C. elegans establishes germline versus soma by balancing inherited histone methylation

  1. Brandon S. Carpenter
  2. Teresa W. Lee
  3. Caroline F. Plott
  4. Jovan S. Brockett
  5. Dexter A. Myrick
  6. David J. Katz

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Abstract

Embryos undergo extensive reprogramming at fertilization to prevent the inappropriate inheritance of histone methylation. In C. elegans, this reprogramming is mediated by the H3K4me2 demethylase, SPR-5, and the H3K9 methyltransferase, MET-2. In contrast to this reprogramming, the H3K36 methyltransferase, MES-4, maintains H3K36me2/3 at germline genes between generations to help re-establish the germline. To determine whether the MES-4 germline inheritance system antagonizes spr-5; met-2 reprogramming, we examined the interaction between these two systems. We find that the developmental delay of spr-5; met-2 mutant progeny is associated with ectopic H3K36me2/3 and the ectopic expression of MES-4 targeted germline genes in somatic tissues. Furthermore, the developmental delay is dependent upon MES-4 and the H3K4 methyltransferase, SET-2. We propose that the MES-4 inheritance system prevents critical germline genes from being repressed by maternal spr-5; met-2 reprogramming. Thus, the balance of inherited histone modifications is necessary to distinguish germline versus soma and prevent developmental delay.

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    Reply to the reviewers

    We thank the reviewers for their close reading and constructive comments on our manuscript. We believe that their insight has substantially strengthened our manuscript. Please find our response/revision plan for each comment below (in blue). Note, because of the substantial changes to the figures and the additional experiments that are we are undertaking, we have not initially revised the text. The proposed textual revisions will be included in the full revision.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The Katz lab has contributed greatly to the field of epigenetic reprogramming over the years, and this is

    another excellent paper on the subject. I enjoyed reviewing this manuscript and don't have any major

    comments/suggestions for improving it. The findings presented are novel and important, the results are clear

    cut, and the writing is clear.

    It's important to stress the novelty of the findings, which build upon previous studies from the same lab (upon

    a shallow look one might think that some of the conclusions were described before, but this is not the case).

    Despite the fact that this system has been studied in depth before, it remained unclear why and how

    germline genes are bookmarked by H3K36 in the embryo, and it wasn't known why germline genes are not

    expressed in the soma.

    To study these questions Carpenter et al. examine multiple phenotypes (developmental aberrations,

    sterility), that they combine with analysis of multiple genetic backgrounds, RNA-seq, CHIP-seq, single

    molecule FISH, and fluorescent transgenes.

    Previous observations from the Katz lab suggested that progeny derived from spr-5;met-2 double mutants

    can develop abnormally. They show here that the progeny of these double mutants (unlike spr-5 and met-2

    single mutants) develop severe and highly penetrate developmental delays, a Pvl phenotype, and sterility.

    They show also that spr-5; met-2 maternal reprogramming prevents developmental delay by restricting

    ectopic MES-4 bookmarking, and that developmental delay of spr-5;met-2 progeny is the result of ectopic

    expression of MES-4 germline genes. The bottom line is that they shed light on how SPR-5, MET-2 and

    MES-4 balance inter-generational inheritance of H3K4, H3K9, and H3K36 methylation, to allow correct

    specification of germline and somatic cells. This is all very important and relevant also to other organisms.

    **(very) Minor comments:**

    -Since the word "heritable" is used in different contexts, it could be helpful to elaborate, perhaps in the

    introduction, on the distinction between cellular memory and transgenerational inheritance.

    We are happy to elaborate on this in the revised manuscript.

    -It might be interesting in the Discussion to expand further about the links between heritable chromatin

    marks and heritable small RNAs. The do hint that the result regarding the silencing of the somatic transgene

    are especially intriguing.

    We are happy to expand this in the revised manuscript.

    Reviewer #1 (Significance (Required)):

    This is an exciting paper which build upon years of important work in the Katz lab. The novelty of the paper

    is in pinpointing the mechanisms that bookmark germline genes by H3K36 in the embryo, and explaining

    why and how germline genes are prevented from being expressed in the soma.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Katz and colleagues examine the interaction between the methyltransferase MES-4 and spr-5; met-2 double

    mutants. Their prior analysis (PNAS, 2014) showed the dramatic enhancement in sterility and development

    for spr-5; met-2; this paper extends that finding by showing these effects depend on MES-4. The results are

    interesting and the genetic interactions dramatic. The examination by RNAseq and ChIP helps move the

    phenotypes into a more molecular analysis. The authors hypothesize that SPR-5 and MET-2 modify

    chromatin of germline genes (MES-4 targets) in somatic cells, and this is required to silence germline genes

    in the soma. A few issues need to be resolved to test these ideas and rule out others.

    **Main comments:**

    The authors' hypothesis is that SPR-5 and MET-2 act directly, to modify chromatin of germline genes (MES-

    4 targets), but alternate hypothesis is that the key regulated genes are i) MES-4 itself and/or ii) known

    regulators of germline gene expression e.g. the piwi pathway. Mis regulation of these factors in the soma

    could be responsible for the phenotypes. Therefore, the authors should analyze expression (smFISH and

    where possible protein stains) for MES-4 and PIWI components in the embryo and larvae of wildtype, double

    and triple mutant strains. These experiments are essential and not difficult to perform.

    In our RNA-seq analysis we see a small elevation of MES-4 itself (average 1.18 log2 fold change across 5 replicates). This does not seem likely to be solely driving such a dramatic phenotype. Nevertheless, it is possible that the small increase in expression of MES-4 itself could be contributing. To determine if MES-4 is being ectopically expressed in spr-5; met-2 double mutants, we have obtained a tag version of MES-4 from Dr. Susan Strome and will use this to examine the localization of MES-4 protein in spr-5; met-2 double mutants. We are definitely interested in the potential interaction between PIWI components and the histone modifying enzymes that we have explored in this study. However, since RNAi of MES-4 is sufficient to rescue the developmental delay of spr-5; met-2 mutants, we have chosen to focus on that interaction in this paper. In the future, we hope to examine the role of PIWI components in this system.

    A second aspect of the hypothesis is that spr-5 and met-2 act before mes-4 and that while these genes are

    maternally expressed, they act in the embryo. There really aren't data to support these ideas - the timing and

    location of the factors' activities have not been pinned down. One way to begin to address this question

    would be to perform smFISH on the target genes and on mes-4 in embryos and determine when and where

    changes first appear. smFISH in embryos is critical - relying on L1 data is too late. If timing data cannot be

    obtained, then I suggest that the authors back off of the timing ideas or at least explain the caveats.

    Certainly, figure 8 should be simplified and timing removed. (note: Typical maternal effect tests probably

    won't work because if the genes' RNAs are germline deposited, then a maternal effect test will reflect when

    the RNA is expressed but not when the protein is active. A TS allele would be needed, and that may not be

    available.)

    To determine the timing of the ectopic expression of MES-4 targets, we have performed smFISH on two MES-4 targets in embryos. Thus far, these experiments show that MES-4 targets are ectopically expressed in the embryo, but only after the maternal to zygotic transition. This is consistent with our proposed model. A figure containing this data will be added to the revised manuscript. In addition, our model is predicated on the known embryonic protein localization of SPR-5 and MES-4. Maternal SPR-5 protein is present in the early embryo up to around the 8-cell stage, but absent in later embryos (Katz et al., 2009). In addition, in mice, the SPR-5 ortholog LSD1 is required maternally prior to the 2-cell stage (Wasson et al., 2016 and Ancelin et al., 2016). In contrast, MES-4 continues to be expressed in the embryo until later embryonic stages where it is concentrated into the germline precursors Z2 and Z3 (Fong et al., 2002). This is consistent with SPR-5 establishing a chromatin state that continues to be antagonized by MES-4. There is evidence that MET-2 is expressed both in early embryos and later embryos. However, since the phenotype of MET-2 so closely resembles the phenotype of SPR-5 (Kerr et al., 2014), we have included it in our model as working with SPR-5. Further experimentation will be required to substantiate the model, but we believe the model is consistent with all of the current data.

    Writing/clarity:

    -It would be helpful to include a table that lists the specific genes studied in the paper and how they behaved

    in the different assays e.g. RNAseq 1, RNAseq 2, MES-4 target, ChIP. That way, readers will understand

    each of the genes better.

    We are happy to include a table in the revised manuscript.

    -At the end of each experiment, it would be helpful to explain the conclusion and not wait until the

    Discussion. For readers not in the field, the logic of the Results section is hard to follow.

    This seems like a stylistic choice. Traditionally, papers did not include any conclusions in the results section, and it is our preference to keep our paper organized this way. However, if the reviewer would still like us to change this, we are happy to do so.

    -The model is explained over three pages in the Discussion. It would be great to begin with a single

    paragraph that summarizes the model/point of the paper simply and clearly.

    The discussion in the revised manuscript will altered to include this.

    **Specific comments:**

    -Figure 1 has been published previously and should be moved to the supplement.

    In our original paper (Kerr et al.) we reported in the text that spr-5; met-2 mutants have a developmental delay. However, we did not characterize this developmental delay. Nor did we include any images of the double mutants, except for one image of the adult germline phenotype. As a result, we believe that the inclusion of the developmental delay in the main body of this manuscript is warranted.

    -Cite their prior paper for the vulval defects e.g. page 6 or show in supplement.

    We are happy to include a citation of our previous paper for the vulval defects in the revised manuscript.

    -The second RNAseq data should be shown in the Results since it is much stronger. The first RNAseq,

    which is less robust, should be moved to supplement.

    The revised manuscript will include this alteration.

    -Figure 3 is very nice. Please explain why the RNAs were picked (+ the table, see comment above), and

    please add here or in a new figure mes-4 and piwi pathway expression data in wildtype vs double/triple

    mutants.

    We performed RT-PCR on 9 MES-4 targets. These 9 targets were picked because they had the highest ectopic expression in spr-5; met-2 mutants and largest change in H3K36me3 in spr-5; met-2 mutants versus Wild Type. Amongst these 9 genes, we performed smFISH on htp-1 and cpb-1 because they are relatively well characterized as germline genes.

    The revised manuscript will include added panels to supplemental figure 2 showing the expression of PIWI pathway components.

    -Figure 3 here or later, please show if mes-4 RNAi removes somatic expression of target genes.

    We are currently carrying out this experiment. Once it is completed, the data will hopefully be added to the paper.

    -Is embryogenesis delayed?

    Embryogenesis seems to be sped up in spr-5; met-2 mutants. A supplemental figure will be added to the revised manuscript showing this. It is unclear why embryogenesis is sped up. However, this confirms that the developmental delay is unique to the L1/L2 stages.

    -Figure 4 since htp-1 smFISH is so dramatic, it would be helpful to include htp-1 in the lower panels.

    htp-1 will be added to the lower panels in the revised manuscript.

    -Figure 4, please add an extra 2 upper panels showing all the genes in N2 vs spr-5;met-2, for comparison to

    the mes-4 cohort.

    As a control, we will add panels showing a comparison to all germline genes, excluding MES-4 targets. This new data shows that germline genes that are not MES-4 targets do not have ectopic H3K36me3. This data, which further suggests that the phenomenon is confined to MES-4 targets, is consistent with our results showing that MES-4 RNAi is sufficient to suppress the developmental delay.

    -Figure 6. Please show a control that met-1 RNAi is working.

    We performed RT-PCR to try and confirm that met-1 RNAi was working. Despite controls repeating the MES-4 suppression and verifying that RNAi was working, we were unable to demonstrate that met-1 was knocked down. As a result, we will remove this result from the paper. Importantly, this does not affect the conclusion of the paper.

    -To quantify histone marks more clearly, it would be wonderful to have a graph of the mean log across the

    gene. showing the mean numbers would help clarify the degree of the effect. we had an image as an

    example but it does not paste into the reviewer box. Instead, see figure 2 or figure 4

    here: https://www.nature.com/articles/ng.322

    We will attempt to include this analysis in the revised manuscript.

    Reviewer #2 (Significance (Required)):

    Katz and colleagues examine the interaction between the methyltransferase MES-4 and spr-5; met-2 double

    mutants. Their prior analysis (PNAS, 2014) showed the dramatic enhancement in sterility and development

    for spr-5; met-2; this paper extends that finding by showing these effects depend on MES-4. The results are

    interesting and the genetic interactions dramatic. The examination by RNAseq and ChIP helps move the

    phenotypes into a more molecular analysis.

    This work will be of interest to people following transgenerational inheritance, generally in the C. elegans

    field. People using other organisms may read it also, although some of the worm genetics may be

    complicated. Some of the writing suggestions could make a difference.

    I study C. elegans embryogenesis, chromatin and inheritance.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In the paper entitled "C. elegans establishes germline versus soma by balancing inherited histone

    methylation" Carpenter BS et al examined a double mutant worm strain they had previously produced of the

    H3K4me1/2 demethylase spr-5 and the predicted H3K9me1/me2 methylase met-2. These mutant worms

    have a developmental delay that arises by the L2 larval stage. They performed an analysis of what genes

    get misexpressed in these double mutants by performing RNAseq and compare this to datasets generated

    from other labs on an H3K36me2/me3 methylase MES-4 where they see a high degree of overlap. They

    validate the misexpression of some germline specific genes in the soma by in situ and validate that there is a

    dysregulation of H3K36me3 in their double mutant worms. They further find that knocking down mes-4

    reverts the developmental delay.

    I think that the authors need to make more of an effort to be a bit more scholarly in terms of placing their

    work in the context of the field as a whole and also need to add a few additional experiments as well as

    reorganize a bit before this is ready for publication. Remember that the average reader is not necessarily an

    expert in C. elegans or this particular field and you really want to try and make the manuscript as accessible

    to everyone as possible.

    **Major Points**

    1)It would be good to see western blots or quantitative mass spec examining H3K36me3 in the WT and spr-

    5;met-2 double mutant worms. I believe this was also previously reported by Greer EL et al Cell Rep 2014 in

    the single spr-5 mutant worm so that work should be cited here in addition to the identification of JMJD-2 as

    an enzyme involved in the inheritance of H3K4me2 phenotype.

    The ectopic H3K36me3 is confined to a small set of MES-4 targets. We don’t even see ectopic H3K36me3 at non-MES-4 germline genes (see above). Therefore, we don’t expect to see any global differences in bulk H3K36me3. Greer et al reported that there are elevated H3K36me3 levels in spr-5 mutants. This discrepancy may be due to different stages (embryos, germline) present in their bulk preparation. Alternatively, the met-2 mutant may counteract the effect of the spr-5 mutation on H3K36me3. Regardless, we believe that the genome-wide ChIP-seq is more informative than bulk H3K36me3 levels.

    We will add a citation for the Greer paper in the revised manuscript.

    2)Missing from Fig.5 is mes-4 KD by itself. This is needed to determine whether these effects are specific to

    the spr-5;met-2 double mutants or more general effects that KD of mes-4 would decrease the expression of

    all these genes to a similar extent. Then statistics should be done to see if the decrease in the WT context is

    the same or greater than the decrease in the double mutants.

    The MES-4 targets are generally expressed only in the germline and defined by having mes-4 dependent H3K36me3. Knocking down mes-4 would be expected to prevent the expression of these genes in the germline, but this is difficult to test because mes-4 mutants basically don’t make a germline. Regardless, knocking down mes-4 by itself would only assess the role of MES-4 in germline transcription, not the ectopic expression that is being assayed in spr-5; met-2 mutants in Fig 5. Importantly, it remains possible that spr-5; met-2 mutants might also result in an increase in the expression of MES-4 targets in the germline. However, the experiments performed in this manuscript were conducted on L1 larvae, which do not have any germline expression, to eliminate this potential confounding contribution.

    **Minor Points**

    1)A greater attempt needs to be made to be more scholarly for citing previously published literature. This

    includes work on the inheritance of H3K27 and H3K36 methylation in C. elegans and other species as well.

    A few papers which seem germane to this story which should be cited in the intro are (Nottke AC et al PNAS

    2011, Gaydos LJ et al Science 2014, Ost A et al Cell 2014, Greer EL et al Cell Rep 2014, Siklenka K et al

    Science 2015, Tabuchi TM et al Nat Comm 2018, Kaneshiro KR et al Nat Comm 2019). This problem is not

    restricted to the intro.

    Although many of these excellent papers are broadly relevant to this current work, they are not necessarily directly relevant to this paper. For this reason, they were not originally cited. Nevertheless, we will attempt to cite these papers in the revised version when possible.

    2)I think that the authors need to be a little less definitive with your language. Theories should be introduced

    as possibilities rather than conclusions. Should remove "comprehensive" from intro as there are many other

    methods which could be done to test this.

    Throughout the manuscript, we have tried to be clear what the data suggests versus what is model based on the data. Nevertheless, to further clarify this, we are happy to remove “comprehensive” from the intro.

    3)The authors should describe what PIE-1 is. Is this a transcription factor?

    PIE-1 is a transcriptional inhibitor that is thought to block RNA polII elongation by mimicking the CTD of RNA polII and competing for phosphorylation. We are happy to add a reference to this function in the revised manuscript.

    4)The language needs clarification about MES-4 germline genes and bookmark genes. Are these bound by

    MES-4 or marked with K36me2/3?

    The revised manuscript will be modified to make this definition more clear.

    5)I think Fig S1 E+F should be in the main figure 1 so readers can see the extent of the phenotype.

    The original single image of the spr-5; met-2 adult germline phenotype (including the protruding vulva) was included in our previous publication. In this manuscript, we have now quantified this phenotype, which is why it is included in the supplement here. However, because the original picture was included in our original publication, we prefer to leave it as supplemental.

    6)For Fig S2 it would be good to do the same statistics that is done in Fig 2 and mention them in the text so

    the readers can see that the overlap is statistically significant.

    We are happy to include these statistics in the revised manuscript.

    7)Fig S2.2 should be yellow blue rather than red green for the colorblind out there.

    Thanks for pointing this out. We are happy to change the colors in the revised manuscript.

    8)When saying "Many of these genes involved in these processes..." the authors need to include numbers

    and statistics.

    We will amend the revised text to make the definition of the MES-4 genes more clear.

    9)Should use WT instead of N2 and specify what wildtype is in methods.

    We will use WT instead of N2 in the revised manuscript.

    10)Fig. 2A + B could be displayed in a single figure. And Fig 2D seems superfluous and could be combined

    with 2C or alternatively it could be put in supplementary.

    Figure 2A and 2B were purposely separated to make it clear how many of the overlapped changes are up versus down. In the revised manuscript, Figure

    2D will be moved to the supplement.

    11)Non-C. elegans experts won't understand what balancers are. An effort should be made to make this

    accessible to all. Explaining when genes are heterozygous or homozygous mutants seems relevant

    here.

    The text of the revised manuscript will be amended to make it more accessible for non-C. elegans readers.

    12)The GO categories (Fig. S2) should be in the main figure and need to be made to look more scientific

    rather than copied and pasted from a program.

    The GO categories were included to be comprehensive and do not contribute substantially to the main conclusion of the paper. This is why they are supplemental. In the revised manuscript, we will edit the GO results so that they look more scientific.

    13)Fig. 7 seems a bit out of place. If the authors were to KD mes-4 and similarly show that the phenotype

    reverts that would help justify its inclusion in this paper. Without it seems like a bit of an add on that belongs

    elsewhere.

    We believe that the somatic expression of a transgene in spr-5; met-2 mutants adds to our potential understanding of how this double mutant may lead to developmental delay. This is true, regardless of whether of whether the somatic transgene expression is mes-4 dependent or not.

    Reviewer #3 (Significance (Required)):

    I think this is an interesting and timely piece of work. A little more effort needs to be put in to make sure it is

    accessible to the average reader and has sufficient inclusion of more of the large body of work on

    inheritance of histone modifications. I think C. elegans researchers as well as people interested in

    inheritance and the setup of the germline will be interested in this work.

    REFEREES CROSS COMMENTING

    I agree with Reviewer #2's comments on experiments to include or exclude alternative models. I also agree

    about their statement about rewriting to make it more accessible to others who aren't experts in this

    specialized portion of C. elegans research. All in all it seems like the experiments which are required by

    reviewer #2 and myself as well as the rewriting should be quite feasible.

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    Referee #3

    Evidence, reproducibility and clarity

    In the paper entitled "C. elegans establishes germline versus soma by balancing inherited histone methylation" Carpenter BS et al examined a double mutant worm strain they had previously produced of the H3K4me1/2 demethylase spr-5 and the predicted H3K9me1/me2 methylase met-2. These mutant worms have a developmental delay that arises by the L2 larval stage. They performed an analysis of what genes get misexpressed in these double mutants by performing RNAseq and compare this to datasets generated from other labs on an H3K36me2/me3 methylase MES-4 where they see a high degree of overlap. They validate the misexpression of some germline specific genes in the soma by in situ and validate that there is a dysregulation of H3K36me3 in their double mutant worms. They further find that knocking down mes-4 reverts the developmental delay.

    I think that the authors need to make more of an effort to be a bit more scholarly in terms of placing their work in the context of the field as a whole and also need to add a few additional experiments as well as reorganize a bit before this is ready for publication. Remember that the average reader is not necessarily an expert in C. elegans or this particular field and you really want to try and make the manuscript as accessible to everyone as possible.

    Major Points

    1)It would be good to see western blots or quantitative mass spec examining H3K36me3 in the WT and spr-5;met-2 double mutant worms. I believe this was also previously reported by Greer EL et al Cell Rep 2014 in the single spr-5 mutant worm so that work should be cited here in addition to the identification of JMJD-2 as an enzyme involved in the inheritance of H3K4me2 phenotype.

    2)Missing from Fig.5 is mes-4 KD by itself. This is needed to determine whether these effects are specific to the spr-5;met-2 double mutants or more general effects that KD of mes-4 would decrease the expression of all these genes to a similar extent. Then statistics should be done to see if the decrease in the WT context is the same or greater than the decrease in the double mutants.

    Minor Points

    1)A greater attempt needs to be made to be more scholarly for citing previously published literature. This includes work on the inheritance of H3K27 and H3K36 methylation in C. elegans and other species as well. A few papers which seem germane to this story which should be cited in the intro are (Nottke AC et al PNAS 2011, Gaydos LJ et al Science 2014, Ost A et al Cell 2014, Greer EL et al Cell Rep 2014, Siklenka K et al Science 2015, Tabuchi TM et al Nat Comm 2018, Kaneshiro KR et al Nat Comm 2019). This problem is not restricted to the intro.

    2)I think that the authors need to be a little less definitive with your language. Theories should be introduced as possibilities rather than conclusions. Should remove "comprehensive" from intro as there are many other methods which could be done to test this.

    3)The authors should describe what PIE-1 is. Is this a transcription factor?

    4)The language needs clarification about MES-4 germline genes and bookmark genes. Are these bound by MES-4 or marked with K36me2/3?

    5)I think Fig S1 E+F should be in the main figure 1 so readers can see the extent of the phenotype.

    6)For Fig S2 it would be good to do the same statistics that is done in Fig 2 and mention them in the text so the readers can see that the overlap is statistically significant.

    7)Fig S2.2 should be yellow blue rather than red green for the colorblind out there.

    8)When saying "Many of these genes involved in these processes..." the authors need to include numbers and statistics.

    9)Should use WT instead of N2 and specify what wildtype is in methods.

    10)Fig. 2A + B could be displayed in a single figure. And Fig 2D seems superfluous and could be combined with 2C or alternatively it could be put in supplementary.

    11)Non-C. elegans experts won't understand what balancers are. An effort should be made to make this accessible to all. Explaining when genes are heterozygous or homozygous mutants seems relevant here.

    12)The GO categories (Fig. S2) should be in the main figure and need to be made to look more scientific rather than copied and pasted from a program.

    13)Fig. 7 seems a bit out of place. If the authors were to KD mes-4 and similarly show that the phenotype reverts that would help justify its inclusion in this paper. Without it seems like a bit of an add on that belongs elsewhere.

    Significance

    I think this is an interesting and timely piece of work. A little more effort needs to be put in to make sure it is accessible to the average reader and has sufficient inclusion of more of the large body of work on inheritance of histone modifications. I think C. elegans researchers as well as people interested in inheritance and the setup of the germline will be interested in this work.

    REFEREES CROSS COMMENTING

    I agree with Reviewer #2's comments on experiments to include or exclude alternative models. I also agree about their statement about rewriting to make it more accessible to others who aren't experts in this specialized portion of C. elegans research. All in all it seems like the experiments which are required by reviewer #2 and myself as well as the rewriting should be quite feasible.

  3. 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 #2

    Evidence, reproducibility and clarity

    Katz and colleagues examine the interaction between the methyltransferase MES-4 and spr-5; met-2 double mutants. Their prior analysis (PNAS, 2014) showed the dramatic enhancement in sterility and development for spr-5; met-2; this paper extends that finding by showing these effects depend on MES-4. The results are interesting and the genetic interactions dramatic. The examination by RNAseq and ChIP helps move the phenotypes into a more molecular analysis. The authors hypothesize that SPR-5 and MET-2 modify chromatin of germline genes (MES-4 targets) in somatic cells, and this is required to silence germline genes in the soma. A few issues need to be resolved to test these ideas and rule out others.

    Main comments:

    The authors' hypothesis is that SPR-5 and MET-2 act directly, to modify chromatin of germline genes (MES-4 targets), but alternate hypothesis is that the key regulated genes are i) MES-4 itself and/or ii) known regulators of germline gene expression e.g. the piwi pathway. Mis regulation of these factors in the soma could be responsible for the phenotypes. Therefore, the authors should analyze expression (smFISH and where possible protein stains) for MES-4 and PIWI components in the embryo and larvae of wildtype, double and triple mutant strains. These experiments are essential and not difficult to perform.

    A second aspect of the hypothesis is that spr-5 and met-2 act before mes-4 and that while these genes are maternally expressed, they act in the embryo. There really aren't data to support these ideas - the timing and location of the factors' activities have not been pinned down. One way to begin to address this question would be to perform smFISH on the target genes and on mes-4 in embryos and determine when and where changes first appear. smFISH in embryos is critical - relying on L1 data is too late. If timing data cannot be obtained, then I suggest that the authors back off of the timing ideas or at least explain the caveats. Certainly, figure 8 should be simplified and timing removed. (note: Typical maternal effect tests probably won't work because if the genes' RNAs are germline deposited, then a maternal effect test will reflect when the RNA is expressed but not when the protein is active. A TS allele would be needed, and that may not be available.)

    Writing/clarity:

    -It would be helpful to include a table that lists the specific genes studied in the paper and how they behaved in the different assays e.g. RNAseq 1, RNAseq 2, MES-4 target, ChIP. That way, readers will understand each of the genes better.

    -At the end of each experiment, it would be helpful to explain the conclusion and not wait until the Discussion. For readers not in the field, the logic of the Results section is hard to follow.

    -The model is explained over three pages in the Discussion. It would be great to begin with a single paragraph that summarizes the model/point of the paper simply and clearly.

    Specific comments:

    -Figure 1 has been published previously and should be moved to the supplement.

    -Cite their prior paper for the vulval defects e.g. page 6 or show in supplement.

    -The second RNAseq data should be shown in the Results since it is much stronger. The first RNAseq, which is less robust, should be moved to supplement.

    -Figure 3 is very nice. Please explain why the RNAs were picked (+ the table, see comment above), and please add here or in a new figure mes-4 and piwi pathway expression data in wildtype vs double/triple mutants.

    -Figure 3 here or later, please show if mes-4 RNAi removes somatic expression of target genes.

    -Is embryogenesis delayed?

    -Figure 4 since htp-1 smFISH is so dramatic, it would be helpful to include htp-1 in the lower panels.

    -Figure 4, please add an extra 2 upper panels showing all the genes in N2 vs spr-5;met-2, for comparison to the mes-4 cohort.

    -Figure 6. Please show a control that met-1 RNAi is working.

    -To quantify histone marks more clearly, it would be wonderful to have a graph of the mean log across the gene. showing the mean numbers would help clarify the degree of the effect. we had an image as an example but it does not paste into the reviewer box. Instead, see figure 2 or figure 4 here: https://www.nature.com/articles/ng.322

    Significance

    Katz and colleagues examine the interaction between the methyltransferase MES-4 and spr-5; met-2 double mutants. Their prior analysis (PNAS, 2014) showed the dramatic enhancement in sterility and development for spr-5; met-2; this paper extends that finding by showing these effects depend on MES-4. The results are interesting and the genetic interactions dramatic. The examination by RNAseq and ChIP helps move the phenotypes into a more molecular analysis.

    This work will be of interest to people following transgenerational inheritance, generally in the C. elegans field. People using other organisms may read it also, although some of the worm genetics may be complicated. Some of the writing suggestions could make a difference.

    I study C. elegans embryogenesis, chromatin and inheritance.

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    Referee #1

    Evidence, reproducibility and clarity

    The Katz lab has contributed greatly to the field of epigenetic reprogramming over the years, and this is another excellent paper on the subject. I enjoyed reviewing this manuscript and don't have any major comments/suggestions for improving it. The findings presented are novel and important, the results are clear cut, and the writing is clear.

    It's important to stress the novelty of the findings, which build upon previous studies from the same lab (upon a shallow look one might think that some of the conclusions were described before, but this is not the case). Despite the fact that this system has been studied in depth before, it remained unclear why and how germline genes are bookmarked by H3K36 in the embryo, and it wasn't known why germline genes are not expressed in the soma.

    To study these questions Carpenter et al. examine multiple phenotypes (developmental aberrations, sterility), that they combine with analysis of multiple genetic backgrounds, RNA-seq, CHIP-seq, single molecule FISH, and fluorescent transgenes.

    Previous observations from the Katz lab suggested that progeny derived from spr-5;met-2 double mutants can develop abnormally. They show here that the progeny of these double mutants (unlike spr-5 and met-2 single mutants) develop severe and highly penetrate developmental delays, a Pvl phenotype, and sterility. They show also that spr-5; met-2 maternal reprogramming prevents developmental delay by restricting ectopic MES-4 bookmarking, and that developmental delay of spr-5;met-2 progeny is the result of ectopic expression of MES-4 germline genes. The bottom line is that they shed light on how SPR-5, MET-2 and MES-4 balance inter-generational inheritance of H3K4, H3K9, and H3K36 methylation, to allow correct specification of germline and somatic cells. This is all very important and relevant also to other organisms.

    (very) Minor comments:

    -Since the word "heritable" is used in different contexts, it could be helpful to elaborate, perhaps in the introduction, on the distinction between cellular memory and transgenerational inheritance.

    -It might be interesting in the Discussion to expand further about the links between heritable chromatin marks and heritable small RNAs. The do hint that the result regarding the silencing of the somatic transgene are especially intriguing.

    Significance

    This is an exciting paper which build upon years of important work in the Katz lab. The novelty of the paper is in pinpointing the mechanisms that bookmark germline genes by H3K36 in the embryo, and explaining why and how germline genes are prevented from being expressed in the soma.

  5. Version published to 10.1101/2020.01.22.914580 on bioRxiv