Med12 cooperates with multiple differentiation signals to facilitate efficient lineage transitions in embryonic stem cells

  1. Max Fernkorn
  2. Christian Schröter

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Abstract

Cell differentiation results from coordinated changes in gene transcription in response to combinations of signals. FGF, Wnt, and mTOR signals regulate the differentiation of pluripotent mammalian cells towards embryonic and extraembryonic lineages, but how these signals cooperate with general transcriptional regulators is not fully resolved. Here, we report a genome-wide CRISPR screen that reveals both signaling components and general transcriptional regulators for differentiation-associated gene expression in mESCs. Focusing on the Mediator subunit Med12 as one of the strongest hits in the screen, we show that it regulates gene expression in parallel to FGF and mTOR signals. Loss of Med12 is compatible with differentiation along both the embryonic epiblast and the extraembryonic primitive endoderm lineage, but impairs pluripotency gene expression and slows down transitions between pluripotency states. These findings suggest that Med12 helps pluripotent cells to efficiently execute transcriptional changes during differentiation, thereby modulating the effects of a broad range of signals.

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  1. Version published to 10.1101/2024.01.22.576603v3 on bioRxiv
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    The authors describe a genome-wide CRISPR screen in mouse ES cells to identify factors and genes that regulate positively and negatively FGF/ERK signaling during differentiation. Out of known and potentially novel regulating signals, Mediator subunit Med12 was a strong hit in the screen and it was clearly and extensively shown by that the loss of Med12 results in impaired FGF/ERK signal responsiveness, modulation of mRNA levels and disturbed cell differentiation leading to reduced stem cell plasticity.
    This is a very concise and well written manuscript that demonstrates for the first time the important role of Med12 in ES cells and during early cell differentiation. The results support data that had been previously observed in Med12 mouse models and in addition show that Med12 cooperates with various signaling systems to control gene expression during early lineage decision.

    We thank the reviewer for their positive evaluation of our work.

    Fig. 3 Supp1A-B:
    The loci of all three independent Med12 mutant clones and the absence of Med12 should be included. Are all three Med12 loss-of-function mutants?

    In the revised version of the manuscript, we have updated the scheme in Fig. 3 Supp 1A to represent both deletions that were obtained with the CRISPR guides used. Both the more common 97 bp deletion as well as the 105 bp deletion that occurred in one clonal line result in a complete loss of the protein on the western blot (Fig. 3 Supp. 1B), suggesting that all mutant clones used for further experiments are loss-of-function mutants.

    Minor:
    Line 466: Should be Fig. 6F, not 6E.

    We have removed this figure panel and the corresponding text in response to the other reviewers' comments.

    Reviewer #1 (Significance):

    The CRISPR screen identified list of some novel interesting factors that regulate FGF/ERK signaling in ES cells. Med12 was then analyzed in very detail on various levels and under various differentiation conditions, resulting in a complex picture how Med12 controls stem cell plasticity. These data support results observed in mouse models and identified novel regulating mechanisms of Med12.

    Reviewer #2 (Evidence, reproducibility and clarity):

    In the manuscript "Med12 cooperates with multiple differentiation signals to enhance embryonic stem cell plasticity" Ferkorn and Schröter report on the role of Med12 in mouse embryonic stem cells. The perform an elegant genetic screen to identify regulators of Spry4 in mouse ESCs, screening for mutations that increase and decrease Spr4-reporter expression in serum/LIF conditions. They find that Med12 deletion results in defects in the exit from naïve pluripotency and in PrE-formation upon Gata-TF overexpression. Using scRNAseq experiments they report a reduction in biological noise in Med12 KO cells differentiating towards PrE upon Gata6 OE.

    Major points:

    1. The title might not exactly reflect the scientific findings of the manuscript. There is little direct evidence for a decrease in plasticity upon Med12 depletion.

    We have changed the title to "Med12 cooperates with multiple differentiation signals to facilitate efficient lineage transitions in embryonic stem cells". In addition, we have toned down claims that Med12 regulates plasticity throughout the manuscript.

    1. Fig 1G: From the data provided it is not entirely clear how well screen results can be validated. Did some of the mutants identified in the screen also produce no detectable phenotypes? What would be the phenotype of knocking out an unrelated gene? In other words, are some of the weak phenotypes really showing Spry4 downregulation or are they withing the range of biological variance?

    Fluorescence levels in Fig. 1G have been normalized to control wild-type cells (dashed red line). Absence of a detectable phenotype would have resulted in normalized fluorescence values around 1. Fluorescence values of all tested mutants were significantly different from 1, as indicated in the statistical analysis given in the figure legend. Furthermore, H2B-Venus fluorescence of cells transfected with a non-targeting control vector are shown in Fig. 1F, and are not different from that of untransfected control wild-type cells. We have now added an explicit explanation how we normalized the data to the figure legend of Fig. 1G, and hope that this addresses the reviewer's concern.

    1. Rescue experiments by re-expressing Med12 in Med12 KO ESCs are missing. Can the differentiation and transcriptional phenotypes be rescued?

    We agree with the reviewer that a rescue experiment re-expressing Med12 would be ideal to ensure that the observed phenotypes are specifically due to loss of Med12. However, we could not identify commercially available full-length Med12 cDNA clones. Even though we managed to amplify full-length Med12 cDNA after reverse transcription, we were unable to clone it into expression vectors. These observations suggest that specific properties of the Med12 cds make the construction of expression vectors by conventional means difficult, and solving these issues is beyond the scope of this study.

    Throughout the study we used multiple independent clonal lines in multiple experimental readouts and obtained congruent results. The reduced expression of pluripotency genes for example was observed in bulk sequencing of the lines introduced in Fig. 3, and by single-cell sequencing of independently generated _Med12-_mutant GATA6-mCherry inducible lines (Fig. 5 Supp. 1B). We argue that this congruence makes it unlikely that the results are dominated by off-target effects.

    1. L365: The subheading "Transitions between embryonic... buffered against loss of Med12" is confusing. The data simply shows that Med12 KOs can still, albeit less efficiently generate PrE upon Gata TF OE. Is there evidence for some active buffering? I think the authors could simply report the data as is, stating that the phenotypes are not a complete block but an impairment of differentiation.

    Prompted by the reviewer's comment as well as remarks along similar lines by reviewer #4, we have completely reorganized this section and now present all the analysis pertaining to PrE differentiation in a new figure 4. In the revised text (lines 316 - 378), we refrain from any speculations about possible buffering and simply report the data as is, as suggested by the reviewer.

    1. L386: Would it not make more sense to reduce dox concentrations in control cells to equalize Gata6 OE to equalize levels between Med12 KO and controls? A shorter pulse of Gata6 does not really directly address unequal expression levels due to loss of Med12. Different pulse length of OE might have consequences that the authors do not control for. This also impacts scRNAseq experiments which suffer from the same, in my opinion, suboptimal experimental setup. This is a point that needs to be addressed.

    We agree with the reviewer that it would have been desirable to equalize GATA6 overexpression levels between wild-type and Med12-mutant cells while keeping induction time the same. In our experience however, reducing the dox concentration is not suitable to achieve this: Rather than reducing transgene expression levels across the board, lower dox concentrations tend to increase the variability within the population - see Fig. 2 in PMID: 16400644 for an example. Since we agree with the reviewer that the setup of the scRNAseq experiment limits our ability to draw conclusion regarding the separation of cell states, we have decided remove these analyses in the revised manuscript. In doing so, we have reorganized the previous figures 5 and 6 into a new single figure 4. This has made the manuscript more concise and allowed us to focus on the main phenotype of the Med12 mutant cells, namely their delayed exit from pluripotency.

    1. The reduced transcript number in Med12 KOs is interesting, but how does it come about. Is there indeed less transcriptional activity or is reduced transcript numbers a side effect of slower growth or the different cell states between WT and Med12 mutants. Appropriate experiments to address this should be performed.

    To address this point, we have performed EU labeling experiments, to compare RNA synthesis rates between wild-type and Med12-mutant during the exit from pluripotency. These experiments confirmed an increase in the mRNA production upon differentiation for both wild-type and Med12 mutant cells, but the method was not sensitive enough to detect any differences between wild-type and Med12 mutant cells within the same condition. The EU labeling thus supports the notion that overall transcriptional rate increases during differentiation, but leaves open the possibility that reduced mRNA levels in Med12 mutant cells arise from effects other than reduced transcriptional output. These new analyses areshown in Fig. 4 Supp. 3 and described in the main text in lines 373 - 378.

    1. I the proposed reduction of biological noise a feature of the PrE differentiation experiments or can it also be observed in epiblast differentiation.

    To address this question, we have carried out single-cell measurements of Spry4 and Nanog mRNA numbers to compare transcriptional variability between wild-type and _Med12-_mutant cells during epiblast differentiation (new Fig. 3 Supp. 1G, H). These measurements confirmed the differences between genotypes in mean expression levels detected by RNA sequencing. However, this analysis did not reveal strong differences in mRNA number distributions. Furthermore, as discussed in point 6 above, our interpretations of noise levels in the PrE differentiation paradigm could have been influenced by the unequal GATA6 induction times. Finally, reviewer #4 pointed out that 10x genomics scRNAseq is not ideal to compare noise levels when total mRNA content differ between samples, as is the case in our dataset. We therefore decided to tone down our conclusions regarding altered noise levels in Med12-mutant cells.

    1. I cannot follow the authors logic that Med12 loss results in enhanced separation between lineages. How is this experimentally supported.

    As discussed in point 6 above, this result could have been influenced by the unequal induction times between wild type and Med12-mutant cells. We have therefore decided to remove this analysis in the revised version of the manuscript.

    Minor points:
    Fig 3, Supp1 A: What exactly are the black and blue highlighted letters?

    The black and blue highlighted letters indicate whether bases are part of an intron or an exon. Exon 7 is now explicitly labelled in the figure, and the meaning of the highlighting is explained in the figure legend.

    Reviewer #2 (Significance):

    Overall, this is an interesting study. The screen has been performed to a high technical standard and differentiation defects were appropriately analyzed. The manuscript has some weaknesses in investigating the molecular mode of action of Med12 which could be improved to provide more significant insights.

    Reviewer #3 (Evidence, reproducibility and clarity):

    The authors sought to identify genes important for the transcriptional changes needed during mouse ES cell differentiation. They identified a number of genes and focussed on Med12, as it was the strongest hit from a cluster of Mediator components.

    Using knockout ES cells, differentiation assays, bulk and scRNAseq, they clearly show that Med12 is important for transgene activation and for gene activation generally during exit from self-renewal, but it is not specifically influencing differentiation efficacy per se. Rather, cells lacking Med12 display "a reduced ability to react to changing culture conditions" and, by inference, to environmental changes. They conclude that Med12 "contributes to the maintenance of cellular plasticity during differentiation and lineage transitions."

    Med12 is a structural component of the kinase module of Mediator, but it is not clear what this study tells us about Mediator function. The authors state that their results contrast with those obtained using a Cdk8 inhibitor, which resulted in increased self-renewal (lines 577-580). I'm not sure where their results show "...that loss of Med12 leads to reduced pluripotency." (lines 579-580). They do not test potency of these cells. There is reduced expression of some pluripotency-associated markers and fewer colonies formed in a plating assay, but these assays to not test cellular potency.

    We agree with the reviewer that our RNA sequencing and colony formation assays do not exhaustively test cellular potency. We have therefore changed the wording in the paragraphs that describe these assays and now talk about "reduced pluripotency gene expression" (e.g. lines 20, 228, 461, 512).

    While their phenotype certainly appears different from that reported in cells treated with Cdk8 inhibitor, it's not clear to me what to make of it, or what it might tell us about the function of the Mediator Kinase module or of Mediator. That a co-activator is important for gene expression in general, or even for gene activation upon receipt of some signal, is not really surprising.

    We believe that reporting differences in the phenotypes obtained with Cdk8 inhibition versus knock-out of Med12 is relevant, because it yields new insight into the different functions that the components of the Mediator kinase module have in pluripotent cells. We have previously discussed possible reasons for these functional differences (discussion line 519 - 528), and further expand on them in the revised manuscript.

    Minor points:

    It is surprising they don't relate their work to that of Hamilton et al (https://doi.org/10.1038/s41586-019-1732-z) who conclude that differentiation from the ES cell state towards primitive endoderm is compromised without Med24.

    Thank you for pointing out this omission. We now cite the work of Hamilton et al., in line 317 (related to new Fig. 4) and 537 - 538 in the discussion.

    Stylistic point: please make the separation between paragraphs more obvious. With no indentation or extra spacing between paragraphs it looks like one solid mass of words.

    Reviewer #3 (Significance):

    There is a lot of careful work here, but I'm not getting a big conclusion here. Perhaps the authors could argue their main points somewhat more stridently and what we've learned beyond this current system.

    Prompted by the reviewer's comment, we have re-organized the functional analyses of Med12 function in the manuscript by condensing the previous figures 5 and 6 into a new single figure 4. We have removed all discussions of transcriptional noise and plasticity, and now focus more strongly on the slowed pluripotency transitions as the main phenotype of the Med12 mutant cells. These changes make the manuscript more concise, and we hope that they help to deliver a single, clear message to the reader.

    Reviewer #4 (Evidence, reproducibility and clarity):

    Fernkorn and Schröter report the results of a screen in mESCs based on modulation of the fluorescent intensity of the Spry4:H2B-Venus reporter. They identify candidate genes that both positively and negatively modulate the expression of the reporter. Amongst those, are several known regulators of the FGF pathway (transcriptional activator of Spry4) that serve as a positive control for the screen. The manuscript focuses on characterisation of Med12, and the authors conclude that Med12 does not specifically affect FGF-targets. Paradoxically, the authors show that based on the expression of key naïve markers Med12 cells show delayed differentiation. Functionally, however, Med12 mutant cells at 48hrs can form less colonies when plated back in naïve conditions (that would normally indicate accelerated differentiation ). The authors conclude that Med12 mutants have "a reduced ability to react to changing culture conditions". Next, they examine the Med12 mutation affects embryonic/extraembryonic differentiation using an inducible Gata6 expression system. They show that transgene induction is slower and dampened in mutant cells and that overall the balance of fates is skewed towards embryonic cells. Finally, they use single cell RNA sequencing and observe differences in the number of mRNAs detected, as well as the separation between clusters in the mutant cells. They conclude that the mutants have reduce transcriptional noise levels.

    Overall, it was an interesting article exploring the molecular consequences of knocking out a subunit of the mediator complex. The characterisation focuses primarily on the description of the screen and the more functional consequences of the KO, rather than delving onto the molecular aspects (e.g. whether mediator complex assembly is affected, or it's binding etc). The analysis of the transcriptional noise will be of particular interest to the community, although I have some suggestions to exclude the possibility that the analysis simply reflects changes in global transcription levels. I have a small number of concerns and requests for clarification on the data but all of them should be relatively easy to address.

    Mayor points:

    • Med12, transcription levels and noise (Figure 6G, J-L). This is an intriguing observation. The labelling and multiplexing helped resolve many of the issue typically associated with comparing 10x dataset. I have two observations about this analysis:
    1. Clarify how number of mRNA counts per cell is calculated (figure 6F) - the methods only described a value normalised by the total number of counts per cell.

    The mRNA counts shown in the figure correspond to the raw number of UMIs detected per cell. We now explicitly state this in the figure legend. Please note that after re-organizing the manuscript, former Fig. 6F has become Fig. 4 Supp. 3A.

    I feel this observation is key and has repercussions for the interpretation of the data (see point below) and should be independently validated (although I recognise it's difficult!). Since the authors observed differences in a randomly integrated transgene (iGata experiments), it's possible/likely that the dysregulation of transcription output is more generic. A possible suggestion is measuring global mRNA synthesis and degradation rates, either using inhibitors or by adding modified nucleotides and measuring incorporation rate and loss through pulse/chase labelling.

    We have performed an EU labeling experiment to address this point, which is shown in Fig. 4 Supp. 3 and described in the main text in lines 373 - 378 of the revised manuscript. Please refer to our response to reviewer #2, point 6 for a short description of the results.

    1. 10x is not the ideal for looking at heterogeneity/noise since it has a low capture efficiency and there are a lot of gaps/zeros in the lower expression range. Therefore, it's simply possible that mutant cells have dampened transcriptional output, meaning lowly expressed genes which in the WT contribute to the apparent heterogeneity (because there is a higher chance of not being captured), are below the 10x detection range in the mutant. This can be seen by plotting the cumulative sum of the mean gene count across each sample - the 50% mark (=mean gene count at 50% detection) reflects a measure of the "capture efficiency" (either because of technical reasons or lower mRNA input). Generally (e.g. also seen across technical repeats), the mean coefficient of variation, entropy and other measures of population heterogeneity directly scale with this "mean gene count at 50% detection", while the cell-cell correlation inversely scales with the "mean gene count at 50% detection". If this scaling relationships are observed for the WT and mutant, then it is impossible to say from the single cell RNA-seq whether the differences in heterogeneity are due to biological or technical reasons. Unfortunately, down-sampling the reads does not generally correct or normalise for this type of technical noise since the technical errors accumulate at every step of sample prep. Of course, it's possible that the technical noise in the RNAseq obfuscates real differences in the level of noise. The failure of mutant cells to re-establish the naïve network certainly suggest there is something going on. Therefore, I suggest performing the analysis of capture efficiency vs CV2 mentioned above and adjusting the discussion accordingly, and potentially perform single molecule FISH of key variable genes at the interface of the two clusters to validate the difference in heterogeneity.

    As suggested by the reviewer, we have performed single molecule FISH measurements of variable genes (Fig. 3 Supp. 1 G, H), but these did not provide independent evidence for increased noise levels in Med12 mutant cells. In light of the caveats raised by reviewer #4 when estimating noise levels from 10x scRNAseq data, and the suggestion of reviewer #3 to sharpen the focus of the manuscript, we have decided to remove any strong conclusions about different noise levels between the genotypes. Instead, we focus on the slowed pluripotency transitions as the main phenotype of the Med12 mutant cells to make the manuscript more concise, to deliver a single, clear message.

    • Are Oct4 levels affected? Reduction of Oct4 is sufficient to block differentiation (Radzisheuskaya et al. 2013 - PMID: 23629142).

    We thank the reviewer for this idea. We measured OCT4 expression levels in single cells via quantitative immunostaining and found that that there is no difference between wild-type and Med12-mutant cells. It is therefore unlikely that lowered OCT4 levels block differentiation in the mutant. These new results are shown in Fig. 5, Supp. 1 D, E.

    • Med12 mutants showing transcriptionally delayed differentiation (related to figure 4C). Is this delay also reflected in the expression of formative genes? If I understand correctly, Figure 4C is made from a panel of naïve markers. It would be good to determine if the formative network is equally affected (and in the same direction - suggesting a delay), or if the transcriptional changes speak to a global dysregulation/dampened expression.

    Prompted by the reviewer's suggestion, we have extended our analysis of the differentiation delays to genes that are upregulated during differentiation, such as formative genes. Rather than trying to come up with an new set of formative markers to produce a variation of the original Fig. 4C (Fig. 5C in the revised manuscript), we have taken an unbiased approach and extended Fig. 5E with a panel showing the distribution of expression slopes of the 100 most upregulated genes determined as in Fig. 5D. This analysis demonstrates a lower upregulation slope in Med12-mutant cells. This result confirms that both the upregulation and downregulation of genes is less efficient upon the loss of MED12, in line with our conclusion of delayed differentiation.

    • Control for the re-plating experiments in 2i/LIF (Figure 4B). Replating in 2iLIF + FBS can have a large selective effect in certain mutant backgrounds (e.g. Nodal mutants) which don't accurately reflect the differentiation status. To exclude such effects, it would be good to repeat the replating assays in serum-free conditions (laminin coating can help with attachment) and include undifferentiated controls to ensure that the mutant doesn't have a clonal disadvantage.

    The reason we have included FBS in the re-plating assays is that in our experience, _Fgf4-_mutant cells show strongly impaired growth standard in 2i+LIF medium. We anticipate that using laminin coating to help with attachment would not overcome this requirement. We have therefore decided against repeating the re-plating assays. Instead, we state the reason why we used FBS in the main text, and also explicitly acknowledge the reviewers' concern of the risk of selective effects of the FBS and the possible clonal disadvantages of the Med12 mutant line.

    Minor points:

    • I found figure 3D and the corresponding text and caption difficult to understand. It is unclear what a "footprint", "relative pathway activity" or "spearman correlation of footprint" mean. Were all the genes listed below Med12 knocked out and sequenced in this study? I suggest re-working and maybe simplifying the text and figure.

    We re-worked the description about the pathway analysis and stated more clearly that:

    • The footprint is a quantitative measure of the differences in gene expression change of a defined list of target genes between wild-type and perturbation.
    • Only the Med12 mutant data is new data produced in this manuscript and all examples below are from Lackner et al., 2021.

    We think that a more extensive explanation of the terms "relative pathway activity" and "spearman correlation of footprint" would disturb the flow of the manuscript too much. Therefore, we now cite the original paper just next to the sentence these terms are mentioned.

    In figure S1 Sup1 the authors report the dose response of targets to FGF - are those affected in the mutant?

    In this manuscript we have not tested if the dose response of FGF target genes changes upon perturbation of Med12. We argue that such an experiment would be beyond the scope of the current manuscript, since - as acknowledged by the reviewer - "Med12 does not specifically affect FGF-targets".

    • Similarly, it would be helpful to guide the reader through figure 5H-I and the corresponding text and caption since it's not immediately obvious how the analysis/graphs lead to the conclusion stated.

    As a consequence of our reorganization of the manuscript, the original figure 5H-I has been moved to Fig. 4, Supp. 1 in the revised version. The analysis strategy has been described in more detail in one of our previous publications (PMID: 26511924). In keeping with our general decision to make the manuscript more focused and concise, we have decided against further expanding on these data, but instead refer the reader to the original publication.

    • Role of Med12 in regulating FGF signalling. There are two observations that seems a bit at odds with the text description and it would be helpful to clarify: "ppERK levels were indistinguishable between wild-type and Med12-mutant lines" (line 222) - 5/6 datapoints show an increase. "[...] overall these results argue against a strong and specific role of Med12 in regulation of FGF target genes." (line 274). If I understood correctly, ~50% of genes are differentially transcribed because of Med12 KO.

    To address the reviewers' first question, we have performed a statistical test on the quantifications of the western blots. This test indicates that there is no significant change of ppERK levels upon loss-of MED12, which now stated clearly in the text (line 217).

    Second, to clarify why our data argues against a strong and specific role of Med12 in regulation of FGF target genes, we now formulate an expectation (lines 276 - 277): If MED12 specifically regulated FGF target genes, the number of differentially expressed genes would be higher in the wild-type than in the Med12-mutant upon stimulation with FGF. This however is not the case.

    • "[...] as well as transitions between different pluripotent states" (line 41) - references missing.

    We have added a reference to PMID: 28174249 (line 39).

    • Line 447: "differentiation conditions" - it's unclear what it's mean by differentiation and how it relates to the diagram in figure 6A. Are those the 20hr cells? Do the -8h, -4hr and 0hr cells (if I understand the meaning of the diagram) cluster all together?

    We now specify in the text that pluripotency conditions refer to cells maintained in 2i + LIF medium, whereas differentiation refers to cells switched to N2B27 after the doxycycline pulse (lines 341 - 342).

    • The difference in dynamics of mCherry activation as a consequence of Med12 KO are not apparent from figure 5E. It might be easier to visualise this observation if x-axis was normalised to the starting point plotting "time from start of induction".

    We agree with the reviewer that the current alignment has not been optimized to compare GATA6 induction dynamics between wild-type and Med12-mutant cells. If we changed the alignment however, it would not be clear any longer that both genotypes were in N2B27 for the same amount of time before analyzing Epi and PrE differentiation. Since our focus is on the differentiation of the two lineages rather than GATA6-mCherry induction dynamics, we decided to keep the original alignment.

    • Figure 3H/I - what does "gene expression changes" and "fold change ratio" mean?

    In Fig. 3H, we plot the the fold change of gene expression upon FGF4 stimulation in _Med12-_mutant versus that in wild-type cells; in Fig. 3I we plot the distribution of the ratio of these two fold changes across all genes. To make this strategy clearer, we have changed the axis label in Fig. 3H to "expression fold change upon FGF", to make it consistent with the axis label "fold-change ratio" in Fig. 3I.

    • Line 579-580 - please clarify what is meant by "reduced pluripotency".

    Prompted by a similar concern raised by reviewer #3, we have changed the wording throughout this paragraph and now talk of "reduced pluripotency gene expression". See also our response to reviewer #3 above.

    • Title: "enhance ESC plasticity". not sure enhance is the right word? There is no evidence that the plasticity of cells is affected.

    We have changed the title; see also our response to reviewer #2, point 1.

    Reviewer #4 (Significance):

    Overall, it was an interesting article exploring the molecular consequences of knocking out a subunit of the mediator complex. The characterisation focuses primarily on the description of the screen and the more functional consequences of the KO, rather than delving onto the molecular aspects (e.g. whether mediator complex assembly is affected, or it's binding etc). The analysis of the transcriptional noise will be of particular interest to the community, although I have some suggestions to exclude the possibility that the analysis simply reflects changes in global transcription levels. I have a small number of concerns and requests for clarification on the data but all of them should be relatively easy to address.

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    Referee #4

    Evidence, reproducibility and clarity

    Fernkorn and Schröter report the results of a screen in mESCs based on modulation of the fluorescent intensity of the Spry4:H2B-Venus reporter. They identify candidate genes that both positively and negatively modulate the expression of the reporter. Amongst those, are several known regulators of the FGF pathway (transcriptional activator of Spry4) that serve as a positive control for the screen. The manuscript focuses on characterisation of Med12, and the authors conclude that Med12 does not specifically affect FGF-targets. Paradoxically, the authors show that based on the expression of key naïve markers Med12 cells show delayed differentiation. Functionally, however, Med12 mutant cells at 48hrs can form less colonies when plated back in naïve conditions (that would normally indicate accelerated differentiation ). The authors conclude that Med12 mutants have "a reduced ability to react to changing culture conditions". Next, they examine the Med12 mutation affects embryonic/extraembryonic differentiation using an inducible Gata6 expression system. They show that transgene induction is slower and dampened in mutant cells and that overall the balance of fates is skewed towards embryonic cells. Finally, they use single cell RNA sequencing and observe differences in the number of mRNAs detected, as well as the separation between clusters in the mutant cells. They conclude that the mutants have reduce transcriptional noise levels.

    Overall, it was an interesting article exploring the molecular consequences of knocking out a subunit of the mediator complex. The characterisation focuses primarily on the description of the screen and the more functional consequences of the KO, rather than delving onto the molecular aspects (e.g. whether mediator complex assembly is affected, or it's binding etc). The analysis of the transcriptional noise will be of particular interest to the community, although I have some suggestions to exclude the possibility that the analysis simply reflects changes in global transcription levels. I have a small number of concerns and requests for clarification on the data but all of them should be relatively easy to address.

    Major points:

    • Med12, transcription levels and noise (Figure 6G, J-L). This is an intriguing observation. The labelling and multiplexing helped resolve many of the issue typically associated with comparing 10x dataset. I have two observations about this analysis:
    1. Clarify how number of mRNA counts per cell is calculated (figure 6F) - the methods only described a value normalised by the total number of counts per cell. I feel this observation is key and has repercussions for the interpretation of the data (see point below) and should be independently validated (although I recognise it's difficult!). Since the authors observed differences in a randomly integrated transgene (iGata experiments), it's possible/likely that the dysregulation of transcription output is more generic. A possible suggestion is measuring global mRNA synthesis and degradation rates, either using inhibitors or by adding modified nucleotides and measuring incorporation rate and loss through pulse/chase labelling.
    2. 10x is not the ideal for looking at heterogeneity/noise since it has a low capture efficiency and there are a lot of gaps/zeros in the lower expression range. Therefore, it's simply possible that mutant cells have dampened transcriptional output, meaning lowly expressed genes which in the WT contribute to the apparent heterogeneity (because there is a higher chance of not being captured), are below the 10x detection range in the mutant. This can be seen by plotting the cumulative sum of the mean gene count across each sample - the 50% mark (=mean gene count at 50% detection) reflects a measure of the "capture efficiency" (either because of technical reasons or lower mRNA input). Generally (e.g. also seen across technical repeats), the mean coefficient of variation, entropy and other measures of population heterogeneity directly scale with this "mean gene count at 50% detection", while the cell-cell correlation inversely scales with the "mean gene count at 50% detection". If this scaling relationships are observed for the WT and mutant, then it is impossible to say from the single cell RNA-seq whether the differences in heterogeneity are due to biological or technical reasons. Unfortunately, down-sampling the reads does not generally correct or normalise for this type of technical noise since the technical errors accumulate at every step of sample prep. Of course, it's possible that the technical noise in the RNAseq obfuscates real differences in the level of noise. The failure of mutant cells to re-establish the naïve network certainly suggest there is something going on. Therefore, I suggest performing the analysis of capture efficiency vs CV2 mentioned above and adjusting the discussion accordingly, and potentially perform single molecule FISH of key variable genes at the interface of the two clusters to validate the difference in heterogeneity.
    • Are Oct4 levels affected? Reduction of Oct4 is sufficient to block differentiation (Radzisheuskaya et al. 2013 - PMID: 23629142).
    • Med12 mutants showing transcriptionally delayed differentiation (related to figure 4C). Is this delay also reflected in the expression of formative genes? If I understand correctly, Figure 4C is made from a panel of naïve markers. It would be good to determine if the formative network is equally affected (and in the same direction - suggesting a delay), or if the transcriptional changes speak to a global dysregulation/dampened expression.
    • Control for the re-plating experiments in 2i/LIF (Figure 4B). Replating in 2iLIF + FBS can have a large selective effect in certain mutant backgrounds (e.g. Nodal mutants) which don't accurately reflect the differentiation status. To exclude such effects, it would be good to repeat the replating assays in serum-free conditions (laminin coating can help with attachment) and include undifferentiated controls to ensure that the mutant doesn't have a clonal disadvantage.

    Minor points:

    • I found figure 3D and the corresponding text and caption difficult to understand. It is unclear what a "footprint", "relative pathway activity" or "spearman correlation of footprint" mean. Were all the genes listed below Med12 knocked out and sequenced in this study? I suggest re-working and maybe simplifying the text and figure. In figure S1 Sup1 the authors report the dose response of targets to FGF - are those affected in the mutant?
    • Similarly, it would be helpful to guide the reader through figure 5H-I and the corresponding text and caption since it's not immediately obvious how the analysis/graphs lead to the conclusion stated.
    • Role of Med12 in regulating FGF signalling. There are two observations that seems a bit at odds with the text description and it would be helpful to clarify: "ppERK levels were indistinguishable between wild-type and Med12-mutant lines" (line 222) - 5/6 datapoints show an increase. "[...] overall these results argue against a strong and specific role of Med12 in regulation of FGF target genes." (line 274). If I understood correctly, ~50% of genes are differentially transcribed because of Med12 KO.
    • "[...] as well as transitions between different pluripotent states" (line 41) - references missing .
    • Line 447: "differentiation conditions" - it's unclear what it's mean by differentiation and how it relates to the diagram in figure 6A. Are those the 20hr cells? Do the -8h, -4hr and 0hr cells (if I understand the meaning of the diagram) cluster all together?
    • The difference in dynamics of mCherry activation as a consequence of Med12 KO are not apparent from figure 5E. It might be easier to visualise this observation if x-axis was normalised to the starting point plotting "time from start of induction".
    • Figure 3H/I - what does "gene expression changes" and "fold change ratio" mean?
    • Line 579-580 - please clarify what is meant by "reduced pluripotency".
    • Title: "enhance ESC plasticity". not sure enhance is the right word? There is no evidence that the plasticity of cells is affected.

    Significance

    Overall, it was an interesting article exploring the molecular consequences of knocking out a subunit of the mediator complex. The characterisation focuses primarily on the description of the screen and the more functional consequences of the KO, rather than delving onto the molecular aspects (e.g. whether mediator complex assembly is affected, or it's binding etc). The analysis of the transcriptional noise will be of particular interest to the community, although I have some suggestions to exclude the possibility that the analysis simply reflects changes in global transcription levels. I have a small number of concerns and requests for clarification on the data but all of them should be relatively easy to address.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors sought to identify genes important for the transcriptional changes needed during mouse ES cell differentiation. They identified a number of genes and focussed on Med12, as it was the strongest hit from a cluster of Mediator components.

    Using knockout ES cells, differentiation assays, bulk and scRNAseq, they clearly show that Med12 is important for transgene activation and for gene activation generally during exit from self-renewal, but it is not specifically influencing differentiation efficacy per se. Rather, cells lacking Med12 display "a reduced ability to react to changing culture conditions" and, by inference, to environmental changes. They conclude that Med12 "contributes to the maintenance of cellular plasticity during differentiation and lineage transitions."

    Med12 is a structural component of the kinase module of Mediator, but it is not clear what this study tells us about Mediator function. The authors state that their results contrast with those obtained using a Cdk8 inhibitor, which resulted in increased self-renewal (lines 577-580). I'm not sure where their results show "...that loss of Med12 leads to reduced pluripotency." (lines 579-580). They do not test potency of these cells. There is reduced expression of some pluripotency-associated markers and fewer colonies formed in a plating assay, but these assays to not test cellular potency. While their phenotype certainly appears different from that reported in cells treated with Cdk8 inhibitor, it's not clear to me what to make of it, or what it might tell us about the function of the Mediator Kinase module or of Mediator. That a co-activator is important for gene expression in general, or even for gene activation upon receipt of some signal, is not really surprising.

    Minor points:

    It is surprising they don't relate their work to that of Hamilton et al (https://doi.org/10.1038/s41586-019-1732-z) who conclude that differentiation from the ES cell state towards primitive endoderm is compromised without Med24.

    Stylistic point: please make the separation between paragraphs more obvious. With no indentation or extra spacing between paragraphs it looks like one solid mass of words.

    Significance

    There is a lot of careful work here, but I'm not getting a big conclusion here. Perhaps the authors could argue their main points somewhat more stridently and what we've learned beyond this current system.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript "Med12 cooperates with multiple differentiation signals to enhance embryonic stem cell plasticity" Ferkorn and Schröter report on the role of Med12 in mouse embryonic stem cells. The perform an elegant genetic screen to identify regulators of Spry4 in mouse ESCs, screening for mutations that increase and decrease Spr4-reporter expression in serum/LIF conditions. They find that Med12 deletion results in defects in the exit from naïve pluripotency and in PrE-formation upon Gata-TF overexpression. Using scRNAseq experiments they report a reduction in biological noise in Med12 KO cells differentiating towards PrE upon Gata6 OE.

    Major points:

    1. The title might not exactly reflect the scientific findings of the manuscript. There is little direct evidence for a decrease in plasticity upon Med12 depletion.
    2. Fig 1G: From the data provided it is not entirely clear how well screen results can be validated. Did some of the mutants identified in the screen also produce no detectable phenotypes? What would be the phenotype of knocking out an unrelated gene? In other words, are some of the weak phenotypes really showing Spry4 downregulation or are they withing the range of biological variance?
    3. Rescue experiments by re-expressing Med12 in Med12 KO ESCs are missing. Can the differentiation and transcriptional phenotypes be rescued?
    4. L365: The subheading "Transitions between embryonic... buffered against loss of Med12" is confusing. The data simply shows that Med12 KOs can still, albeit less efficiently generate PrE upon Gata TF OE. Is there evidence for some active buffering? I think the authors could simply report the data as is, stating that the phenotypes are not a complete block but an impairment of differentiation.
    5. L386: Would it not make more sense to reduce dox concentrations in control cells to equalize Gata6 OE to equalize levels between Med12 KO and controls? A shorter pulse of Gata6 does not really directly address unequal expression levels due to loss of Med12. Different pulse length of OE might have consequences that the authors do not control for. This also impacts scRNAseq experiments which suffer from the same, in my opinion, suboptimal experimental setup. This is a point that needs to be addressed.
    6. The reduced transcript number in Med12 KOs is interesting, but how does it come about. Is there indeed less transcriptional activity or is reduced transcript numbers a side effect of slower growth or the different cell states between WT and Med12 mutants. Appropriate experiments to address this should be performed.
    7. I the proposed reduction of biological noise a feature of the PrE differentiation experiments or can it also be observed in epiblast differentiation.
    8. I cannot follow the authors logic that Med12 loss results in enhanced separation between lineages. How is this experimentally supported.

    Minor points:

    Fig 3, Supp1 A: What exactly are the black and blue highlighted letters?

    Significance

    Overall, this is an interesting study. The screen has been performed to a high technical standard and differentiation defects were appropriately analyzed. The manuscript has some weaknesses in investigating the molecular mode of action of Med12 which could be improved to provide more significant insights.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors describe a genome-wide CRISPR screen in mouse ES cells to identify factors and genes that regulate positively and negatively FGF/ERK signaling during differentiation. Out of known and potentially novel regulating signals, Mediator subunit Med12 was a strong hit in the screen and it was clearly and extensively shown by that the loss of Med12 results in impaired FGF/ERK signal responsiveness, modulation of mRNA levels and disturbed cell differentiation leading to reduced stem cell plasticity.
    This is a very concise and well written manuscript that demonstrates for the first time the important role of Med12 in ES cells and during early cell differentiation. The results support data that had been previously observed in Med12 mouse models and in addition show that Med12 cooperates with various signaling systems to control gene expression during early lineage decision.

    Fig. 3 Supp1A-B:
    The loci of all three independent Med12 mutant clones and the absence of Med12 should be included. Are all three Med12 loss-of-function mutants?

    Minor:

    Line 466: Should be Fig. 6F, not 6E.

    Significance

    The CRISPR screen identified list of some novel interesting factors that regulate FGF/ERK signaling in ES cells. Med12 was then analyzed in very detail on various levels and under various differentiation conditions, resulting in a complex picture how Med12 controls stem cell plasticity. These data support results observed in mouse models and identified novel regulating mechanisms of Med12.

  7. Version published to 10.1101/2024.01.22.576603v2 on bioRxiv
  8. Version published to 10.1101/2024.01.22.576603v1 on bioRxiv