Maternal H3K36 and H3K27 HMTs protect germline development via regulation of the transcription factor LIN-15B

  1. Chad Steven Cockrum
  2. Susan Strome

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    This study provides a compelling and significant advance on the understanding of how gene regulation by the histone methyltransferase MES-4 underlies germ cell survival in C. elegans, with the major claims being nicely substantiated. The critical and surprising finding is that the degeneration of mes-4 mutant primordial germ cells is due to inappropriate upregulation of genes on the silenced X chromosome, and not failure to activate germline-expressed genes, though reduced levels of germline gene expression were observed. An X-linked target of mes-4, lin-15b, is necessary for the degeneration phenotype. The work could be improved by clarification of the relationship between X and autosomal gene expression, especially in consideration with the action of the other histone methyltransferase MET-1, but otherwise it is excellent.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Maternally synthesized products play critical roles in the development of offspring. A premier example is the Caenorhabditis elegans H3K36 methyltransferase MES-4, which is essential for germline survival and development in offspring. How maternal MES-4 protects the germline is not well understood, but its role in H3K36 methylation hinted that it may regulate gene expression in primordial germ cells (PGCs). We tested this hypothesis by profiling transcripts from nascent germlines (PGCs and their descendants) dissected from wild-type and mes-4 mutant (lacking maternal and zygotic MES-4) larvae. mes-4 nascent germlines displayed downregulation of some germline genes, upregulation of some somatic genes, and dramatic upregulation of hundreds of genes on the X chromosome. We demonstrated that upregulation of one or more genes on the X is the cause of germline death by generating and analyzing mes-4 mutants that inherited different endowments of X chromosome(s). Intriguingly, removal of the THAP transcription factor LIN-15B from mes-4 mutants reduced X misexpression and prevented germline death. lin-15B is X-linked and misexpressed in mes-4 PGCs, identifying it as a critical target for MES-4 repression. The above findings extend to the H3K27 methyltransferase MES-2/3/6, the C. elegans version of polycomb repressive complex 2. We propose that maternal MES-4 and PRC2 cooperate to protect germline survival by preventing synthesis of germline-toxic products encoded by genes on the X chromosome, including the key transcription factor LIN-15B.

Article activity feed

  1. Version published to 10.7554/elife.77951 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This is an extremely well-done study, revealing a fascinating phenotype of mes-4 mutant, which they show upregulates X-linked genes, leading to PGC death. These X-linked genes are mostly oogenesis genes, upregulation of which likely impedes normal proliferation of PGCs. The results are very concrete and supports their conclusion, and contribute significantly to the field. I do not have any major concerns except for a couple of conceptual issues. First, the title 'germline immortality' does not seem to be well aligned with the results. It is not wrong that PGCs die in mes-4 mutant, and thus the germline is 'mortal': however, the term 'germline immortality' implies multi-generational passages of germline, and the data in the present study, where mutant PGCs just die in the offspring, do not necessarily point to 'germline immortality' per se. So, I suggest to change the title to reflect the contents of the paper better.

    Good point. We changed germline immortality to germline survival and/or development throughout the paper.

    Second, although the authors speculate (in the discussion) why X activation is toxic to germ cells (discussing that upregulated X-linked genes are oogenesis genes, whose precocious activation is toxic to PGCs), there is not sufficient discussion as to why the effect is mostly limited to X chromosome, and why mes-4 is specifically involved in this. Is it because all oogenesis genes are concentrated on X chromosome? (likely not). Are autosomal genes that are upregulated in mes-4 mutant also oogenesis genes? Is this related to dosage compensation? I would like to see fuller discussions as to why X chromosome requires special regulation, also discussing the role of mes-4 in this context. I understand that the authors might have refrained from expanding discussions on matters that do not have any data, but without this discussion, I feel that many readers will be left wondering 'why?'.

    As noted in Point #5 above, we added to Discussion whether up-regulation of X genes in mes-4 mutant PGCs and EGCs reflects a defect in dosage compensation or a defect in keeping the oogenesis program (which is enriched for X-linked genes) quiet in the nascent germline (see lines 604-630). Based on new analyses showing up-regulation of oogenesis genes (on the X and autosomes) in mes-4 and PRC2 nascent germlines and the points in Discussion, we favor the view that the essential function of MES-4 and PRC2 is to repress X-linked oogenesis genes in PGCs and EGCs (see Figures 6 and 7, associated figure supplements, and lines 389-417).

    Reviewer #2 (Public Review):

    This manuscript makes substantial progress in resolving a long-standing mystery regarding the precise role of the histone methyltransferase MES-4 in promoting germline development. MES-4 maintains the histone modification H3K36me3 and germ cell survival, but prior evidence was unable to distinguish among several possibilities for target pathways. This paper utilizes a transcriptional profiling approach at the critical time of germline development to definitively demonstrate that the essential function of MES-4 is to repress X gene expression in germ cells. This result is surprising because X repression is an indirect effect of MES-4 activity (MES-4 does not localize to the X), while the direct effect of maintaining germline gene expression is not essential. To buttress this finding, the authors also utilize a series of elegant genetic experiments to independently test whether expression from the X is sufficient to cause germ cell degeneration. They then go further to identify a single X-linked target, lin-15b, as a primary contributor to the inappropriate X-linked gene expression in mes-4 mutants, by showing that loss of lin-15b activity rescues both the germline degeneration and X mis-expression of mes-4 mutants. Finally, the authors demonstrate that PRC2, the H3K27me3 histone methyltransferase and MRG-1, a candidate H3K36me3 effector protein, are also involved in promoting X silencing through lin-15b.

    The manuscript's strengths lie in the development or application of novel techniques, including the profiling of individual pairs of PGCs (a non-trivial advancement), as well as some very well-designed and conceptually innovative genetic assays. These were used to address specific and important gaps in knowledge regarding the phenotype of mes-4, which had been elusive despite having been studied for almost 30 years. Although specific to C. elegans in some ways, the findings are clearly relevant to conserved regulatory events, such as epigenetic memory mechanisms and establishment of opposing chromatin states. Thus, this work provides a substantial advance in the field overall.

    One limitation of this study is the lack of clarity about the conclusions regarding the relationship between the two H3K36me3 histone methyltransferases mes-4 and met-1, and between X vs autosomal gene expression. The authors do not precisely state what genes (X or A) are affected in the met-1 and mes-4 mutants. Ultimately, this confusion muddles the final message of X chromosome upregulation being the critical contributor to the mes-4 germline degeneration phenotype. The experiment presented in figure 3B indicates that loss of mes-4 or met-1 is sufficient to prevent germline development even when the Xs are repressed, indicating that failure to activate autosomal gene expression is also an underlying cause of the degeneration. Perhaps this cannot be definitively concluded without directly assessing met-1 and met-1;mes-4 mutant PGCs (or EGCs) for gene expression changes. If technically possible, this would be a very valuable experiment to directly examine autosomal gene expression changes in the double mutant.

    We profiled met-1 PGCs and observed very few mis-regulated genes (Figure 7 – supplemental figure 1). We tried to profile met-1; mes-4 double mutant PGCs, which completely lack both MET-1 and MES-4 and inherit chromosomes that lack H3K36me3. That was not feasible, due to the high level of embryonic lethality and rapid deterioration of PGCs dissected from met-1; mes-4 double mutant larvae. Notably, this demonstrates that germlines that lack both maternal K36me3 HMTs are sicker than those that lack just 1 of the HMTs. The high degree of embryo lethality suggests an essential function for MET-1 and MES-4 in the soma. As requested, we generated and included a list of X and autosomal genes mis-regulated in met-1, mes-4, and other mutant PGCs (see Figure 7—figure supplement 1).

    The sterility of hermaphrodites with a met-1; mes-4 mutant XspXsp germline and lacking either maternal MES-4 or maternal MET-1 may be due to mis-regulation of autosomal genes, or it may reflect that the X chromosomes are not repressed in met-1; mes-4 XspXp germlines that lack H3K36me3. To test that, we would need to profile those XspXsp PGCs. It is not feasible to identify mutant F1 larvae with Xsp/Xsp PGCs immediately after hatching, which is required for transcript profiling. We think that the main message from analyzing met-1; mes-4 mutant XspXsp germlines -- that inherited H3K36me3 marking is not critical for germline development but re-establishment of marking is important and requires both enzymes – does not require our delving into the cause of sterility of mutant XspXsp germlines lacking MET-1.

  3. eLife

    Evaluation Summary:

    This study provides a compelling and significant advance on the understanding of how gene regulation by the histone methyltransferase MES-4 underlies germ cell survival in C. elegans, with the major claims being nicely substantiated. The critical and surprising finding is that the degeneration of mes-4 mutant primordial germ cells is due to inappropriate upregulation of genes on the silenced X chromosome, and not failure to activate germline-expressed genes, though reduced levels of germline gene expression were observed. An X-linked target of mes-4, lin-15b, is necessary for the degeneration phenotype. The work could be improved by clarification of the relationship between X and autosomal gene expression, especially in consideration with the action of the other histone methyltransferase MET-1, but otherwise it is excellent.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This is an extremely well-done study, revealing a fascinating phenotype of mes-4 mutant, which they show upregulates X-linked genes, leading to PGC death. These X-linked genes are mostly oogenesis genes, upregulation of which likely impedes normal proliferation of PGCs.

    The results are very concrete and supports their conclusion, and contribute significantly to the field. I do not have any major concerns except for a couple of conceptual issues. First, the title 'germline immortality' does not seem to be well aligned with the results. It is not wrong that PGCs die in mes-4 mutant, and thus the germline is 'mortal': however, the term 'germline immortality' implies multi-generational passages of germline, and the data in the present study, where mutant PGCs just die in the offspring, do not necessarily point to 'germline immortality' per se. So, I suggest to change the title to reflect the contents of the paper better. Second, although the authors speculate (in the discussion) why X activation is toxic to germ cells (discussing that upregulated X-linked genes are oogenesis genes, whose precocious activation is toxic to PGCs), there is not sufficient discussion as to why the effect is mostly limited to X chromosome, and why mes-4 is specifically involved in this. Is it because all oogenesis genes are concentrated on X chromosome? (likely not). Are autosomal genes that are upregulated in mes-4 mutant also oogenesis genes? Is this related to dosage compensation? I would like to see fuller discussions as to why X chromosome requires special regulation, also discussing the role of mes-4 in this context. I understand that the authors might have refrained from expanding discussions on matters that do not have any data, but without this discussion, I feel that many readers will be left wondering 'why?'.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript makes substantial progress in resolving a long-standing mystery regarding the precise role of the histone methyltransferase MES-4 in promoting germline development. MES-4 maintains the histone modification H3K36me3 and germ cell survival, but prior evidence was unable to distinguish among several possibilities for target pathways. This paper utilizes a transcriptional profiling approach at the critical time of germline development to definitively demonstrate that the essential function of MES-4 is to repress X gene expression in germ cells. This result is surprising because X repression is an indirect effect of MES-4 activity (MES-4 does not localize to the X), while the direct effect of maintaining germline gene expression is not essential. To buttress this finding, the authors also utilize a series of elegant genetic experiments to independently test whether expression from the X is sufficient to cause germ cell degeneration. They then go further to identify a single X-linked target, lin-15b, as a primary contributor to the inappropriate X-linked gene expression in mes-4 mutants, by showing that loss of lin-15b activity rescues both the germline degeneration and X mis-expression of mes-4 mutants. Finally, the authors demonstrate that PRC2, the H3K27me3 histone methyltransferase and MRG-1, a candidate H3K36me3 effector protein, are also involved in promoting X silencing through lin-15b.

    The manuscript's strengths lie in the development or application of novel techniques, including the profiling of individual pairs of PGCs (a non-trivial advancement), as well as some very well-designed and conceptually innovative genetic assays. These were used to address specific and important gaps in knowledge regarding the phenotype of mes-4, which had been elusive despite having been studied for almost 30 years. Although specific to C. elegans in some ways, the findings are clearly relevant to conserved regulatory events, such as epigenetic memory mechanisms and establishment of opposing chromatin states. Thus, this work provides a substantial advance in the field overall.

    One limitation of this study is the lack of clarity about the conclusions regarding the relationship between the two H3K36me3 histone methyltransferases mes-4 and met-1, and between X vs autosomal gene expression. The authors do not precisely state what genes (X or A) are affected in the met-1 and mes-4 mutants. Ultimately, this confusion muddles the final message of X chromosome upregulation being the critical contributor to the mes-4 germline degeneration phenotype. The experiment presented in figure 3B indicates that loss of mes-4 or met-1 is sufficient to prevent germline development even when the Xs are repressed, indicating that failure to activate autosomal gene expression is also an underlying cause of the degeneration. Perhaps this cannot be definitively concluded without directly assessing met-1 and met-1;mes-4 mutant PGCs (or EGCs) for gene expression changes. If technically possible, this would be a very valuable experiment to directly examine autosomal gene expression changes in the double mutant.

  6. eLife

    Reviewer #3 (Public Review):

    The H3K36 histone methyltransferase MES-4 and the Polycomb system in C. elegans are essential for the viability of germ cells. The cause of inviability has not been determined, but it has been hypothesized that germ cell death in mutants may be due to a need for MES-4 to promote germline gene expression and prevente somatic gene expression. In addition, it had been previously observed that the MES-4/Pc system represses X chromosome gene expression in the adult germ line. Here the authors directly investigate the cause of early germ cell death in mes-4 and Pc mutants by conducting an impressive set of gene expression profiling experiments on dissected primordial germ cells and slightly later germ cells. The found that the most striking defect in primordial germ cells is the upregulation of X-linked gene expression. They demonstrated that decreasing X chromosome dosage in mes-4 and Pc mutants substantially rescues germ cell development, showing that upregulation of X-linked gene expression is a major contributor to germ cell death. These are important findings that further understanding of the roles of the key germline regulation systems in the C. elegans germline. The profiling experiments in primordial germ cells did not show downregulation of germ line gene expression, leading the authors to conclude that MES-4 is not needed for turn on of germline gene expression. However, as it was not clear whether wild-type germ cells had fully activated zygotic gene expression at the early larval time point assayed, and profiling in later germ cells did show reduced germline gene expression, this conclusion needs further support. It would also be of interest to determine whether upregulation of X-linked gene expression in the mutants occurs before or after the major wave of zygotic gene expression activation in germ cells.

  7. Version published to 10.1101/2022.03.17.484692v1 on bioRxiv