The MES-2/MES-3/MES-6 complex and regulation of histone H3 methylation in C. elegans

The C. elegans proteins MES-2 and MES-6, orthologs of the Polycomb group (PcG) chromatin repressors E(Z) and ESC, exist in a complex with their novel partner MES-3. The MES system participates in silencing the X chromosomes in the hermaphrodite germline. Loss of maternal MES function leads to germline degeneration and sterility. We report here that the MES complex is responsible for di- and trimethylation of histone H3 Lys27 (H3-K27) in the adult germline and in early embryos and that MES-dependent H3-K27 marks are concentrated on the X's. Another H3-K27 HMT functions in adult somatic cells, oocytes, and the PGCs of embryos. In PGCs, the MES complex may specifically convert dimethyl to trimethyl H3-K27. The HMT activity of the MES complex appears to be dependent on the SET domain of MES-2. MES-2 thus joins its orthologs Drosophila E(Z) and human EZH2 among SET domain proteins known to function as HMTs (reviewed in ). Methylation of histones is important for long-term epigenetic regulation of chromatin and plays a key role in diverse processes such as X inactivation and oncogenesis. Our results contribute to understanding the composition and roles of E(Z)/MES-2 complexes across species.     

Caenorhabditis elegans dosage compensation regulates histone H4 chromatin state on X chromosomes

Dosage compensation equalizes X-linked gene expression between the sexes. This process is achieved in Caenorhabditis elegans by hermaphrodite-specific, dosage compensation complex (DCC)-mediated, 2-fold X chromosome downregulation. How the DCC downregulates gene expression is not known. By analyzing the distribution of histone modifications in nuclei using quantitative fluorescence microscopy, we found that H4K16 acetylation (H4K16ac) is underrepresented and H4K20 monomethylation (H4K20me1) is enriched on hermaphrodite X chromosomes in a DCC-dependent manner. Depletion of H4K16ac also requires the conserved histone deacetylase SIR-2.1, while enrichment of H4K20me1 requires the activities of the histone methyltransferases SET-1 and SET-4. Our data suggest that the mechanism of dosage compensation in C. elegans involves redistribution of chromatin-modifying activities, leading to a depletion of H4K16ac and an enrichment of H4K20me1 on the X chromosomes. These results support conserved roles for histone H4 chromatin modification in worm dosage compensation analogous to those seen in flies, using similar elements and opposing strategies to achieve differential 2-fold changes in X-linked gene expression.     

Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line

The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54-60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20-24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.     

P granules extend the nuclear pore complex environment in the C. elegans germ line

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed 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 for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.     

C. elegans pro-1 activity is required for soma/germline interactions that influence proliferation and differentiation in the germ line

Strict spatial and temporal regulation of proliferation and differentiation is essential for proper germline development and often involves soma/germline interactions. In C. elegans, a particularly striking outcome of defective regulation of the proliferation/differentiation pattern is the Pro phenotype in which an ectopic mass of proliferating germ cells occupies the proximal adult germ line, a region normally occupied by gametes. We describe a reduction-of-function mutation in the gene pro-1 that causes a highly penetrant Pro phenotype. The pro-1 mutant Pro phenotype stems from defects in the time and position of the first meiotic entry during early germline development. pro-1(RNAi) produces a loss of somatic gonad structures and concomitant reduction in germline proliferation and gametogenesis. pro-1 encodes a member of a highly conserved subfamily of WD-repeat proteins. pro-1(+) is required in the sheath/spermatheca lineage of the somatic gonad in its role in the proper establishment of the proliferation/differentiation pattern in the germline. Our results provide a handle for further analysis of this soma-to-germline interaction.     

Functions of the novel RhoGAP proteins RGA-3 and RGA-4 in the germ line and in the early embryo of C. elegans

We have identified two redundant GTPase activating proteins (GAPs) - RGA-3 and RGA-4 - that regulate Rho GTPase function at the plasma membrane in early Caenorhabditis elegans embryos. Knockdown of both RhoGAPs resulted in extensive membrane ruffling, furrowing and pronounced pseudo-cleavages. In addition, the non-muscle myosin NMY-2 and RHO-1 accumulated on the cortex at sites of ruffling. RGA-3 and RGA-4 are GAPs for RHO-1, but most probably not for CDC-42, because only RHO-1 was epistatic to the two GAPs, and the GAPs had no obvious influence on CDC-42 function. Furthermore, knockdown of either the RHO-1 effector, LET-502, or the exchange factor for RHO-1, ECT-2, alleviated the membrane-ruffling phenotype caused by simultaneous knockdown of both RGA-3 and RGA-4 [rga-3/4 (RNAi)]. GFP::PAR-6 and GFP::PAR-2 were localized at the anterior and posterior part of the early C. elegans embryo, respectively showing that rga-3/4 (RNAi) did not interfere with polarity establishment. Most importantly, upon simultaneous knockdown of RGA-3, RGA-4 and the third RhoGAP present in the early embryo, CYK-4, NMY-2 spread over the entire cortex and GFP::PAR-2 localization at the posterior cortex was greatly diminished. These results indicate that the functions of CYK-4 are temporally and spatially distinct from RGA-3 and RGA-4 (RGA-3/4). RGA-3/4 and CYK-4 also play different roles in controlling LET-502 activation in the germ line, because rga-3/4 (RNAi), but not cyk-4 (RNAi), aggravated the let-502(sb106) phenotype. We propose that RGA-3/4 and CYK-4 control with which effector molecules RHO-1 interacts at particular sites at the cortex in the zygote and in the germ line.     

Processing bodies and germ granules are distinct RNA granules that interact in C. elegans embryos

In somatic cells, untranslated mRNAs accumulate in cytoplasmic foci called processing bodies or P-bodies. P-bodies contain complexes that inhibit translation and stimulate mRNA deadenylation, decapping, and decay. Recently, certain P-body proteins have been found in germ granules, RNA granules specific to germ cells. We have investigated a possible connection between P-bodies and germ granules in Caenorhabditis elegans. We identify PATR-1, the C. elegans homolog of the yeast decapping activator Pat1p, as a unique marker for P-bodies in C. elegans embryos. We find that P-bodies are inherited maternally as core granules that mature differently in somatic and germline blastomeres. In somatic blastomeres, P-bodies recruit the decapping activators LSM-1 and LSM-3. This recruitment requires the LET-711/Not1 subunit of the CCR4-NOT deadenylase and correlates spatially and temporally with the onset of maternal mRNA degradation. In germline blastomeres, P-bodies are maintained as core granules lacking LSM-1 and LSM-3. P-bodies interact with germ granules, but maintain distinct dynamics and components. The maternal mRNA nos-2 is maintained in germ granules, but not in P-bodies. We conclude that P-bodies are distinct from germ granules, and represent a second class of RNA granules that behaves differently in somatic and germline cells.     

Linker histone HIS-24 (H1.1) cytoplasmic retention promotes germ line development and influences histone H3 methylation in Caenorhabditis elegans

RNA interference with one of the eight Caenorhabditis elegans linker histone genes triggers desilencing of a repetitive transgene and developmental defects in the hermaphrodite germ line. These characteristics are similar to the phenotype of the C. elegans Polycomb group genes mes-2, mes-3, mes-4, and mes-6 (M. A. Jedrusik and E. Schulze, Development 128:1069-1080, 2001; I. Korf, Y. Fan, and S. Strome, Development 125:2469-2478, 1998). These Polycomb group proteins contribute to germ line-specific chromatin modifications. Using a his-24 deletion mutant and an isoform-specific antibody, we characterized the role of his-24 in C. elegans germ line development. We describe an unexpected cytoplasmic retention of HIS-24 in peculiar granular structures. This phenomenon is confined to the developing germ lines of both sexes. It is strictly dependent on the activities of the chromatin-modifying genes mes-2, mes-3, mes-4, and mes-6, as well as on the C. elegans sirtuin gene sir-2.1. A temperature shift experiment with a mes-3(ts) mutant revealed that mes gene activity is required in a time window ranging from L3 to the early L4 stage before the onset of meiosis. We find that the his-24(ok1024) mutant germ line is characterized by an increased level of the activating H3K4 methylation mark concomitant with a decrease of the repressive H3K9 methylation. In the germ line of his-24(ok1024) mes-3(bn35) double mutant animals, the repressive H3K27 methylation is more reduced than in the respective mes single mutant. These observations distinguish his-24 as an unusual element in the developmental regulation of germ line chromatin structure in C. elegans.     

glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans

In the wild-type C. elegans germ line there are both mitotic and meiotic germ cells. Mutations in glp-1 cause germ cells that would normally divide mitotically to enter meiosis. This mutant phenotype mimics the effect of killing the distal tip cell, a somatic cell that interacts with the germ line to regulate the mitotic/meiotic decision. In addition, wild-type glp-1 product is required maternally for embryogenesis. Temperature-shift experiments indicate that the temporal requirement for glp-1 activity in the germ line is the same as that for distal tip cell regulation. Mosaic analyses suggest that glp-1 is produced in the germ line. We propose that glp-1 acts as part of the receiving mechanism in the interaction between the distal tip cell and germ line.     

More mog genes that influence the switch from spermatogenesis to oogenesis in the hermaphrodite germ line of Caenorhabditis elegans

The Caenorhobditis elegans XX animal possesses a hermaphrodite germ line, producing first sperm, then oocytes. In this paper, we report the genetic identification of five genes, mog-2, mog-3, mog-4, mog-5, and mog-6, that influence the hermaphrodite switch from sper-matogenesis to oogenesis. In mcg-2-mog-6 mutants, spermatogenesis continues past the time at which hermaphrodites normally switch into oogenesis and no oocytes are observed. Therefore, in these mutants, germ cells are transformed from a female fate (oocyte) to a male fate (sperm). The fem-3 gene is one of five genes that acts at the end of the germline sex determination pathway to direct spermatogenesis. Analyses of mog;fem-3 double mutants suggest that the mog-2-mog-6 genes act before fem-3; thus these genes may be in a position to negatively regulate fem-3 or one of the other terminal regulators of germline sex determination. Double mutants of fem-3 and any one of the mog mutations make oocytes. Using these double mutants, we show that oocytes from any mog;fem-3 double mutant are defective in their ability to support embryogenesis. This maternal effect lethality indicates that each of the mog genes is required for embryogenesis. The two defects in mog-2-mog-6 mutants are similar to those of mog-1: all six mog genes eliminate the sperm/oocyte switch in hermaphrodites and cause maternal effect lethality. We propose that the mog-2-mog-6 mutations identify genes that act with mog-1 to effect the sperm/oocyte switch. We further speculate that the mog-1-mog-6 mutations all interfere with translational controls of fem-3 and other maternal mRNAs. © 1993 Wiley-Liss, Inc.     

Genetic control of sex determination in the germ line of Caenorhabditis elegans

The nematode Caenorhabditis elegans normally exists as one of two sexes: self-fertilizing hermaphrodite or male. Development as hermaphrodite or male requires the differentiation of each tissue in a sex-specific way. In this review, I discuss the genetic control of sex determination in a single tissue of C. elegans: the germ line. Sex determination in the germ line depends on the action of two types of genes:--those that act globally in all tissues to direct male or female development and those that act only in the germ line to specify either spermatogenesis or oogenesis. First, I consider a tissue-specific sex-determining gene, fog-1, which promotes spermatogenesis in the germ line. Second, I consider the regulation of the hermaphrodite pattern of germ-line gametogenesis where first sperm and then oocytes are produced.     

The role of cell-cell interactions in postembryonic development of the Caenorhabditis elegans germ line.

This review addresses the role of cell-cell interactions in the development of the Caenorhabditis elegans germ line: specifically, the relative contributions of germ-line-soma interactions versus autonomous processes are considered. Current knowledge of the interacting cell types and the genes essential for various aspects of germ-line development is discussed.