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

Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation 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 a 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 claim is compellingly supported. https://doi.org/10.7554/eLife.77951.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Many critical events during early development are orchestrated by maternally synthesized gene products. Mutations in genes that encode such products in the mother can cause 'maternal-effect' phenotypes in offspring. These phenotypes are usually severe developmental defects. Maternal-effect lethal genes, which cause maternal-effect death of offspring, encode products that guide crucial events in early embryo development, such as pattern formation and embryonic genome activation (e.g., the PAR proteins in Caenorhabditis elegans, BICOID in Drosophila, and Mater in mouse) (Nüsslein-Volhard et al., 1987; Tong et al., 2000; Kemphues et al., 1988). Maternal-effect sterile genes encode products needed for fertility of the offspring. A few genes in this category encode proteins in germ granules (e.g., PGL-1 in C. elegans and VASA in Drosophila) (Nüsslein-Volhard et al., 1987; Rongo and Lehmann, 1996; Kawasaki et al., 1998). Another fascinating set of genes in this category encode chromatin regulators, which are the focus of this article. The C. elegans MES proteins were identified in genetic screens for maternal-effect sterile mutants, hence their name (MES for maternal-effect sterile) (Capowski et al., 1991). MES-2, MES-3, and MES-6 assemble into a trimeric complex that is the C. elegans version of Polycomb Repressive Complex 2 (PRC2) (Xu et al., 2001; Bender et al., 2004). PRC2 is a histone methyltransferase (HMT) that methylates Lys 27 on histone H3 (H3K27me) to repress genes that are packaged by those methylated nucleosomes (Ketel et al., 2005; Margueron and Reinberg, 2011; Pengelly et al., 2013). MES-4 is an HMT that methylates Lys 36 on H3 (H3K36me), which marks actively transcribed genes and has context-dependent roles in transcriptional regulation (Bender et al., 2006; Furuhashi et al., 2010; Rechtsteiner et al., 2010; Kreher et al., 2018). Although PRC2 and MES-4 catalyze opposing flavors of histone marking, the loss of either causes nearly identical mutant phenotypes (Capowski et al., 1991). Worms that inherit a maternal load of gene product but cannot synthesize zygotic product (referred to as mes M+Z- mutants) are fertile. Worms that do not inherit a maternal load or produce zygotic gene product (mes M-Z- mutants) are sterile due to death of nascent germ cells in early- to mid-stage larvae. In mes M-Z+ mutants, zygotically synthesized product does not rescue fertility, highlighting the critical importance of maternal product. PRC2's roles in transcriptional repression and development have been intensively studied and are well defined across species, including roles in C. elegans germline development (Bender et al., 2004; Patel et al., 2012; Gaydos et al., 2014; Kaneshiro et al., 2019; Delaney et al., 2019). In contrast, how MES-4 ensures the survival of nascent germ cells is unknown and particularly puzzling. One possibility for MES-4 function is that maternal MES-4 promotes expression in offspring of genes required for germline development. Support for this comes from analyses of mutants that ectopically express germline genes in their soma (e.g., mep-1, lin-15B, lin-35, and spr-5; met-2 mutants), and as a result have developmental defects (Unhavaithaya et al., 2002; Wang et al., 2005; Cui et al., 2006; Petrella et al., 2011; Wu et al., 2012; Carpenter et al., 2021). Concomitant loss of MES-4 from these mutants prevents ectopic expression of germline genes and restores worm health (Wang et al., 2005; Cui et al., 2006; Petrella et al., 2011; Wu et al., 2012; Carpenter et al., 2021). In wild-type (wt) early embryos, MES-4 and methylated H3K36 associate with genes that were transcribed in the maternal germline, regardless of whether they are transcribed in embryos (Furuhashi et al., 2010; Rechtsteiner et al., 2010). Focusing on H3K36me3, genetic tests showed that MES-4 strictly maintains pre-existing patterns of H3K36me3 and is unable to catalyze de novo H3K36me3 marking of genes (Furuhashi et al., 2010); the other H3K36 HMT in C. elegans, MET-1, like H3K36 HMTs in other systems, catalyzes de novo H3K36me3 on genes in response to transcriptional turn-on (Kizer et al., 2005; Furuhashi et al., 2010; Kreher et al., 2018). Taken together, these findings suggested the appealing model that in embryos maternal MES-4 maintains H3K36me3 marking of germline-expressed genes and in that way transmits an epigenetic 'memory of germline,' a developmental blueprint, to the primordial germ cells (PGCs) of offspring. Two findings challenge the model that MES-4 somehow promotes expression of germline genes. First, among mes-4 M-Z- mutants, hermaphrodites (with two X chromosomes) are always sterile, while males (with one X chromosome) can be fertile (Garvin et al., 1998). This suggested that the dosage of X-linked genes matters for the Mes-4 mutant phenotype. Second, profiling transcripts in the germlines of fertile mes-4 M+Z- mutant hermaphrodites revealed that the most dramatic effect of losing zygotic MES-4 is upregulation of genes on the X (Bender et al., 2006; Gaydos et al., 2012). Notably, the X chromosomes are normally kept globally repressed during all stages of germline development in males and during most stages of germline development in hermaphrodites (Kelly et al., 2002; Reinke et al., 2004; Wang et al., 2009; Arico et al., 2011; Strome et al., 2014; Tzur et al., 2018), and likely as a consequence, most germline-expressed genes are located on the autosomes. These findings focused attention on the X chromosome and raised the question – what role does maternal MES-4 serve to ensure that PGCs survive and develop into a full and healthy germline? To investigate the role of MES-4 in nascent germlines, which critically rely on maternal MES-4 for survival, and to formally test the model that MES-4 activates a germline transcription program, we performed transcript profiling in dissected single pairs of PGCs and in dissected single sets of their descendants (which we term 'early germ cells' [EGCs]) from mes-4 M-Z- mutant versus wt larvae. We asked whether the absence of maternal MES-4 causes nascent germlines to (1) fail to express germline genes, (2) inappropriately express somatic genes, and/or (3) inappropriately express X genes. We found evidence of all three of those effects in mes-4 nascent germlines, the most striking of which was upregulation of many X genes. Our genetic analysis of mes-4 mutants with different X-chromosome endowments from the oocyte and sperm demonstrated that upregulation of one or more X genes is the cause of germline death in mes-4 M-Z- mutant larvae. This finding clarifies that MES-4's role in promoting expression of some germline genes is not its essential role in germline development and focused our analysis on the X. We identified the transcription factor LIN-15B, an X-linked gene upregulated in mes-4 mutants, as a major cause of X misexpression and germline death in mes-4 mutants. Our analysis also revealed that the X genes misexpressed in mes-4 nascent germlines are oogenesis genes, suggesting that MES-4 ensures survival and proliferation of nascent germlines by repressing a toxic oogenesis program. Performing similar tests of PRC2 (mes-3) M-Z- mutant larvae revealed that their nascent germlines upregulate many X-linked genes in common with mes-4 nascent germlines, and that removal of LIN-15B restores the health of their germline, as it does for mes-4 mutants. This study revealed that maternal MES-4 and PRC2 cooperate to ensure germline survival and health in offspring by antagonizing a key X-encoded transcription factor. Results MES-4 promotes the expression of some germline genes in EGCs MES-4 propagates an epigenetic 'memory' of a germline gene expression program during embryogenesis by maintaining H3K36me3 on genes that were previously transcribed in parental germlines (Rechtsteiner et al., 2010; Furuhashi et al., 2010; Kreher et al., 2018). A popular model predicts that delivery of this memory to offspring PGCs instructs them to launch a gene expression program that promotes germline proliferation and development. To test this model, we performed RNA-sequencing to determine whether PGCs from mes-4 M-Z- (Maternal MES-4 minus and Zygotic MES-4 minus) mutant larvae, which completely lack MES-4, fail to express a germline program (Figure 1A). We developed a hand-dissection strategy to isolate single sets of two PGCs from L1 larvae within 30 min for RNA-seq library preparation. We identified PGCs using a specifically and highly expressed germline marker (GLH-1::GFP). To test for gene expression defects in mes-4 mutant PGCs as they begin to launch their germline program, we profiled transcripts in PGCs from newly hatched wt or mes-4 M-Z- mutant L1 larvae that were fed for 30 min. We performed differential expression analysis to identify genes that are significantly downregulated (DOWN) or upregulated (UP) in mes-4 mutant PGCs compared to wt PGCs. Our analysis identified 176 DOWN genes and 450 UP genes (Figure 1B–D). Figure 1 Download asset Open asset Transcriptome defects in mes-4 M-Z- primordial germ cells (PGCs) are dominated by upregulation of genes on the X. (A) Cartoon illustrating the maternal-effect sterile (Mes) phenotype in mes-4 mutants. mes-4 M+Z- (M for maternal, Z for zygotic) mutants cannot synthesize MES-4 (Z-) but are fertile because they inherited maternal MES-4 gene product (M+). Removal of maternal MES-4 renders mes-4 M-Z- mutant adults sterile. Germline is green, and soma is white. Transcripts were profiled from single sets of two sister PGCs and from single sets of two early germ cells (EGCs) (red boxes) hand-dissected from mes-4 M-Z- (hereafter called mes-4) mutant and wild-type (wt) L1 and L2 larvae, respectively. (B–D) MA plots showing log2(average expression) versus log2(fold change) of transcript abundance for 20,258 protein-coding genes (circles) between mes-4 and wt PGCs. Numbers of biological replicates (sets of two cells): 19 wt PGCs and 11 mes-4 PGCs. Genes that exceeded one or both plot scales (triangles) were set at the maximum value of the scale. Genes belonging to a specific gene set are colored: (B) 168 germline-specific genes are blue, (C) 861 soma-specific genes are green, and (D) 2808 X-chromosome genes are red. Differentially expressed genes in mes-4 vs. wt PGCs were identified using Wald tests in DESeq2 and by setting a q-value < 0.05 significance threshold. Numbers of all misregulated genes (black) and numbers of those in gene sets (colored) are indicated in the corners; top is upregulated (UP) and bottom is downregulated (DOWN) in mes-4 vs. wt. (E–G) Bar plots showing the expected number (gray) and observed number (black) of misregulated genes that are members of the indicated gene sets. Hypergeometric tests were performed in R to test for gene-set enrichment. p-value designation is ****<1e-5. (E) Enrichment analyses for DOWN genes were restricted to 5858 protein-coding genes that we defined as 'expressed' (minimum average read count of 1) in wt PGCs. Gene-set sizes: germline-specific (140), germline-enriched (1867), MES-4-bound (3702), soma-specific (73). (F, G) Enrichment analyses for UP genes included all 20,258 protein-coding genes in the transcriptome. Gene-set sizes: germline-specific (168), germline-enriched (2176), MES-4-bound (4132), soma-specific (861), chrI (2888), chrII (3508), chrIII (2670), chrIV (3300), chrV (5084), and chrX (2808). To determine whether the DOWN genes include germline genes, we analyzed transcript levels and fold changes (mes-4 vs. wt) for genes that are members of three 'germline' gene sets: (1) a 'germline-specific' set containing 168 genes that are expressed in germline tissue but not in somatic tissues, (2) a 'germline-enriched' set containing 2176 genes that are expressed at higher levels in adults with a germline compared to adults that lack a germline, and (3) a 'MES-4-bound' set containing 4132 genes that are bound by MES-4 in embryos (see 'Materials and methods' for gene sets). We found that some germline-specific genes (6 of 168 genes, or 4%), germline-enriched genes (65 of 2176 genes, or 3%), and MES-4-bound genes (124 of 4132, or 3%) are significantly DOWN in mes-4 PGCs (Figure 1B and E). However, the numbers of DOWN genes for each gene set are not more than expected by chance (Figure 1E). Transcription of some germline genes turns on in PGCs during embryogenesis (e.g., Kawasaki et al., 1998; Subramaniam and Seydoux, 1999; Spencer et al., 2011), but the major wave of zygotic genome activation in germ cells commences after L1s hatch and feed for ~1.5 hr and before the PGCs start proliferating (Schaner et al., 2003; Butuči et al., 2015). Since some gene expression defects in mes-4 larvae may not manifest until after that time, we used our hand-dissection strategy to isolate sets of two EGCs from mes-4 mutant and wt L2 larvae 20 hr after hatching and feeding and profiled their transcripts (Figure 1A, Figure 2). We found that mes-4 EGCs downregulated more germline-specific genes (21 of 168, or 13%), more germline-enriched genes (247 of 2176, or 11%), and more MES-4-bound genes (396 of 4132, or 10%) than mes-4 PGCs (Figure 2A, D and G). The numbers of DOWN genes for each gene set are more than expected by chance (Figure 2G). Figure 2 with 2 supplements see all Download asset Open asset mes-4 M-Z- early germ cells (EGCs) have more severe transcriptome defects than mes-4 M-Z- primordial germ cells (PGCs). (A–C) MA plots as described for Figure 1B–D showing differential expression analysis for mes-4 vs. wild-type (wt) EGCs. Numbers of biological replicates (sets of two cells): 13 wt EGCs and 15 mes-4 EGCs. (D–F) Venn diagrams comparing sets of misregulated genes (mes-4 vs. wt) between PGCs and EGCs: (D) germline-specific downregulated (DOWN) genes, (E) soma-specific upregulated (UP) genes, and (F) X UP genes. (G–I) Bar plots showing the expected number (gray) and observed number (black) of misregulated genes in mes-4 EGCs that are members of the indicated gene sets. Hypergeometric tests were performed in R to test for gene-set enrichment. p-value designations are *<0.01 and ****<1e-5. (G) Enrichment analyses for DOWN genes were restricted to 6682 protein-coding genes that we defined as 'expressed' (minimum average read count of 1) in wt EGCs. Gene-set sizes: germline-specific (143), germline-enriched (1857), MES-4-bound (3675), soma-specific (126). (H, I) Enrichment analyses for UP genes included all 20,258 protein-coding genes in the transcriptome. Gene-set sizes: germline-specific (168), germline-enriched (2176), MES-4-bound (4132), soma-specific (861), chrI (2888), chrII (3508), chrIII (2670), chrIV (3300), chrV (5084), and chrX (2808). As an independent test of differential expression in PGCs, we selected three genes and performed single-molecule fluorescence in situ hybridization (smFISH) to measure and compare their transcript levels between mes-4 and wt PGCs. We analyzed PGCs from L1 larvae that were fed for 5 hr prior to smFISH to ensure that the PGCs had sufficient time to fully turn on their germline program. Two of the genes we tested, cpg-2 and pgl-3, are members of our germline-specific set and by RNA-seq analysis were DOWN or not DOWN, respectively, in mes-4 PGCs (Figure 1B, Figure 3—source data 1). Corroborating our RNA-seq analysis, smFISH analysis showed that the transcript abundance of cpg-2 was significantly lower in mes-4 vs. wt PGCs, while the transcript abundance of pgl-3 was not significantly different (Figure 3A–C, Figure 3—source data 1). The other gene we tested by smFISH, chs-1, is a member of our germline-enriched set and was consistently not misexpressed by RNA-seq or smFISH analysis (Figure 3C, Figure 3—source data 1). Together, our RNA-seq and smFISH analysis showed that mes-4 nascent germ cells fail to normally express a small number of germline genes, but display near-normal expression of the majority of germline genes analyzed. Figure 3 Download asset Open asset Transcript quantification in mes-4 M-Z- mutant primordial germ cells (PGCs) by single-molecule fluorescence in situ hybridization (smFISH) corroborates RNA-seq results. (A, D) UCSC Genome Browser images showing average sequencing read coverage over Ensembl gene models for wild-type (wt) PGCs (top track) and mes-4 PGCs (bottom track). (B, E) Representative maximum-intensity Z-projection images of smFISH experiments in L1 larvae collected after 5 hr of feeding. DAPI-stained nuclei are red. GLH-1::GFP is green. The dashed lines circumscribe PGCs marked by GLH-1::GFP. The second and third images in each set are zoomed insets of the yellow box in the first image. Foci in the mRNA channel (third image in each set) represent individual transcripts. Scale bars are 10 µm. (A, B) Two germline-specific genes: cpg-2 (left) and pgl-3 (right). cpg-2 was downregulated (DOWN) and pgl-3 was not DOWN in transcript profiling of mes-4 vs. wt PGCs. (D, E) Two X-linked UP genes in transcript profiling of mes-4 vs. wt PGCs and/or early germ cells (EGCs): lsd-1 (left) and lin-15B (right). (C, F) Transcript quantification in smFISH 3D images of PGCs. Each circle represents one quantified image. The number of quantified images for each combination of probe and genotype is indicated. Boxplots show the median, the 25th and 50th percentiles (boxes), and the 2.5th and 97.5th percentiles (whiskers). Mann–Whitney tests were used to compare a gene's transcript counts between mes-4 and wt PGCs. p-value designations are NS >0.01 and ****<1e-5. (C) Quantification of cpg-2, pgl-3, and chs-1 transcripts. chs-1 is in the germline-enriched gene set and is not DOWN in transcript profiling of mes-4 vs. wt PGCs and EGCs. (F) Quantification of lsd-1, mbk-1, pek-1, and lin-15B transcripts, four X-linked UP genes in transcript profiling of mes-4 vs. wt PGCs and/or EGCs. Figure 3—source data 1 Comparison of single-molecule fluorescence in situ hybridization (smFISH) and transcript profiling data. Table showing differential expression statistics for the seven genes tested by smFISH. Log2(fold change) values were calculated by comparing mean transcript counts in mes-4 primordial germ cells (PGCs) to wild-type (wt) PGCs, and the p-value is from a Mann–Whitney test. For transcript profiling analysis, log2(fold change) values were calculated using DESeq2 and in Download MES-4 some somatic genes in EGCs can protect transcription patterns by as a to transcription factor of MES-4 and PRC2 has been to misexpression of target genes ectopic expression of the transcription factor in the germline et al., 2012; et al., loss of PRC2 in the C. elegans germline was to misexpression of some genes and et al., 2019). MES-4 may offspring germline development by preventing germ cells from on a somatic gene expression program. To test this we whether UP genes in mes-4 vs. wt PGCs and EGCs are members of a gene set that 861 genes expressed in soma but not in We found that 19 and UP genes in mes-4 PGCs and mes-4 respectively, are the of which is a higher number than expected by chance (Figure and Figure and We that mes-4 germ cells begin to inappropriately express some somatic genes as they MES-4 genes on the X chromosome in PGCs and EGCs of the X chromosomes in the C. elegans germline is essential for germline health in Strome et al., We found that of the 2808 protein-coding X genes were UP in mes-4 vs. wt PGCs. more than of all UP genes are on the X chromosome of 450 and this number is significantly higher than expected by chance (Figure and G). mes-4 EGCs misexpressed X genes, including all of the X genes that were misexpressed in mes-4 PGCs and an X genes (Figure and These data show that mes-4 PGCs many genes on the X chromosome and that X misexpression more severe in their EGCs. As an independent test of differential we selected four X UP genes and performed smFISH to compare their transcript levels between mes-4 vs. wt PGCs from larvae that were fed for 5 smFISH analysis showed that all four X genes had higher transcript abundance in mes-4 PGCs than in wt PGCs (Figure our transcriptome analysis (Figure 3—source data 1). These data that the X chromosome is the focus of MES-4 regulation in PGCs. of genes on the X causes germline death in mes-4 mutants Since X misexpression is the transcriptome in mes-4 PGCs, we that misexpression of the two X chromosomes in germlines of mes-4 mutant hermaphrodites causes germline death. To test our we asked whether mes-4 mutant which inherit a single X chromosome from the have healthy We wt and mes-4 mutant and males that express a germline-specific and their germline health as either germline, germline, or mes-4 mutant hermaphrodites a germline (Figure In contrast, some mes-4 mutant males that inherited their single X from an oocyte had either a or full germline (21 and Since X chromosomes turn on during oogenesis (Kelly et al., 2002; Arico et al., 2011; Tzur et al., 2018), males inherited an X with a of a we wt and mes-4 mutant males that inherited their X from the sperm which has a of repression because the X was not on previously during We tested whether mes-4 mutant males that inherited a single X with a of repression have germlines than mes-4 mutant males that inherited a single X with a of of mes-4 mutant males full germlines compared to of mes-4 mutant Figure with 3 supplements see all Download asset Open asset Maternally MES-4 promotes germline development by repressing the X chromosomes from H3K36me3 across (A, B) Bar plots showing of germline and in with different X-chromosome in their Numbers of offspring and the genotype of their germlines are indicated (mes-4 mes-4 embryos (left) and some somatic the germline and some somatic blue, and X-chromosome is is and is one X and one X. To offspring that inherited two and two from one were with that a in tests were used to test whether the of adults with a germline significantly between p-value designations are *<0.01 and ****<1e-5. (A) To offspring that inherited their single X from the were that the which causes X-chromosome during oogenesis in the (B) or absence of H3K36me3 marking, maternal MES-4, and maternal in the germline of offspring are indicated in the of each and each A and genetic to that a germline of two inherited from the sperm or two inherited from the oocyte and et al., 2019). is a in a newly embryo causes on the and sperm which can those from in turn of the maternal and sets of In the the chromosomes from one and the chromosomes from the other Since the and sperm each in the all cells inherit two full from one this we tested whether mes-4 mutant hermaphrodites germline inherited two sets of sperm including 2 X chromosomes with a of repression and 10 have germlines than mes-4 mutant hermaphrodites germline inherited two sets of oocyte including 2 X chromosomes with a of expression and 10 all mes-4 mutant hermaphrodites a germline, some mes-4 mutant hermaphrodites had a or full germline and (Figure Our genetic analysis that misexpression of one or more X genes causes germline death in mes-4 mutants. also that mes-4 mutant PGCs can launch a transcription program that germline development. MES-4 promotes germline health from its role in H3K36me3 patterns across of epigenetic information across can the health of offspring. We that maternally MES-4's role in H3K36me3 patterns from to offspring is essential for X repression and offspring germline development. of parental chromosomes H3K36me3 to offspring cause their germline to they maternal To test our we used the genetic and the GLH-1::GFP germline marker to offspring PGCs inherited two from the sperm and either or not inherit maternal To completely H3K36me3 from sperm we used mes-4 mutant which lack the of both HMTs in C. elegans, and We found that over of offspring had a full germline their PGCs inherited two from the sperm and maternal MES-4 (Figure In contrast, of offspring had a full germline their PGCs inherited two from the sperm and not inherit maternal MES-4 (Figure This result that maternally MES-4 is critical for offspring
Source: eLife
DOI: 10.7554/elife.77951.sa2
Language: English
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4289545881
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