors, antagonistic functions on the same targe genes is also a possibility. The classical approach to test this would be through expression profiling. I understand that RNA-seq on single Y cells cannot be carried out for technical reasons and that bulk RNA-seq would not be informative. Importantly, the same reasoning applies to the ChIP-seq data that is presented in support for common regulatory functions of a subset of synMuvs and LIN-15A (Figure 6 and S6), which was obtained from whole animals. The relevance of these results to the Y to PDA Td process is therefore extremely limited, as the claim that LIN-15A restricts lin-35/DREAM binding on a subset of target genes is based on a reported decrease in DREAM binding in lin-15 mutants in bulk chromatin. This is especially true as both DREAM and LIN-15A are widely expressed proteins.

    We agree with the general limitation highlighted here. As the reviewer notes, neither expression profiling nor chromatin profiling can currently be performed specifically in the Y cell due to the extremely small number of cells involved and the lack of suitable purification strategies. Consequently, the ChIP-seq experiments were performed on synchronized - and fed - whole-animal L1 populations. These data do not directly establish the mechanism operating during Y-to-PDA transdifferentiation. Rather, our conclusions are based on two distinct observations. First, the genetic analyses demonstrate an antagonistic relationship between LIN-15A and multiple SynMuvB factors during transdifferentiation. Second, the ChIP-seq experiments provide independent evidence that LIN-15A can influence DREAM chromatin occupancy at the organismal level. We interpreted these observations together as supporting a model in which the genetic antagonism may involve modulation of SynMuvB/DREAM chromatin activity. We agree, however, that the ChIP-seq data do not demonstrate that these chromatin changes occur in the Y cell itself, nor do they identify the relevant target genes involved in Y-to-PDA transdifferentiation. We have therefore revised the manuscript to more clearly distinguish between the conclusions supported directly by the genetic analyses and the mechanistic interpretation suggested by the ChIP-seq experiments. Throughout the revised version, and in the discussion in particular, we present the chromatin-level model as a hypothesis consistent with the available data rather than as a demonstrated mechanism operating in Y (see p2, l7 ; p6, l110-112 ; p18-19, l405-420 ; p23-24, l523-542 and p25 l566-575).

    In addition there are specific issues with Figure 6, which is mislabeled: upregulated and downregulated applies to gene expression, while the numbers refer to binding peaks. Why are some numbers in red (not mentioned in the legend). An example of the corresponding genome browser tracks should be shown in supplementary. Was a spike-in used to normalize data?

    We thank the reviewer for these helpful suggestions. We agree that the terminology "upregulated" and "downregulated" is potentially confusing in the context of ChIP-seq peaks. In the revised manuscript, we have replaced these terms with "up-bound" and "down-bound" in Figure 6. Regarding the red numbers, these were originally highlighted to emphasise the relatively small number of peaks showing decreased occupancy in lin-15A mutants compared to the other genotypes analyzed. However, as this information was not explained in the legend and may be confusing to readers, we have removed the color coding in the revised figure. Following the reviewer's suggestion, we have also added representative genome browser tracks in the Figure S6E to illustrate the binding changes described in Figure 6. No exogenous spike-in controls were used in these experiments. The ChIP-seq workflow was intentionally designed to closely follow that used by Gal et al. (2022), to allow direct comparison with the published LIN-15B and LIN-35 datasets. However, several observations suggest that the patterns reported here are unlikely to result from normalization artifacts alone. First, the genome-wide binding profiles obtained for LIN-15B and LIN-35 in wild-type animals closely recapitulate those reported previously, providing an independent validation of the overall quality of the datasets. In addition, the different mutant backgrounds exhibit distinct peak gain/loss profiles rather than a common directional shift that would be expected from a systematic technical bias. Nevertheless, we acknowledge the absence of spike-in controls as a limitation of the dataset and have clarified this point in the revised manuscript in the Material and Methods section (see p36 l84-805).

    Overall the discussion is highly speculative and could be shortened and refocused on the actual findings reported. For example, the fact that GO terms associated LIN-15B targets are associated with membrane processes (mentioned above) is not sufficient to speculate that LIN-15A could increase the delaminating capacities of Y by alleviating SynMuvB repression of membrane process genes.

    Our intention was to discuss possible mechanisms that could connect the observed genetic interactions to the cellular events underlying Y-to-PDA transdifferentiation. We fully agree that some of these interpretations, such as the impact of the DREAM/LIN-15A antagonisms on membrane remodeling, are purely speculative in nature. We have removed the following sentence : "In brief, the role of the Licensers would be to provide a favorable chromatin context for cellular processes that favor/install a plastic state, possibly through the modulation of membrane processes as suggested by our ChIP-seq analyses (Fig. S6). » and changed it to "In this framework, Td Licensers would facilitate transdifferentiation by alleviating identity-safeguarding chromatin states, thereby creating a permissive context for the Drivers to execute the Td program. », and have removed the paragraph describing Y delamination. More generally, we have substantially shortened and refocused the Discussion section to answer the referee's comment.

    The classical definition of a licensing factor is a protein (or complex) that allows the start of DNA replication from a replication origin. In the field of reprogramming, the term "licenser" has been applied to pioneer factors which 'license' transcriptional reprogramming by accessing chromatin to initiate a series of events, including binding of additional, non-pioneer transcription factors and additional chromatin regulators. Here the authors apply the term 'Licensers' to LIN-15A and LIN-56 as factors that facilitate the Td process. This may lead to confusion (and implications) as to what these factors are actually doing.

    We thank the reviewer for raising this point. We agree that the term "licensing" has been used in several biological contexts, including DNA replication and, more recently, cellular reprogramming, where it is often associated with pioneer factors that initiate chromatin remodeling and transcriptional changes. However, our use of the term "Licenser" is intended to describe a distinct functional concept emerging from the genetic analyses presented here. We introduced this terminology to distinguish a class of factors that facilitate transdifferentiation by alleviating identity-safeguarding mechanisms from the previously identified "Driver" factors that actively promote the cell-fate transition itself. In this framework, LIN-15A and LIN-56 are not proposed to act as pioneer factors or direct initiators of transcriptional reprogramming. Rather, the genetic data support a role in creating a permissive context for transdifferentiation by antagonizing mechanisms that oppose cell-fate change. We agree, however, that this distinction was not sufficiently defined in the original manuscript and may lead to confusion. We have therefore revised the Results and Discussion to explicitly frame it in the context of transdifferentiation ("Td Licenser"), define what we mean, and to clearly distinguish this usage from previous applications of the term in DNA replication and reprogramming studies (for instance, see p10, l211-217; p13, l283-289 and p14 l312-315).

    __Minor comments: __

    __ Abstract: why are Drivers and Licensers in capitals? How is Driver defined? __

    __- __We use capital letters to signal that these represent two conceptual categories. However, this could be changed if that impairs reading. Drivers are defined in this study as plasticity factors whose loss completely prevents Td initiation (see p10, l211-217 and p14 l312-315).

    __Figure Aii: no PDE, ajm-1::GFP positive Y cells. It is not clear how the Y cell is identified-isn't ajm-1 supposed to surround the cell? The difference between the top and bottom ajm-1:egl-5 panels is not clear to a non expert. LIN-26 panel is missing. __

    • The Y cell is identified by its location at the ventral-most position on the anterior side of the rectal slit. AJM-1 is a component of the apical junctions, hence it is expressed at the apical domain of the Y cell. The LIN-26 typo has been corrected, the marker used in this experiment is the rectal-specific gene egl-5 which labels the nucleus of the Y cell.

    2F color scheme : licensers are not in yellow but pink

    • Addressed : they now are yellow in the revised version

    Fig S1: need to provide more details about experimental conditions for WB-stage, conditions (reducing agents?), nature of Q2015 antibody. In the absence of this information hard to substantiate claim of a LIN-15/LIN-56 heterodimer in the text -

    See answer to reviewer #1 : we agree that this experiment is dispensable for the results presented in this manuscript and adds more questions than useful information, and it has been removed from the revised version.

    __Line 130. What is the nature of the LIN-56 protein? This would be useful information __

    • Addressed. We have indicated this early on in the introduction (p5, l95-96) and in the Results section (p7, l132-134). Note that little is known about LIN-56 except its association with LIN-15A in VPC specification and that is equally possesses a THAP-like C2CH motif.

    __ Line 38 yielding__

    • Addressed

    __Line 48 identities suggested by Blau and Baltimore (1991). __

    • Addressed
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    Referee #3

    Evidence, reproducibility and clarity

    Through classical genetic analysis and the use of markers for different cell fates the authors identify the THAP domain protein LIN-15A as a novel factor in rectal-to-neuronal Y-to-PDA transdifferentiation (Td) in Caenorhabditis elegans. They show that lin-15A is not a core plasticity factor per se, but acts specifically in the Y cell to initiate Td by antagonizing a subset of chromatin-modifying complexes of the synmuvB class. Based on their data, the authors propose that lin-15A acts as a "licenser", as opposed to their previously described "drivers", to facilitate transdifferentiation.

    Most of the key conclusions are convincing, and experiments overall well executed and controlled.

    The lab previously showed that NODE-like complex components SEM-4/SALL4, CEH-6/OCT, EGL-27/MTA1 act together with the HOX TF EGL-5, SOX-2/SOX2 and HLH-16 to drive transdifferentiation of Y to PDA: in their absence Td does not initiate. The genetic evidence they provide here supports a model in which LIN-15A (together with another factor LIN-56) contributes (but is not essential) to the Td process: its loss results in a 50% decrease in Td (Figure 1A).

    Genetic rescue experiments are consistent with lin-15A acting specifically in rectal cells (Figure 1D), and genetic interaction studies support a role in parallel to "driver" genes (Figure 2). The genetic experiments showing that lin-15A does not act in a second natural transdifferentiation process (K-to-DVB), and actually restricts plasticity in blastomeres, are also well executed and support a specific role for LIN-15A in the rectal Y cell.

    The genetic data in Fig 4 is consistent with SynMuvB genes and LIN-15A acting antagonistically in the same pathway or on the same targets (figure 5). This is interesting, since in vulval cell fate specification lin-15A and synMuvB genes are redundant.

    Major comments:

    Based on the observation that LIN-15A does not affect SynMuvB expression in Y (figure S4), the authors conclude that antagonism of the SynMuvBs by LIN-15A is not likely mediated by a negative control of their expression, but rather by impacting their activity.

    However, as suggested by the authors, antagonistic functions on the same targe genes is also a possibility. The classical approach to test this would be through expression profiling. I understand that RNA-seq on single Y cells cannot be carried out for technical reasons and that bulk RNA-seq would not be informative. Importantly, the same reasoning applies to the ChIP-seq data that is presented in support for common regulatory functions of a subset of synMuvs and LIN-15A (Figure 6 and S6), which was obtained from whole animals. The relevance of these results to the Y to PDA Td process is therefore extremely limited, as the claim that LIN-15A restricts lin-35/DREAM binding on a subset of target genes is based on a reported decrease in DREAM binding in lin-15 mutants in bulk chromatin. This is especially true as both DREAM and LIN-15A are widely expressed proteins.

    In addition there are specific issues with Figure 6, which is mislabeled: upregulated and downregulated applies to gene expression, while the numbers refer to binding peaks. Why are some numbers in red (not mentioned in the legend). An example of the corresponding genome browser tracks should be shown in supplementary. Was a spike-in used to normalize data?

    Overall conclusions based on ChIP-seq data should be significantly toned down throughout - eg line 445 in the discussion: a role in the modulation of membrane processes based on ChIP-seq would require some type of validation using available cell membrane markers. The GO term analysis (Figure S1) identifies many very broad classes.

    Overall the discussion is highly speculative and could be shortened and refocused on the actual findings reported. For example, the fact that GO terms associated LIN-15B targets are associated with membrane processes (mentioned above) is not sufficient to speculate that LIN-15A could increase the delaminating capacities of Y by alleviating SynMuvB repression of membrane process genes.

    A general comment: The classical definition of a licensing factor is a protein (or complex) that allows the start of DNA replication from a replication origin. In the field of reprogramming, the term "licenser" has been applied to pioneer factors which 'license' transcriptional reprogramming by accessing chromatin to initiate a series of events, including binding of additional, non-pioneer transcription factors and additional chromatin regulators. Here the authors apply the term 'Licensers' to LIN-15A and LIN-56 as factors that facilitate the Td process. This may lead to confusion (and implications) as to what these factors are actually doing.

    Minor comments:

    Abstract: why are Drivers and Licensers in capitals? How is Driver defined?

    Figure Aii: no PDE, ajm-1::GFP positive Y cells. It is not clear how the Y cell is identified-isn't ajm-1 supposed to surround the cell? The difference between the top and bottom ajm-1:egl-5 panels is not clear to a non expert. LIN-26 panel is missing.

    2F color scheme : licensers are not in yellow but pink

    Fig S1: need to provide more details about experimental conditions for WB-stage, conditions (reducing agents?), nature of Q2015 antibody. In the absence of this information hard to substantiate claim of a LIN-15/LIN-56 heterodimer in the text

    Line 130. What is the nature of the LIN-56 protein? This would be useful information

    Line 38 yielding

    Line 48 identities suggested by Blau and Baltimore (1991).

    Significance

    The present work builds upon previous work from the lab identifying drivers of the Y to PDA transdifferentiation process and additional players. This is the only group working on this specific Td process. The main limitation of this study is that it relies almost exclusively on classical genetic analysis and reporter gene expression. LIN-15A and SynMuvB proteins are broadly expressed chromatin associated factors and no LIN-15A homolog has been identified outside nematodes; technical difficulties have hindered the implementation of single cell expression data or chromatin binding profiles of the individual cells studied, which would constitute a major brekthrough. In addition the notion of chromatin factors as reprogramming barriers is already well documented. The novelty here lies in the study of natural developmentally regulated transdifferentiation process.

    The work may nonetheless be of broad interest in the reprogramming field by providing an example of the complexity of interaction driving a natural transdifferentiation process, highlighting the activity of parallel pathways and the central role of chromatin associated proteins.

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    Referee #2

    Evidence, reproducibility and clarity

    The study describes the implication of LIN-15A, which is a THAP zinc-finger-like protein, in the Y-to-PDA conversion. This cell fate conversion is an intriguing type of direct reprogramming, as it is a developmentally programmed transdifferentiation process in the nematode C. elegans and offers a unique model for investigating cell fate conversion in vivo. The Jarriault research group has demonstrated in the past how powerful this cell-fate conversion model is for identifying novel players in transdifferentiation. This is a well-executed genetic study that establishes lin-15A as a new player in the Y-to-PDA transdifferentiation phenomenon and introduces a useful conceptual distinction between Driver and Licenser factors. The genetic interactions with class B SynMuvs are clean and informative. LIN-15A is proposed to act as a Licenser in a context-dependent manner because it is also expressed in other cell types; mechanistic insights are indispensable for understanding the nature of this context dependence. Overall, I support the publication of this study, but have some comments prior to its acceptance.

    Main comments:

    The conclusions derived from the presented data are generally comprehensible but should be phrased more carefully to grant full legitimacy. The reason is that the central mechanistic claim that LIN-15A licenses Td by antagonizing most of the SynMuvBs chromatin factors, including DREAM, rests on whole-animal ChIP-seq that cannot resolve the Y cell. The authors acknowledge that "it was not technically feasible to purify sufficient Y cells for analysis" and therefore use synchronized unstarved L1 whole-animal lysates. This is certainly legitimate, but demands more tact when using such a conclusion as the headline claim.

    Also, in the context of the ChIP-Seq experiments, it is understandable that it could not be conducted in a cell-specific manner, but two duplicates in some ChIP-Seq experiments (as stated in the material and methods) is below standard.

    Regarding the genetic interactions with met-2: as MET-2 works in concert with other SET domain proteins, such as SET-25, and also HPL-2, is there a possibility they may be implicated?

    The fact that Y-to-PDA in males (which involves a cell division) shows the same lin-15A dependence as in hermaphrodites is informative and a bit underplayed. Since this argues against a cell-cycle-coupled mechanism (an important aspect of the reprogramming field) for LIN-15A, it is worth elaborating on this in the discussion.

    Minor:

    • in the legend of Figure 1 and other places, it should be "Fisher's exact" instead of "Fisher exact"
    • line 31; exhibits instead of exhibit
    • line 85: results instead of result
    • line 228: involvement instead of involvment
    • line 293: "of missing" in loss of lin-36 had no effect while loss ... lin-53 further
    • lines 297 - 299: check sentence; reads not correct
    • line 395: "with an increase"
    • line 484: "with regard"

    Significance

    This is a well-executed genetic study that establishes lin-15A as a new player in Y-to-PDA transdifferentiation and introduces a useful conceptual distinction between Driver and Licenser factors. The genetic interactions are clean and informative.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    This manuscript from Sophie Jarriault's lab investigates the genetic mechanisms underpinning Y-to-PDA transdifferentiation in the nematode Caenorhabditis elegans. Transdifferentiation is the process by which one differentiated cell type converts to a different differentiated cell type without passing through a pluripotent stem cell stage. Understanding the biology and molecular mechanisms of transdifferentiation in an in vivo context will ultimately aid in engineering transdifferentiation for regenerative medical applications.

    Through a forward genetic screen the authors isolated a mutant allele of the lin-15A gene, which encodes a THAP domain chromatin factor. lin-15A has been well-studied for its role in redundant chromatin pathways that repress the expression of LIN-3/EGF during vulval development. Hence it is referred to as a SynMuvA gene. The authors show that lin-15A null mutants exhibit an incompletely penetrant defect in Y-to-PDA transdifferentiation, with approximately 50% of the animals exhibiting the defect. They show that lin-15A functions in rectal cells, a group of cells in the vicinity of Y and they provide evidence that LIN-15A functions in the initiation of transdifferentiation. Genetic evidence supports a model in which LIN-15A functions in parallel to "Drivers" of transdifferentiation, which include the transcriptional regulators, CEH-6, SOX-2, EGL-5, SEM-4, EGL-27, and SEM-4. An interesting genetic result is that LIN-15A, together another synMuvA gene LIN-56, functions antagonistically to synMuvB genes. Through an analysis of ChIP-seq data, they suggest a model in which LIN-15A antagonizes SynMuvB function in the transdifferentiation decision.

    Critique

    This is a well-written manuscript on an interesting topic. The work provides genetic insights that will be useful for setting "boundary conditions" and predictions for subsequent molecular studies. The authors should consider the following points.

    Major Points

    1. As the authors acknowledge in the section at the end of the discussion (Limitations of this study) it is not established that LIN-15A has a cell-autonomous function in Y-to-PDA transdifferentiation. Given that LIN-15A has a cell non-autonomous function in vulval development (Herman and Hedgecock, 1990) it is possible that its function here could also be. The authors have used an egl-5 promoter to rescue lin-15A through expression in rectal cells; however, all these cells are in a neighborhood. The lack of a promoter that is specfic for Y has impeded answering this question (a standard genetic mosaic analysis would be problematic because of the incomplete penetrance of the mutation). Although this issue is addressed in the section at the end of the Discussion, I think most readers would like to see this acknowledged earlier in the presentation, perhaps after describing the egl-5 rescue experiment.
    2. The experiment shown in Figure S1B is unconvincing. To show that they are detecting a LIN-15A-LIN-56 heterodimer, the authors need to show that antibody tags to both proteins detect the band. Mass spectrometry or biochemical purifications would also be helpful. As it is, they show the protein(s) detected depend genetically on lin-56 and lin-15A. It was also unclear what the other bands were in the mutant backgrounds
    3. Lines 207-212. The authors are making an argument that LIN-15A and LIN-56 function as "Licensers" not "Drivers" because they are not strictly required but appear to facilitate the process. Could it be that LIN-15A and LIN-56 function as "Drivers" but that in their absence the fidelity of the process is compromise? There are many ways that genetic redundancy can be manifested at biochemical levels and the concern is that there are other interpretations of the data. In this regard, the authors should consider rewriting the Abstract to focus on the genetic results underpinning the work. The current version or the Abstract focuses on an interpretation of the data, not the data itself.

    Minor Points

    1. Line 279. The authors state that "LIN-15A becomes dispensable when member of the SynMuvB factors are absent." This statement is not completely accurate as the suppression is incomplete.
    2. Line 294. The number in Tagble S1 is 58.8% not 65%
    3. Lines 300-301. I couldn't find the data for lin-40.
    4. Line 363. Should be "represses cell cycle genes."
    5. Line 862. AJM-1 is not a tight junction component. AJM-1 is best described as a component of apical junctions.

    David Greenstein

    Significance

    General Assessment

    This is well-written genetic study on an interesting topic-a natural case of transdifferentiation. The experiments are well conducted and properly analyzed. The identification of LIN-15A as a player in Y-to-PDA transdifferentiation will enable experimental tests of the authors' model. The limitations of the study are that for technical reasons the authors acknowledge, it is not established whether the function of LIN-15A is cell autonomous to Y. The authors have undertaken the beginnings of molecular work to get at mechanism, but the downstream targets of the apparent chromatin regulation are not yet apparent.

    Advance

    The identification of LIN-15A as a regulator of Y-to-PDA transdifferentation and the suppression by mutations in synMuvB genes are of interest to the field.

    Audience

    This manuscript will be of keen interest to developmental geneticists focusing on chromatin regulation in genetic model systems as well as developmental biologists studying transdifferentiation processes.

  5. Version published to 10.64898/2026.01.22.701184 on bioRxiv