The conserved phosphatase GSP-2/PP1 promotes germline immortality via small RNA-mediated genome silencing during meiosis

AbstractGenomic silencing can promote germ cell immortality, or transgenerational maintenance of the germ line, via mechanisms that may occur during mitosis or meiosis. Here we report that the gsp-2 PP1/Glc7 phosphatase promotes germ cell immortality. We identified a separation-of-function allele of C. elegans GSP-2 that caused a meiosis-specific chromosome segregation defect and defects in transgenerational small RNA-induced genome silencing. GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. Sterile gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents and histone phosphorylation defects in oocytes, similar to small RNA genome silencing mutants. Epistasis and RNA analysis suggested that GSP-2 functions downstream of small RNAs. We conclude that a meiosis-specific function of GSP-2/LAB-1 ties small RNA-mediated silencing of the epigenome to germ cell immortality. Given that hemizygous genetic elements can drive transgenerational epigenomic silencing, and given that LAB-1 promotes pairing of homologous chromosomes and localizes to the interface between homologous chromosomes during pachytene, we suggest that discontinuities at this interface could promote nuclear silencing in a manner that depends on GSP-2.Author SummaryThe germ line of an organism is considered immortal in its capacity to give rise to an unlimited number of future generations. To protect the integrity of the germ line, mechanisms act to suppress the accumulation of transgenerational damage to the genome or epigenome. Loss of germ cell immortality can result from mutations that disrupt the small RNA-mediated silencing pathway that helps to protect the integrity of the epigenome. Here we report for the first time that the C. elegans protein phosphatase GSP-2 that promotes core chromosome biology functions during meiosis is also required for germ cell immortality. Specifically, we identified a partial loss of function allele of gsp-2 that exhibits defects in meiotic chromosome segregation and is also dysfunctional for transgenerational small RNA-mediated genome silencing. Our results are consistent with a known role of Drosophila Protein Phosphatase 1 in heterochromatin silencing, and point to a meiotic phosphatase function that is relevant to germ cell immortality, conceivably related to its roles in chromosome pairing or sister chromatid cohesion.

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Synaptonemal complex components are required for meiotic checkpoint function in C. elegans

10.1101/052977 ◽

2016 ◽

Author(s):

Tisha Bohr ◽

Guinevere Ashley ◽

Evan Eggleston ◽

Kyra Firestone ◽

Needhi Bhalla

Keyword(s):

Synaptonemal Complex ◽

Chromosome Segregation ◽

Meiotic Chromosome ◽

Checkpoint Response ◽

Homologous Chromosomes ◽

C Elegans ◽

Homolog Pairing ◽

The Stability ◽

Complex Components

AbstractSynapsis involves the assembly of a proteinaceous structure, the synaptonemal complex (SC), between paired homologous chromosomes and is essential for proper meiotic chromosome segregation. In C. elegans, the synapsis checkpoint selectively removes nuclei with unsynapsed chromosomes by inducing apoptosis. This checkpoint depends on Pairing Centers (PCs), cis-acting sites that promote pairing and synapsis. We have hypothesized that the stability of homolog pairing at PCs is monitored by this checkpoint. Here, we report that SC components SYP-3, HTP-3, HIM-3 and HTP-1 are required for a functional synapsis checkpoint. Mutation of these components does not abolish PC function, demonstrating they are bonafide checkpoint components. Further, we identify mutant backgrounds in which the instability of homolog pairing at PCs does not correlate with the synapsis checkpoint response. Altogether, these data suggest that, in addition to homolog pairing, SC assembly may be monitored by the synapsis checkpoint.

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Dynein-dynactin segregate meiotic chromosomes in C. elegans spermatocytes

10.1101/2020.10.19.345041 ◽

2020 ◽

Author(s):

Daniel J. Barbosa ◽

Vanessa Teixeira ◽

Joana Duro ◽

Ana X. Carvalho ◽

Reto Gassmann

Keyword(s):

Chromosome Segregation ◽

Protein Misfolding ◽

Meiotic Chromosome ◽

Spindle Assembly ◽

Cell Types ◽

Early Embryo ◽

Loss Of Function ◽

C Elegans ◽

Spindle Elongation

ABSTRACTThe dynactin complex is an essential co-factor of the microtubule-based motor dynein. Dynein-dynactin have well-documented roles in spindle assembly and positioning during C. elegans female meiosis and embryonic mitosis, while dynein-dynactin’s contribution to male meiosis has not been investigated. Here, we characterize the G33S mutation in DNC-1’s N-terminal microtubule binding domain, which corresponds to G59S in the human dynactin subunit p150/Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents homologous chromosome segregation and generates aneuploid sperm with an extra centrosome. Consequently, embryos produced by dnc-1(G33S) hermaphrodites exhibit a high incidence of tetrapolar mitotic spindles, yet dnc-1(G33S) embryos with bipolar spindles proceed through early mitotic divisions without errors in chromosome segregation. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to DNC-1’s inability to interact with microtubules. Rather, our results suggest that the DNC-1(G33S) protein, which is aggregation-prone in vitro, is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the unusual hypomorphic nature of the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.

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Excess crossovers impede faithful meiotic chromosome segregation in C. elegans

10.1101/2020.01.30.927640 ◽

2020 ◽

Cited By ~ 2

Author(s):

Jeremy A. Hollis ◽

Marissa L. Glover ◽

Aleesa Schlientz ◽

Cori K. Cahoon ◽

Bruce Bowerman ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Chromosome Segregation ◽

High Frequency ◽

Meiotic Chromosome ◽

Dependent Manner ◽

Homologous Chromosomes ◽

Crossover Interference ◽

C Elegans ◽

Functional Consequences ◽

Chromosome Bridges

AbstractDuring meiosis, at least one crossover must form between each pair of homologous chromosomes to ensure their proper partitioning. However, most organisms limit the number of crossovers by a phenomenon called crossover interference; why this occurs is not well understood. Here we investigate the functional consequences of extra crossovers in Caenorhabditis elegans. Using a fusion chromosome that exhibits a high frequency of supernumerary crossovers, we find that essential chromosomal structures are mispatterned, subjecting chromosomes to improper spindle forces and leading to congression and segregation defects. Moreover, we uncover mechanisms that counteract these errors; anaphase I chromosome bridges were often able to resolve in a LEM-3 nuclease dependent manner, and tethers between homologs that persisted were frequently resolved during Meiosis II by a second mechanism. This study thus provides evidence that excess crossovers impact chromosome patterning and segregation, and also sheds light on how these errors are corrected.

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glp-3 Is Required for Mitosis and Meiosis in the Caenorhabditis elegans Germ Line

Genetics ◽

10.1093/genetics/145.1.111 ◽

1997 ◽

Vol 145 (1) ◽

pp. 111-121 ◽

Cited By ~ 1

Author(s):

Lisa C Kadyk ◽

Eric J Lambie ◽

Judith Kimble

Keyword(s):

Caenorhabditis Elegans ◽

Germ Cells ◽

Germ Line ◽

Stem Cell Population ◽

Phenotypic Characterization ◽

Loss Of Function ◽

Dapi Staining ◽

Cell Cycles ◽

C Elegans ◽

Mitosis And Meiosis

The germ line is the only tissue in Caenorhabditis elegans in which a stem cell population continues to divide mitotically throughout life; hence the cell cycles of the germ line and the soma are regulated differently. Here we report the genetic and phenotypic characterization of the glp-3 gene. In animals homozygous for each of five recessive loss-of-function alleles, germ cells in both hermaphrodites and males fail to progress through mitosis and meiosis, but somatic cells appear to divide normally. Germ cells in animals grown at 15° appear by DAPI staining to be uniformly arrested at the G2/M transition with <20 germ cells per gonad on average, suggesting a checkpoint-mediated arrest. In contrast, germ cells in mutant animals grown at 25° frequently proliferate slowly during adulthood, eventually forming small germ lines with several hundred germ cells. Nevertheless, cells in these small germ lines never undergo meiosis. Double mutant analysis with mutations in other genes affecting germ cell proliferation supports the idea that glp-3 may encode a gene product that is required for the mitotic and meiotic cell cycles in the C. elegans germ line.

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Mutations that Affect Channel Opening of Innexin Hemichannels in the C. elegans Gonad

10.20944/preprints202011.0151.v1 ◽

2020 ◽

Author(s):

Todd Starich ◽

David Greenstein

Keyword(s):

Cell Proliferation ◽

Gap Junctions ◽

Germ Cell ◽

Structural Model ◽

Loss Of Function ◽

C Elegans ◽

Somatic Gonad ◽

Channel Opening ◽

Germline Proliferation ◽

Germ Cell Proliferation

In C. elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. We previously characterized a strong loss-of-function mutation (T239I) affecting the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. Here we describe the characterization of mutations that restore germ cell proliferation in the T239I EL2 mutant background. We recovered seven intragenic mutations located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky hemichannels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three other suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. These latter mutations may be useful to probe interactions with the biochemical pathways that produce the molecules transiting through soma-germline gap junctions.

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Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.201909136 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 2

Author(s):

Gisela Cairo ◽

Anne M. MacKenzie ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Mitotic Chromosome ◽

Budding Yeast ◽

Meiotic Chromosome ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

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Intertwined Functions of Separase and Caspase in Cell Division and Programmed Cell Death

10.1101/653584 ◽

2019 ◽

Author(s):

Pan-Young Jeong ◽

Ashish Kumar ◽

Pradeep Joshi ◽

Joel H. Rothman

Keyword(s):

Cell Proliferation ◽

Cell Division ◽

Chromosome Segregation ◽

Meiotic Chromosome ◽

Cysteine Proteases ◽

Embryonic Lethality ◽

Substrate Specificities ◽

C Elegans ◽

Cell Suicide ◽

Chromosome Disjunction

AbstractTimely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report that the C. elegans separase SEP-1, primarily known for its role in cell division, is required for apoptosis when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(−) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation and germline meiotic chromosome disjunction and the normal rate of embryonic development. Moreover, loss of the soma-specific (CSP-3) and germline-specific (CSP-2) caspase inhibitors results in CED-3-dependent suppression of embryonic lethality and meiotic chromosome non-disjunction respectively, when separase function is compromised. Thus, while caspases and separases have evolved different substrate specificities associated with their specialized functions in apoptosis and cell division respectively, they appear to have retained the residual ability to participate in both processes, supporting the view that co-option of components in cell division may have led to the innovation of programmed cell suicide early in metazoan evolution.

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The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

eLife ◽

10.7554/elife.12039 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 11

Author(s):

Saravanapriah Nadarajan ◽

Firaz Mohideen ◽

Yonatan B Tzur ◽

Nuria Ferrandiz ◽

Oliver Crawley ◽

...

Keyword(s):

Map Kinase ◽

Synaptonemal Complex ◽

Chromosome Segregation ◽

Meiotic Chromosome ◽

Late Pachytene ◽

C Elegans ◽

Map Kinase Pathway ◽

Rho Gef ◽

Kinase Signaling Pathway ◽

Kinase Pathway

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.

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A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress

10.1101/112847 ◽

2017 ◽

Author(s):

Alicia N. McMurchy ◽

Przemyslaw Stempor ◽

Tessa Gaarenstroom ◽

Brian Wysolmerski ◽

Yan Dong ◽

...

Keyword(s):

Small Rna ◽

Germ Line ◽

Large Fraction ◽

Repetitive Sequences ◽

Genotoxic Stress ◽

Repetitive Elements ◽

C Elegans ◽

Genes Encoding ◽

Rna Pathways ◽

Nuclear Rnai

AbstractRepetitive sequences derived from transposons make up a large fraction of eukaryotic genomes and must be silenced to protect genome integrity. Repetitive elements are often found in heterochromatin; however, the roles and interactions of heterochromatin proteins in repeat regulation are poorly understood. Here we show that a diverse set of C. elegans heterochromatin proteins act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. Mutants in genes encoding HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 also show functionally redundant sterility, increased germline apoptosis, DNA repair defects, and interactions with small RNA pathways. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting cep-1/p53, endogenous meiotic double strand breaks, or the expression of MIRAGE1 DNA transposons. Functional redundancy among these factors and pathways underlies the importance of safeguarding the genome through multiple means.

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The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans

eLife ◽

10.7554/elife.35478 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 1

Author(s):

Gregory M Davis ◽

Shikui Tu ◽

Joshua WT Anderson ◽

Rhys N Colson ◽

Menachem J Gunzburg ◽

...

Keyword(s):

Small Rna ◽

Rna Binding ◽

Meiotic Chromosome ◽

Rna Stability ◽

Rnai Pathway ◽

Regulatory Pathways ◽

C Elegans ◽

Bona Fide ◽

Rna Pathways ◽

Nuclear Rnai

Proper regulation of germline gene expression is essential for fertility and maintaining species integrity. In the C. elegans germline, a diverse repertoire of regulatory pathways promote the expression of endogenous germline genes and limit the expression of deleterious transcripts to maintain genome homeostasis. Here we show that the conserved TRIM-NHL protein, NHL-2, plays an essential role in the C. elegans germline, modulating germline chromatin and meiotic chromosome organization. We uncover a role for NHL-2 as a co-factor in both positively (CSR-1) and negatively (HRDE-1) acting germline 22G-small RNA pathways and the somatic nuclear RNAi pathway. Furthermore, we demonstrate that NHL-2 is a bona fide RNA binding protein and, along with RNA-seq data point to a small RNA independent role for NHL-2 in regulating transcripts at the level of RNA stability. Collectively, our data implicate NHL-2 as an essential hub of gene regulatory activity in both the germline and soma.

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