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Petrella, Lisa, Strome, Susan, Takasaki, Teruaki, Gaydos, Laura, Rechtsteiner, Andreas, Egelhofer, Thea A, Lieb, Jason D, Ercan, Sevinc
[
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.
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[
Development,
2018]
Susan Strome is Distinguished Professor of Molecular, Cell and Developmental Biology at the University of California, Santa Cruz, USA. Recently appointed an editor at Development, her lab studies the regulation of germ cell development in<i>C. elegans</i>, with a particular focus on the epigenetic transmission of chromatin states. We caught up with Susan to discuss her early career switch from prokaryotes to worms, her experiences of small and big science, and why teaching is so important to her.
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[
WormBook,
2005]
In C. elegans, the germ line is set apart from the soma early in embryogenesis. Several important themes have emerged in specifying and guiding the development of the nascent germ line. At early stages, the germline blastomeres are maintained in a transcriptionally silent state by the transcriptional repressor PIE-1 . When this silencing is lifted, it is postulated that correct patterns of germline gene expression are controlled, at least in part, by MES-mediated regulation of chromatin state. Accompanying transcriptional regulation by PIE-1 and the MES proteins, RNA metabolism in germ cells is likely to be regulated by perinuclear RNA-rich cytoplasmic granules, termed P granules. This chapter discusses the molecular nature and possible roles of these various germline regulators, and describes a recently discovered mechanism to protect somatic cells from following a germline fate.
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[
International C. elegans Meeting,
2001]
Gamma-tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, C. elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics and chromosome segregation in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germline and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth, and the differential behavior of centrosomes destined for germline and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. Gamma-tubulin(RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays, but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous, long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans , but is required for the normal organization and function of kinetochore and interpolar microtubules. (This work will appear in the June, 2001 issue of Molec. Biol. Cell.)
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[
International Worm Meeting,
2009]
Germ granules are large, non-membrane-bound, ribonucleoprotein (RNP) organelles found in the germline cytoplasm of most, if not all, animals[1]. Like germ granules across species, P granules in C. elegans are found at the nuclear periphery[2], and are closely associated with nuclear pores in the germ line[3]. The C. elegans VASA homologs, GLH-1, GLH-2, and GLH-4, which are constitutively associated with P granules, resemble nuclear pore (NUP) proteins in being rich in FG (PheGly) repeats[4]. We hypothesized that the association between P granules and nuclear pores is facilitated by hydrophobic interactions between the FG repeats of the GLHs and NUPs. Consistent with this, P granules are dispersed when hydrophobic interactions are disrupted by aliphatic alcohols. In nuclear pores, hydrophobic interactions between FG repeat domains create a size exclusion barrier. We have shown that P granules impose a similar size exclusion barrier. The integral connection between P granules and nuclear pores is supported by results from a genome-wide RNAi screen for components required for proper P granule assembly and localization, in which we identified several nuclear pore associated factors. We are currently using a sensitive assay to measure interactions between FG repeat domains from GLH-1, GLH-2, GLH-4 and nuclear pore components identified in our screen. We propose that P granules extend the nuclear pore environment and provide a unique cytoplamic domain for post-transcriptional regulation in the germ line. 1.Eddy, E.M., Germ plasm and the differentiation of the germ cell line. Int Rev Cytol, 1975. 43: p. 229-80. 2.Strome, S. and W.B. Wood, Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans. Proc Natl Acad Sci U S A, 1982. 79(5): p. 1558-62. 3.Pitt, J.N., J.A. Schisa, and J.R. Priess, P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA. Dev Biol, 2000. 219(2): p. 315-33. 4.Gruidl, M.E., et al., Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci U S A, 1996. 93(24): p. 13837-42.
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[
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.
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[
International Worm Meeting,
2015]
Maintenance of the germ-soma distinction ensures proper cellular identity and function. A 2009 study reported that long-lived
daf-2 mutants of C. elegans ectopically express a germline program in their somatic cells, including mRNAs for the P-granule factors PGL-1 and PGL-3 (Curran et al. Nature 459: 1079-1084, 2009). Importantly, knock-down of several germline factors via RNAi in adult
daf-2 worms resulted in a slight decrease in those worms' lifespan, suggesting that those proteins confer some lifespan extension to
daf-2 mutants. Our lab's interest in germline development, regulators of the germ-soma distinction, and the possibility that some of the immortality of germ cells can be harnessed to extend somatic lifespan led us to explore and extend the
daf-2 findings. Our results have shown that somatic expression of a germline program does not extend lifespan in C. elegans: 1) We do not detect ectopic expression of P-granule proteins in
daf-2 worms, as assayed by immunostaining for PGL proteins and by expression of a PGL-1::GFP transgene under the control of the
pgl-1 promoter. 2) Simultaneous depletion of 4 constitutive components of P granules (PGL-1, PGL-3, GLH-1, and GLH-4) from
daf-2 mutants did not decrease lifespan but instead slightly extended lifespan. 3) Expressing a PGL-1::GFP fusion protein in the intestine of wild-type worms did not alter lifespan. 4) Six synMuv mutants that ectopically express germline proteins in their somatic cells at 24oC are not long-lived compared to wild type. We also tested the effect of loss of the master germline chromatin regulator MES-4 on the lifespan of
daf-2 worms. Compared to
daf-2 single mutants, fertile
daf-2;
mes-4 double mutants had the same lifespan, while sterile
daf-2;
mes-4 double mutants were dramatically longer-lived. We attribute this hyper-increased longevity to the synergistic effects of reduced insulin-like signaling and absence of a germline. Taken together, our results show that extreme extension of lifespan can be accomplished by combining loss of DAF-2 with absence of germ cells, and that somatic expression of germline proteins does not extend lifespan, arguing against the appealing possibility that expression of a germline progam in somatic cells can provide the somatic body with partial "germline immortality.".
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[
International Worm Meeting,
2015]
Cellular specification during embryogenesis relies on coordinated transcriptional regulatory networks that transition cells from old to new developmental programs. We seek to understand how developing somatic cells avoid expressing the germ cell program. C. elegans Synthetic Multivulval class B genes, which include genes that encode the 8 subunits of the Dp, Rb, and MuvB (DRM) transcriptional repressor complex, are known to protect somatic cells from activating germ-like regulatory networks. Mutations that compromise DRM complex activity, including loss of the Retinoblastoma-like pocket protein LIN-35, result in expression of germline genes in somatic cells. Importantly, many promoters of ectopically expressed germline genes (e.g.,
pgl-1) are not directly targeted by the DRM complex. We hypothesize the DRM complex represses germline genes in somatic cells through direct repression of transcription factor(s) required for germline gene expression. DRM binds to the promoters of 311 predicted transcription factor-encoding genes. Using a
lin-35(
n745) Ppgl-1::
pgl-1::GFP reporter strain that displays somatic
pgl-1::GFP expression, we tested for suppression of ectopic GFP expression following RNA interference of 243 of these candidate trans-acting factors, as compared to empty vector control. This qualitative screen identified 51 candidate trans-acting factors that potentially activate germline genes in somatic cells following loss of DRM. We also evaluated changes in chromatin accessibility, which can identify transcription factor binding sites, following loss of LIN-35. Chromatin accessibility can be measured by sensitivity of genomic regions to DNase I digestion. Changes in DNase I sensitivity as measured by quantitative PCR indicate an increase in chromatin accessibility at the
pgl-1 promoter in
lin-35(
n745) as compared to wild type. This analysis will be linked to high throughput sequencing of DNase I hypersensitive fragments between
lin-35(
n745) and wild type to reveal novel transcription factor binding events enriched upon loss of DRM activity. Together, these complementary approaches will identify transcription factors that drive germline gene expression in somatic cells following loss of DRM complex activity. This study provides a strong foundation for the identification and analysis of potential novel components of the germ cell transcriptional regulatory network required for in vivo germ cell specification.
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[
International Worm Meeting,
2011]
C. elegans germline development relies heavily upon post-transcriptional regulation in the germ plasm. Within the germ plasm, ribonucleoprotein aggregates called P granules overlie nuclear pore clusters and receive mRNAs as they are exported from the nucleus. A number of P-granule components, including the Vasa-related proteins GLH-1, GLH-2, and GLH-4, contain phenylalanine-glycine (FG) repeat domains, which are a common feature of nuclear pore complex (NPC) proteins. Within the NPC, FG-rich domains form a cohesive meshwork of filaments through hydrophobic interactions involving the phenylalanines in the FG motifs, creating a size-exclusion barrier that prevents diffusion of molecules larger than 45 kDa between the nucleus and the cytoplasm. We demonstrated that P granules, like NPCs, are held together by weak hydrophobic interactions and that they also establish a size-exclusion barrier similar to that of NPCs within the germ plasm. By expressing P-granule components in heterologous (intestinal) cells, we show that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient to target intestinal PGL granules to the nuclear periphery. Our results provide insights into the roles of the PGL and GLH families of proteins and suggest that P granules extend the NPC environment in the germ line to create a specialized hydrophobic microenvironment that may facilitate post-transcriptional processing events while selectively excluding large protein complexes from gaining access to mRNAs and endogenous siRNAs.
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[
International Worm Meeting,
2019]
Organization of the genome into domains of euchromatin and heterochromatin is a conserved and essential feature of all eukaryotes. Heterochromatin plays important roles in repression of transcription, chromosome segregation, and maintenance of genome integrity. Heterochromatin can be divided into constitutive heterochromatin and facultative heterochromatin, which can be distinguished by their associated histone modifications. Methylation of lysine 27 on histone H3 (H3K27me) is associated with facultative heterochromatin, while methylation of lysine 9 on histone H3 (H3K9me) is associated with constitutive heterochromatin. In mouse, fungus, and Drosophila, these two marks largely anticorrelate. However, in C. elegans, H3K27me and H3K9me show a surprising positive correlation, suggesting a species-specific difference in the organization of heterochromatin. In C. elegans, H3K27me represses transcription of the X chromosome in the germline, and its loss leads to a maternal-effect sterile (Mes) phenotype. Intriguingly, Gaydos et al. (Science, 2014) showed that mes mutant males that inherited their single X chromosome from the father are usually fertile and that the fertility of those males is dependent on H3K9me. Together, these observations suggest a potential redundant function of H3K27me and H3K9me in repressing the single X chromosome in the male germline to promote fertility in subsequent generations. We are investigating the potential redundant functions of H3K27me and H3K9me in the C. elegans male germline. First, by generating chimeric animals whose germlines inherit only paternal chromosomes, we have shown that mes mutant XX hermaphrodites are rendered fertile when both of their X chromosomes, which lack H3K27me, are inherited from the father. Furthermore, as in males, fertility is dependent on H3K9me. Second, we are comparing misexpression of genes and repetitive elements in hermaphrodite and male germlines lacking either H3K27me or H3K9me or lacking both marks, to investigate cross-talk between those two marks and differential responses of hermaphrodite (XX) and male (XO) germlines. Lastly, we are examining other species of Caenorhabditis. Early results suggest that C. briggsae accomplishes X-chromosome repression differently than C. elegans.