Takasaki, Teruaki et al. (2013) International Worm Meeting "MRG-1 acts as an epigenome interpreter of Lys36 methylation on histone H3."

Sakamoto, Hiroshi, Strome, Susan, Takasaki, Teruaki, Rechtsteiner, Andreas, Egelhofer, Thea

[International Worm Meeting, 2013]

We previously identified the autosome-associated protein MRG-1 as an essential maternal factor for proper germline development, yet its molecular function remains unclear (Takasaki et al., 2007). MRG-1 contains a CHROMO domain, which is found in numerous proteins that associate with methylated histones. In our search for the element(s) that recruits MRG-1 to autosomes, we found that oocyte and sperm chromosomes arrive in the zygote marked with methylated H3K36, and that MRG-1 accumulates predominantly on those methylated chromosomes. Genome-wide chromatin immunoprecipitation analysis in early embryos revealed that methylation of H3K36 is propagated on the body of germline-expressed genes by the histone methyltransferase MES-4 in an epigenetic manner (Rechtsteiner et al., 2010; Furuhashi et al., 2010). The distribution of MRG-1 exhibits a high correlation with the distributions of both MES-4 and H3K36me, and the accumulation of MRG-1 on germline-specific genes depends on MES-4-generated H3K36me. These findings suggest that MRG-1 recognizes germline-expressed genes using the epigenetic mark H3K36me generated by MES-4. Strikingly, we found that germline-specific loci exhibit an atypical H4K16 acetylation pattern, which is dependent on MES-4. By analogy to the Drosophila homolog of MRG-1, which acts in the dosage compensation complex (the MSL complex) to generate H4K16ac on the X chromosome, it is likely that MRG-1 contributes to the germline-gene-specific H4K16ac pattern. Supporting this idea, we identified MYS-2, which is a homolog of another component of the MSL complex, as an H4K16 acetyltransferase. Furthermore, we found that loss of MYS-2, like loss of MES-4 and MRG-1, suppresses ectopic expression of germline genes in somatic cells of mep-1 mutant larvae. This suggests that H4K16 acetylation serves a pivotal role in germline development. Taken together, our results suggest that MRG-1 interprets the epigenetic H3K36me landmark and as part of an MSL-like complex generates H4K16ac specifically at germline-expressed genes.

Tabuchi, Tomoko et al. (2015) International Worm Meeting "Epigenetic landscape of C. elegans sperm chromatin."

Strome, Susan, Tabuchi, Tomoko, Rechtsteiner, Andreas

[International Worm Meeting, 2015]

Information can be passed from parent to child through DNA sequence and through packaging of DNA into distinct types of chromatin. The latter, termed epigenetics, is a critical facet of normal development. In contrast to our growing understanding of maternal contributions to epigenetic control of development, paternal contributions are relatively unexplored. The Strome lab has shown that H3K36me and H3K27me, histone modifications associated with gene expression or gene repression, respectively, are transmitted to embryos from sperm and persist on paternal chromatin for at least a few cell divisions. This finding supports the notion that C. elegans sperm chromatin transmits epigenetic information to embryos. To elucidate paternal epigenetic contributions to progeny, we are defining the epigenetic landscape in C. elegans sperm using chromatin immunoprecipitation followed by sequencing (ChIP-seq). We developed a protocol to efficiently and reproducibly fragment and solubilize tightly packaged C. elegans sperm chromatin, and determined optimal conditions for ChIP from two million sperm. We found that the sperm H3K36me3 landscape looks strikingly similar to the H3K36me3 landscape in early embryos, suggesting retention in embryos of most inherited H3K36me3 marking. Some genes marked with H3K36me3 in sperm lose H3K36me3 in early embryos, which likely reflects erasure by either a histone demethylase or histone replacement. The landscapes of other histone modifications are currently being investigated. Comparing sperm patterns with the embryonic patterns will provide us with a first glimpse of which aspects of sperm patterns are likely to be retained versus erased in early embryos. This study will expand our understanding of transgenerational epigenetics and how sperm epigenetic information may influence embryo development.

Kaneshiro, Kiyomi et al. (2019) International Worm Meeting "Sperm-inherited H3K27me3 impacts offspring transcription and development in C. elegans."

Kaneshiro, Kiyomi, Rechtsteiner, Andreas, Strome, Susan

[International Worm Meeting, 2019]

Do gamete-inherited chromatin states influence gene expression in offspring? This is a burning question in the field and one that remains elusive, in part because it is difficult to determine cause versus consequence when comparing chromatin states with transcriptional status. To test for a cause-effect relationship between inherited chromatin states and offspring transcription we 1) compared genetically identical individuals for different transcriptional outcomes as a result of inheriting different chromatin states, 2) assessed whether transcriptional changes occurred in cis, thus eliminating alternative epigenetic carriers that function in trans (e.g. cytoplasmic ncRNAs), and 3) used the model system C. elegans, which lacks canonical DNA methylation, thus eliminating the alternative epigenetic carrier that also functions in cis. Specifically, we compared transcription in the germline from sperm-inherited and oocyte-inherited alleles in genetically identical worms that inherited the sperm genome with or without the repressive histone modification H3K27me3. Our studies reveal that in C. elegans, sperm-inherited chromatin states directly influence transcription during germline development and protect germ cell identity. Inheritance of a sperm genome without H3K27me3 results in derepression in the germline of many somatic genes, especially neuronal genes, predominantly from sperm-inherited alleles. In a sensitized genetic background, this results in loss of germline-specific markers and reprogramming toward a neuronal fate, as evidenced by expression of neuronal markers and extension of axo-dendritic structures. We found that this reprogramming is progressive and only occurs later in development, indicating that sperm-inherited H3K27me3 serves to protect germ cell identity rather than establish it. Altogether, our findings point to a model in which gamete-inherited patterns of H3K27me3 act as a barrier to inappropriate transcription; in germ cells, this barrier function suppresses transcription of factors that could drive germ cells toward alternative cell identities. These findings demonstrate that histone modifications are one mechanism through which epigenetic information can be passed from father to shape gene expression, development, and fertility in offspring.

Gaydos, Laura et al. (2013) International Worm Meeting "Epigenetic regulation of fertility in C. elegans males depends on the gamete source and chromatin history of the X chromosome."

Gaydos, Laura, Wang, Wenchao, Strome, Susan, Rechtsteiner, Andreas

[International Worm Meeting, 2013]

Organisms with different numbers of X chromosomes in the two sexes have evolved diverse strategies to regulate X-linked gene expression. Somatic cells compensate for the difference in X dosage by inactivating one X in XX mammals, down-regulating both Xs in XX C. elegans, or up-regulating the single X in XY Drosophila. Germ cells in C. elegans display a more extreme type of regulation, near silencing of both Xs in XX hermaphrodites and the single X in XO males. X repression in the germline is achieved at least in part by the MES proteins, epigenetic regulators that must be maternally provided to progeny to ensure survival of the primordial germ cells. In the adult germline, the MES-2/3/6 complex, which is the worm version of Polycomb Repressive Complex 2, concentrates a repressive histone modification (H3K27me) on the Xs. MES-4 participates in X repression in an indirect manner. MES-4 catalyzes a mark of active chromatin (H3K36me) on germline-expressed genes on autosomes; this repels H3K27me from autosomal regions and helps concentrate H3K27me on the Xs. Recent investigation of whether the MES proteins participate in repression of the single X in XO males has revealed that the answer depends on the gamete source of the X. Maternal MES function is required when the X in males is inherited from the oocyte but not when the X is inherited from the sperm. In the latter case, X repression appears to be mediated by enzymes that generate the repressive histone modification H3K9me. Our studies have shown that the opposing activities of MES-2/3/6 and MES-4 repress transcription from the two Xs in XX germ cells, and that in XO germ cells H3K9me can serve as another mode of repression.

Gaydos, Laura et al. (2011) International Worm Meeting "The MES proteins cooperate to influence gene expression patterns in the C. elegans germ line."

Gaydos, Laura, Strome, Susan, Wang, Wenchao, Carroll, Coleen, Rechtsteiner, Andreas

[International Worm Meeting, 2011]

In C. elegans, the MES proteins are required for germline viability, which is necessary for producing the next generation and for the propagation of species. The MES proteins modify histone tails on nucleosomes and influence gene expression. MES-2, MES-3 and MES-6 form a trimeric complex and are responsible for methylation of histone H3 on lysine 27 (H3K27) in the germ line. MES-2 and MES-6 have homologs in Drosophila and mammals, which are part of the Polycomb Repressive Complex 2. MES-4 also has homologs in other species and is responsible for H3K36 methylation in the germ line of C. elegans. We want to figure out the gene targets of MES regulation and whether MES-2/3/6 and MES-4 have the same or different gene targets. To do this, we performed microarray analysis of mes mutant germ lines along with genetic tests. We found that mes-2; mes-4 double mutants are sterile a generation earlier than either single mutant and that MES-2 and MES-4 influence expression of some of the same genes. These results suggest that MES-2/3/6 and MES-4 cooperate to influence gene expression. Genes down-regulated by the MES proteins in the germ line include ubiquitously expressed genes on the X and somatically expressed genes on the autosomes. Genes up-regulated by the MES proteins in the germ line include germline-specific genes on the autosomes. These findings suggest that the MES proteins are important for reducing expression of genes not tolerated at high levels during germline development, and enhancing expression of germline genes. Interestingly, we found that the fertility of mes-2, mes-3, and mes-6 males depends on the gamete source of the X chromosome, which underscores the important role that MES-2/3/6 serve in regulation of genes on the X chromosome.

Knutson, Andrew et al. (2015) International Worm Meeting "P granules maintain germ cell identity in the C. elegans germline."

Strome, Susan, Egelhofer, Thea, Rechtsteiner, Andreas, Knutson, Andrew, Tuckfield, Lynnia

[International Worm Meeting, 2015]

Like stem cells, germ cells are able to self renew and, after the union of 2 gametes, differentiate into a multitude of cell types. The factors and mechanisms employed by germ cells to maintain their broad differentiation potential are only starting to be understood and involve regulation at both the transcriptional and translational levels. In C. elegans germ cells, cytoplasmic ribonucleoprotein structures termed P granules participate in maintaining germ cell totipotency (Updike et al. Current Biology 24: 970-975, 2014). Removal of 4 major P-granule proteins (PGL-1, PGL-3, GLH-1, and GLH-4) via RNAi causes sterility and underproliferated germlines. Remarkably, a fraction of these germlines also express neuronal markers (unc-119::GFP and unc-33::GFP) and a muscle marker (MYO-3). This suggests that P granules prevent somatic development in the germline (Updike et al. 2014). We have found that glh-1 single mutants and pgl-1 single RNAi germlines express the unc-119::gfp transgene and stain for MYO-3, revealing the importance of single P-granule proteins in maintaining germ cell totipotency. Additionally, we see a loss of germline characteristics upon P-granule depletion, including loss of the mitotic factor REC-8 and the meiotic factor HTP-3. We also see cellularization in the normally syncytial region of the germline. Taken together, these findings suggests that P-granule-compromised germ cells are differentiating away from a germ cell fate and toward somatic fates. To better understand the spectrum of somatic genes that are expressed in P-granule-compromised germlines and the extent of somatic development, we are performing RNA-seq analysis of dissected germlines that have been depleted of P granules by RNAi and that express the unc-119::gfp neuronal reporter. This analysis may reveal important master regulator transcripts that must be sequestered/silenced/degraded by P granules to prevent premature somatic development. We hypothesize that P granules prevent the translation of somatic transcripts that are stochastically expressed in the germline. P granules may intercept and/or process these mRNAs as they exit the nucleus, thus maintaining germ cell fate and totipotency. .

Costello, Meghan Elizabeth et al. (2013) International Worm Meeting "synMuv B regulation of chromatin states at high temperature."

Egelhofer, Thea, Petrella, Lisa N, Rechtsteiner, Andreas, Strome, Susan, Costello, Meghan Elizabeth

[International Worm Meeting, 2013]

Most organisms experience variations in temperature throughout their lifespan and thus require mechanisms to buffer gene expression in response to fluctuations in temperature. It has been observed that during development, chromatin states are influenced by temperature. For example, in Drosophila heterochromatin formation and spreading is partially compromised at high temperatures. In order to understand the effect of temperature during development, it is critical to understand how chromatin structure affects the buffering of gene expression at elevated temperatures. We are investigating changes in chromatin state at high temperature through study of the C. elegans synMuv B chromatin regulators. The synMuv B proteins include Retinoblastoma/LIN-35 and other members of the DRM complex and HPL-2, the worm homolog of HP1. Mutations in many synMuv B genes cause high temperature larval arrest (HTA) due to derepression of germline gene expression in somatic cells, most notably the intestine. Loss of germline chromatin modifiers suppresses somatic expression of germline genes in synMuv B mutants and rescues the HTA phenotype. This suggests that changes in chromatin states in the soma underlie the temperature-sensitive gene misexpression in synMuv B mutants. Consistent with this hypothesis, immunostaining of synMuv B mutants revealed altered levels of specific histone modifications in somatic tissues. To investigate changes in active and repressive chromatin marks at high resolution genome-wide in synMuv B mutants, we have performed ChIP-seq experiments in lin-35, lin-37, and lin-15B mutants at both low and high temperature. Additionally, in order to determine if the absence of wild-type synMuv B proteins causes the loss of a closed chromatin state at elevated temperatures, we are using the lacO/LacI system to visualize changes in chromatin compaction in lin-15B and lin-35 mutants.

Petrella, Lisa N. et al. (2011) International Worm Meeting "Antagonizing germline fate in somatic cells: opposing roles of the MES and synMuv B chromatin regulators."

Strome, Susan, Rechtsteiner, Andreas, Spike, Caroline A., Petrella, Lisa N., Wang, Wenchao

[International Worm Meeting, 2011]

The newly fertilized embryo inherits a chromatin state with germline characteristics. As development proceeds, those cells destined to generate somatic tissues must be reprogrammed to abolish this germline chromatin state. Previous studies demonstrated that a subset of synMuv B mutants ectopically express germline-specific P-granule proteins in their somatic cells, suggesting a failure to properly orchestrate a soma/germline fate decision (Unhavaithaya et al. 2002 Cell 111:991; Wang et al. 2005 Nature 436:593). Surprisingly, this fate confusion does not affect viability at low to ambient temperatures. We have shown that, when grown at high temperature, a majority of synMuv B mutants irreversibly arrest at the L1 stage (Petrella et al. 2011 Development 138:1069). High temperature arrest (HTA) is preceded by the expression of germline genes in somatic cells starting in late embryogenesis. This leads to somatic expression of P-granule proteins, components of the synaptonemal complex, and other meiosis proteins. Somatic expression of germline genes is enhanced at elevated temperature, leading to developmentally compromised somatic cells and arrest of newly hatched larvae. HTA is suppressed by loss of global regulators of germline chromatin, including the MES proteins, revealing that arrest is caused by somatic cells possessing a germline-like chromatin state. We propose that synMuv B mutants fail to erase or antagonize an inherited germline chromatin state in somatic cells during embryonic and early larval development. Consistent with this, the somatic cells of synMuv B mutants display elevated levels of chromatin marks that are normally enriched in the primordial germ cells. We are currently exploring how chromatin states are affected in synMuv B mutants and by elevated temperature, using nuclear spot assays.

Robert, Valerie et al. (2017) International Worm Meeting "The SET-2/SET1 histone H3K4 methyltransferase maintains cell fate in the Caenorhabditis elegans germline."

Strome, Susan, Palladino, Francesca, Yvert, Gael, Robert, Valerie, Knutson, Andrew Kekupa'a, Rechtsteiner, Andreas

[International Worm Meeting, 2017]

Maintenance of germ cell fate is of critical importance for species survival. In C. elegans, translational repression, P granules, chromatin factors and histone tail modifications have been shown to be essential to maintain germ cell identity. We previously showed that the conserved SET-2/SET1 histone H3K4 methyltransferase plays a crucial role in maintaining germline immortality and totipotency, two defining features of germline identity. set-2 mutant animals grown at 25 deg C become sterile over several generations, which is reminiscent of what happens in mortal germline (mrt) mutants. This sterility, which is reversible when animals are switched back to 20 deg C, is associated with a loss of germline-specific markers, transcriptional derepression of somatic genes, and transdifferentiation of the germline into soma. To look at how the transcriptional landscape changes in the absence of SET-2 across generations and to identify pathways involved in germline maintenance, we performed transcriptome analysis of germlines dissected from fertile animals grown for 2 generations at 25 deg C and on both fertile and sterile animals grown for 4 generations at 25 deg C. We are currently analyzing these transcriptome data and performing RNAi to identify triggering events of transdifferentiation and transcriptional networks deregulated in transdifferentiated germlines. I will present preliminary results of this analysis.

Strome, Susan et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Transmission and Antagonism of Germline Fate in C. elegans"

Takasaki, Teruaki, Rechtsteiner, Andreas, Gaydos, Laura, Egelhofer, Thea A, Lieb, Jason D, Petrella, Lisa, Ercan, Sevinc, Strome, Susan

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Perpetuation of the C. elegans germ line from generation to generation depends on the MES proteins, named for their maternal-effect sterile phenotype. MES-2, MES-3, and MES-6 form the worm version of the Polycomb Repressive Complex 2 and methylate histone H3 on Lys 27 (H3K27), a modification associated with repression of gene expression. MES-4 is a homolog of the mammalian NSD proteins and methylates H3K36, a modification associated with active gene expression. Numerous studies have linked MES-2/3/6 and MES-4 to global repression of gene expression from the X chromosomes in the germ line, but have also suggested that MES-4 serves an additional critical role in early primordial germ cells (PGCs). Recent chromatin immunoprecipitation analysis of the distribution of MES-4, RNA Polymerase II, and H3K36 methyl marks across the genome in early embryos suggests that MES-4 serves a memory role. In contrast to previously studied H3K36 methyltransferases, which are targeted to genes by association with Pol II, MES-4 can associate with genes in a Pol II-independent manner. The genes that MES-4 binds and methylates in embryos are genes that were previously expressed in the maternal germline, many of which are no longer expressed in embryos. These and other findings suggest that MES-4 transmits the memory of gene expression in the parental germ line to offspring and ensures the ability of the PGCs to execute a proper germline program. Our findings raise the question of how somatic cells in the embryo, which also inherit MES-4 marking of germline genes, avoid following a germline fate. The synMuv B chromatin regulators play a key role, as their loss causes somatic cells to misexpress numerous germline-specific genes and causes larvae grown at elevated temperature to arrest. Concomitant loss of maternal MES-4 suppresses the germline potential of somatic cells and larval arrest. We will discuss the opposing roles of the MES and synMuv B proteins in the germ-soma decision.