Mazzetto, Mariateresa et al. (2021) International Worm Meeting "Analysis of OEF-1 as a potential epigenetic reader of H3K36me3 in the C.elegans germ line"

Mazzetto, Mariateresa, McManus, Cassie, Reinke, Valeria

[International Worm Meeting, 2021]

The trimethylation of Lys 36 residue on H3 (H3K36me3) is a signature histone mark for gene expression in the C.elegans germline. Its genome distribution is mediated specifically by MES-4 and MET-1 histone methyltransferases. This modification regulates several events related to transcription, including promoting splicing, suppressing cryptic transcription initiation, and suppressing DNA damage. H3K36me3 is likely linked to these downstream pathways by epigenetic "readers", proteins that bind a specific epigenetic mark and regulate downstream pathways in response. However, H3K36me3 reader proteins in the C.elegans germline are poorly characterized. We explored the possibility that Oocyte-Excluded Factor 1 (OEF-1), a germline-specific factor discovered in our lab (McManus and Reinke, 2017), might function as a reader of H3K36me3. OEF-1 has a role in germline progression, binds preferentially to autosomal genes, and co-localizes with H3K36me3. To study the functional relationship between OEF-1 and H3K36me3, we developed a computational pipeline comprehensive of ChIP-seq and RNA-seq experiments, which we then analyzed. ChIP-seq data for H3K27me3 and H3K36me3 in oef-1 mutants and wild-type adults were obtained from purified germ nuclei, and binding profiles were then further analyzed. RNA-seq was performed from dissected gonads from oef-1 mutant and wild-type young adults and analyzed for gene expression and splicing differences. From the ChIP-seq data, we found a strong correlation between OEF-1 and H3K36me3 binding profiles. From the RNA-seq data, we found that, even though the loss of OEF-1 has minimal effect on transcript abundance, it greatly increases the frequency of retained introns predominantly at the level of autosomal germline-specific genes. Thus, our data to date implicates OEF-1 in a role in promoting splicing fidelity in the C.elegans germ line, potentially by its association with H3K36me3 at germline-expressed genes.

Alex Lublin et al. (2001) International C. elegans Meeting "An RNAi based screen for regulators of GLP-1 translation and new factors involved in early embryonic polarity from a set of germline enriched genes."

Tom Evans, Alex Lublin

[International C. elegans Meeting, 2001]

In C. elegans embryos, polarity is established prior to the first division by the segregation of maternally derived factors. Polarity in the early embryo leads to the localized expression of many developmental factors that promote distinct cellular fates. The notch protein GLP-1 is localized in anterior cells of the early embryo. GLP-1 is localized through translation control through the 3’ untranslated region (UTR) of the mRNA. Reporter RNA experiments have shown that there are multiple 3' UTR elements that control GLP-1 translation. However, the factors that regulate GLP-1 and those that link GLP-1 translation to early polarity remain largely unknown. We are using an RNA interference (RNAi) based screen to find new factors that either directly or indirectly regulate GLP-1 or function in the control of general developmental polarity. Previously we screened random clones from a cDNA library. Although some interesting genes were identified (see Barbee et. al., abstract), RNAi suppression limits the success of this screen. Thus, we have modified the screen to limit the number of factors being screened by screening a subset of genes that were shown to be germline enriched by gene chip array experiments performed in the Kim lab (Reinke et. al. 2000). We obtained primers pairs, which were kindly provided by Valerie Reinke and Stuart Kim that correspond to the both the germline intrinsic and the oocyte enriched genes. These primers were used to PCR amplify fragments from genomic DNA. Using gene information from the genome project, we have prioritized three groups of genes to screen. Since the regulation of GLP-1 is though the 3’ UTR of the mRNA, the first of these contains any genes that may bind RNA (RNA binding proteins and zinc finger proteins). The second sub-set contains genes with an unknown function but with an interesting domain and/or out of species homologue. Lastly, there is also a set of genes that have no known function or homologue but still may play vital roles in development. Following RNA synthesis of selected genes worms were injected with dsRNA either individually or in pools of three. Using immunoflourescence worms were labeled with antibodies to GLP-1 and P-granules (as a posterior marker of polarity) and screened for mislocalization. At present we have screened 39 factors and have found 14 genes that had varying levels of lethality following RNAi. Two novel genes appear to potentially mislocalized GLP-1in the embryo, although at very low penetrance. The low penetrance could be due to an incomplete RNAi affect, redundancy, or an indirect affect resulting from general disruption of embryonic development. Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, Kim SK, A global profile of germline gene expression in C. elegans Mol Cell 2000 Sep; 6(3): 605-16

Jeffers, Tess E. et al. (2013) International Worm Meeting "Nucleosome organization in C. elegans gamete chromatin."

Lieb, Jason D., Jeffers, Tess E.

[International Worm Meeting, 2013]

We are interested in how the genome is packaged in gametes (sperm and oocytes), and how differences might contribute to the specification of zygotic gene expression programs. Using fem-1 and him-5 mutants, we separately isolated pure populations of C. elegans sperm and oocytes and performed RNA-seq and nucleosome mapping through MNase-seq. Preliminary transcriptome analysis reveals ~700 transcripts with higher relative levels in sperm, and ~2000 transcripts with higher relative levels in oocytes, in agreement with quantities reported by previous microarray and proteomics experiments (Reinke et al., Mol. Cell 2000 & Development 2004, Chu et al., Nature 2006). Sperm are enriched in transcripts encoding proteins that function in the cytoskeleton, as chaperones, and in the mitochondria while oocytes are enriched for transcripts that encode proteins functioning in mRNA processing, mitosis and meiosis. By integrating our nucleosome and transcriptome data, we find nucleosome depletion at the promoters of genes highly transcribed in the germline. Future experiments will investigate the use of differential histone isoforms and histone modifications in gamete genome packaging.

Marian Walhout et al. (2001) International C. elegans Meeting "Integration of functional genomics approaches: protein interaction mapping using genes enriched in the C. elegans germline"

Marian Walhout, Philippe Vaglio, Jerome Reboul, Marc Vidal, Olena Shtanko, Valerie Reinke, Stuart Kim

[International C. elegans Meeting, 2001]

Since the complete genome sequence of C. elegans became available at the end of 1998, several functional genomics projects have been initiated to functionally annotate the many predicted genes that had remained previously uncharacterized. The data generated by these projects can be viewed as hypotheses until validated further. We have proposed that the likelihood of such hypotheses to be biologically meaningful might increase as the data from different projects are integrated. For example, genes that cluster in expression profiling, protein interaction maps and phenotypic analysis might have a relatively high likelihood to functionally interact in the same biological process. Recently, microarray analysis has led to the identification of a set of genes that show increased expression in the germline (Reinke et al, Mol. Cell. 6, 605-616, 2000). We have cloned these ~750 open reading frames and transferred them into yeast two-hybrid vectors using Gateway cloning. Subsequently, we have generated a 750 X 750 protein interaction map. The data obtained and its integration with the RNAi data obtained by Fabio Piano and colleagues will be discussed.

Lydia The et al. (2007) International Worm Meeting "A screen for genes required for P granule localization in embryos."

Geraldine Seydoux, Ekaterina Voronina, Lydia The

[International Worm Meeting, 2007]

P granules are cytoplasmic determinants necessary for germ line development. These ribonucleoprotein complexes are inherited maternally and segregate to the germline blastomeres, which eventually gives rise to all the germ cells in the adult. P granules are located in the cytoplasm during the first few embryonic divisions, but localize around the nucleus at all other stages. The mechanisms that regulate P granule localization are not fully known. To uncover new regulators of P granule localization and integrity, we conducted an RNAi screen using a GFP::PGL-1 transgenic line. PGL-1 is a predicted RNA-binding protein that is constitutively associated with P granules. We are initially starting our search with a set of ~1000 genes selectively enriched in oogenesis (Reinke et al. 2004), utilizing a subset of the Ahringer feeding RNAi library (Kamath et al., 2003). Genes known to affect P granule distribution, such as snr-2 (Barbee et al., 2006), have been identified in this screen, supporting our methodology. In preliminary results, novel factors affecting P granule localization have also been identified, including a predicted nucleotide-binding protein. We will report our progress on the screen at the meeting.

Florencia Pauli et al. (2005) International Worm Meeting "Global profile of intestine-specific gene expression in C. elegans"

Yueyi Liu, Stuart Kim, Florencia Pauli

[International Worm Meeting, 2005]

The simple body plan, many tissues, and complete genome sequence of C. elegans allow for a systems biology approach to the question of which genes specify a tissue. A global profile of tissue-specific gene expression of this organism would expand our knowledge of tissue development and maintenance, regulation of gene expression, and higher order chromosome structure. We used mRNA tagging (Roy et al.) to identify genes expressed in the intestine of the worm. Animals expressing an epitope-tagged protein that binds the poly-A tail of mRNAs (FLAG::PAB-1) from an intestine-specific promoter (ges-1) were used to immunoprecipitate (IP) FLAG::PAB-1/mRNA complexes from the intestine. Genes enriched by intestinal IP were identified with DNA microarrays. The average ranks of enrichment from 8 repeats of this experiment identified 1938 intestine-expressed genes (p<0.001). First, we compared the intestine-expressed genes to those expressed in the muscle (Roy et al.) and germline (Reinke et al.), and identified 510 genes enriched in all 3 tissues and 246 intestine-, 115 muscle-, and 219 germline-enriched genes. Second, we showed that the 1938 intestine genes were physically clustered on the chromosomes, suggesting that the order of genes in the genome is influenced by the effect of chromatin domains on gene expression. Furthermore, the commonly-expressed genes showed more chromosomal clustering than the tissue-enriched genes, suggesting that chromatin domains may influence housekeeping genes more than tissue-specific genes. Third, in order to gain further insight into the regulation of intestinal gene expression, we searched for regulatory motifs. We used a modified Gibbs sampling method called CompareProspector (Liu et al.) to identify motifs that were conserved with C. briggsae and over-represented in the 1 kb upstream region of the 1938 intestine-expressed genes. This analysis found that the promoters of the intestine genes were enriched for the consensus sequence for GATA transcription factors. We experimentally verified these results by showing that GATA motifs are required in cis and that GATA transcription factors are required in trans for expression of these intestinal genes. Previous work had shown that GATA transcription factors (med-1, end-1, etc.) are important for intestinal differentiation, and our work has identified about 800 intestinal genes that may be the direct targets of the GATA transcription factors. Liu, Y. et al. (2004). Genome Research, 14(3), 451-458. Reinke, V. et al. (2004). Development, 131(2), 311-323. Roy, P. J. et al. (2002). Nature, 418(6901), 975-979.

Michael A Miller et al. (2001) International C. elegans Meeting "MSP Signalling: Beyond the Sperm Cytoskeleton."

David Greenstein, Michael A Miller

[International C. elegans Meeting, 2001]

Sophisticated regulatory systems have evolved to coordinate fertilization and oocyte meiotic cell cycle progression. However, the molecular interactions that govern critical oocyte cell cycle transitions and their conservation in the metazoa are not clear. C. elegans oocytes, like those of most animals, arrest during meiotic prophase. Sperm promote the resumption of meiosis (maturation) and contraction of the smooth muscle-like gonadal sheath cells, which are necessary for ovulation. Using an in vivo bioassay, we have shown that the major sperm cytoskeletal protein (MSP) acts as a bipartite signal for both oocyte maturation and sheath contraction. During nematode sperm locomotion, MSP plays a role analogous to actin indicating that this 14 kDa sperm-specific protein has acquired extracellular signaling and intracellular cytoskeletal functions during evolution. Proteins with MSP-like domains have been found in plants, fungi, and other animals raising the possibility that MSP signaling functions may exist in other phyla. We are particularly interested in understanding how MSP promotes the resumption of meiosis in oocytes. A receptor for a maturation-promoting factor has not been identified in any animal. Thus, identifying and characterizing the MSP receptor(s) is a major goal. We are taking a genomic approach to identify receptor candidates. Using an in situ MSP binding assay, candidates can be quickly screened for binding following RNAi. In this assay, MSP-flouroscein specifically binds oocytes and sheath cells of the proximal gonad arm. MSP-flouroscein is active in promoting both oocyte maturation and sheath contraction in vivo . Binding is not observed in distal female gonads, male gonads, or following incubation with BSA-flouroscein. Preincubation with a 20-fold excess of unlabeled MSP completely abrogates MSP-flouroscein binding. Further, binding is not observed in emo-1(oz1) oocytes, which lack a functional secretory system due to a mutation in a Sec61p g homologue (Iwasaki et al., 1996). Using data from DNA microarrays (Reinke et al., 2000), we have identified three conserved transmembrane proteins whose transcripts are highly enriched in oocytes. MSP binding to oocytes is dramatically reduced in two loss of function backgrounds and enhanced in the other. Each gene is tightly linked to one of the two large MSP clusters in the genome--a finding of unknown significance. We currently have mutations in two genes and are doing detailed phenotypic analyses. We are also interested in examining protein localization in situ and evaluating MSP binding in a heterologous system. Our results will be discussed in the context of a new model for oocyte meiotic maturation. Iwasaki et al. (1996). J Cell Biol 134:699-714 Reinke et al. (2000). Mol Cell 6:605-616

Bill Kelly (2001) International C. elegans Meeting "The Role of Chromatin Organization in Germ Line Function and Maintenance"

Bill Kelly

[International C. elegans Meeting, 2001]

The germ line in C. elegans is maintained by a variety of suppressive mechanisms throughout development and during adulthood. In the adult germ line both transcriptional and post-transcriptional mechanisms play roles in maintaining germ cell viability and function. Transcriptional suppression involves chromatin-based mechanisms that are products of a compromise reached by the competing goals of meiotic DNA: the chromosomes must be compacted along their lengths for synapsis and recombination, yet must also be transcriptionally active for maternal product synthesis. One might thus hypothesize that genes essential for germ line function have evolved mechanisms to escape aspects of chromatin silencing that likely accompany the compaction of meiotic DNA. Non-integrated transgenes are efficiently targeted for silencing in the germ line by chromatin-based mechanisms, since mutations that are defective in this silencing exhibit defects in chromatin organization. In addition, Valerie Reinke along with Stuart Kim and Anne Villeneuve, as well as my lab, have observed that the entire X chromosome appears to be silenced. Markers for transcriptionally active chromatin (e.g. histone acetylation) are uniquely excluded from the X chromosome in immature germ cells in both males and hermaphrodites. As germ cells mature past pachytene stage in hermaphrodites, however, the X chromosome becomes increasingly labeled by probes that correlate with transcriptional activation. This "activation" is not observed in male germ cells. Interestingly this correlates well with microarray data from Reinke et al. that indicates a paucity of sperm-specific or germ cell-intrinsic genes on the X, but a normal distribution of putative oocyte-specific genes (Mol. Cell. 6:605). Silenced transgenes strongly mimic the observed underacetylation of X chromosome histones in the early stages of meiotic prophase I, but fail to become "activated" in oocytes. Conversely, a transgene (for a normally autosomal gene) integrated onto the X exhibits a germ line expression that is always strongest in oocytes. This suggests the presence of cis-acting elements peculiar to the X that may be involved in post-pachytene activation of the X chromosome in oocytes. The nature of germ line X-regulation is unknown, but is unlikely to involve components of dosage compensation (which do not assemble onto the X until early embryogenesis). Indeed, an absence of X-linked genes essential for both male and female germ cells would appear to obviate a need for dosage compensation in germ cells, as well as permit X silencing in the immature germ cells of both sexes. This may in turn have played a role in the frequent appearance of hermaphroditism in nematode evolution. The effects of silencing mutants on transgene and X silencing, an analysis of X silencing in other hermaphroditic and gonochoristic nematode species, and the role of chromatin organization in germ line function will be presented and discussed.

Johnathan W. Edmonds et al. (2007) International Worm Meeting "RNAi screen for oocyte genes that control directional sperm motility."

Johnathan W. Edmonds, Michael A. Miller

[International Worm Meeting, 2007]

The mechanism(s) by which motile sperm find oocytes in the female reproductive tract is not understood. To investigate this mechanism, we have developed an in vivo assay for tracking the movement of sperm in the uterus of C. elegans hermaphrodites. Previous results support the hypothesis that oocytes generate sperm-recruiting signals derived from polyunsaturated fatty acids (PUFAs). These signals direct sperm migration to the spermatheca, the site of fertilization. In mammals, PUFAs are synthesized into eicosanoids, which direct T-cell migration to sites of inflammation. I am using RNA-mediated interference (RNAi) to identify genes that function in oocytes to control directional sperm motility. Genome-wide DNA microarray (Reinke et al. 2004) and in situ hybridization (Kohara 2001) databases were screened for genes expressed in oocytes. I have identified eight genes that are required in hermaphrodites for the directional movement of wild-type sperm. Several of these genes are related to human genes implicated in eicosanoid synthesis and transport. I am currently focusing on the genes T28F3.1 and C01F6.1, which encode proteins called copines. Copines are conserved calcium-dependent membrane binding proteins with unknown functions. They are characterized by having two Ca(2+)-binding C2-domains at the N-terminal region and an A-domain at the C-terminal region. We hypothesize that C01F6.1 and T28F3.1 function in oocytes to target PUFA-modifying enzymes to the plasma membrane where they can generate sperm-recruiting signals. I am currently conducting phenotypic analysis of loss of function mutations in C01F6.1 and T28F3.1. Results from this analysis and site of action studies will be presented.

Katherine A Williams et al. (2005) International Worm Meeting "Functional analysis of genes with male germline-enriched expression for possible roles in sperm competition or spermiogenesis regulation."

Valerie Reinke, Katherine A Williams

[International Worm Meeting, 2005]

Both C. elegans hermaphrodites and males produce sperm, but these sperm differ in several ways between the two sexes. Male sperm are larger than hermaphrodite sperm and successfully outcompete hermaphrodite sperm for fertilization of the oocyte. Additionally, spermatid activation (spermiogenesis) occurs only once in hermaphrodites, whereas in males, spermiogenesis occurs with every ejaculation. Mutations in several genes display hermaphrodite-specific defects in spermiogenesis, indicating that spermiogenesis must be under distinct regulation in males.Through microarray analysis, we have identified eight genes whose expression is enriched in the male germline but not in hermaphrodite germlines producing excess sperm (Reinke et al, 2004). We are focusing on investigating possible roles for these male-enriched germline genes, including male-specific aspects of spermiogenesis and the competitive advantage of male sperm over hermaphrodite sperm. We will use two mating tests to distinguish behavioral and fertilization defects from competition defects. Male sperm competition ability will be determined by comparing the ratios of cross-progeny and self-progeny of marked hermaphrodites mated to mutant and wildtype males. Control matings to hermaphrodites lacking sperm [fem-1(hc17ts)] will determine whether the mutant males retain the ability to mate and whether spermatogenesis is normal. Currently, we have obtained deletion mutants for two of these eight genes, F54H5.3(vr8) and pqn-2(ok1149), and will present the ongoing analysis of their phenotypes. From these studies we hope to understand how male sperm can differ from hermaphrodite sperm, and how these differences affect the evolutionary success of the species.