Honors & Awards
American Cancer Society Research Professor Award, American Cancer Society (01/01/2016 - 12/31/2020)
Kirsch Investigator Award, Steven and Michele Kirsch Foundation (2003-2004)
Junior Faculty Scholar Award, HHMI (1999)
Esther Ehrman Lazard Faculty Scholar, Stanford University (1996, 1997, 1998)
Searle Scholars Award, Chicago Community Trust (1996-99)
Beginning Faculty Investigator Award, Baxter Foundation (1995)
B.S., University of Notre Dame, Biochemistry (1981)
Ph.D., M.I.T., Biology (1989)
Current Research and Scholarly Interests
We investigate mechanisms underlying the faithful inheritance of eukaryotic chromosomes. Our primary focus is on elucidating the events required for orderly segregation of homologous chromosomes during meiosis, the crucial process by which diploid germ cells generate haploid gametes. These events are of central importance to sexually reproducing organisms, since errors in meiosis lead to chromosomal aneuploidy, one of the leading causes of miscarriages and birth defects in humans.
Diploid germ cells face several major challenges on the road to reducing their ploidy to generate haploid gametes: 1) Chromosomes must locate, identify and align with their appropriate homologous pairing partners. 2) Chromosomes must acquire a structural organization that will promote controlled breakage of DNA molecules and subsequent recombinational repair using the homologous chromosome as a repair partner to yield interhomolog crossovers. 3) Chromosomes must couple the events of recombination with further structural reorganization to yield an organization in which homologs are connected by chiasmata, yet oriented away from each other in a way that promotes their attachment to and segregation toward opposite poles of the meiosis I spindle. Moreover, the connections afforded by chiasmata must be coupled with a two-step loss of cohesion, such that partial loss of cohesion occurs at meiosis I to permit dissolution of chiasmata and homolog separation while maintaining the connections between sisters required to permit bipolar attachment on the meiosis II spindle. 4) During oocyte meiosis, a bipolar spindle must be assembled and function without the aid of centrosomes. All of these events must be tightly coordinated to achieve a successful outcome.
Despite the fundamental importance of meiosis, the mechanisms underlying many key events remain poorly understood. We are approaching the study of meiosis using the nematode C. elegans, a simple metazoan that is especially amenable to combining genetic, genomic and cytological approaches in a single system, and in which the events of meiosis are particularly accessible. The germ line accounts for more than half of the cell nuclei in the adult worm, with nuclei in all stages of meiosis present simultaneously in a temporal/spatial gradient along the distal-proximal axis of the gonad, so that each gonad represents a complete meiotic time course. These features facilitate visualizing chromosome organization using high-resolution microscopic imaging in the context of intact 3D nuclear architecture.
Topics under investigation include:
HOMOLOGOUS CHROMOSOME PAIRING AND SYNAPSIS:
How do chromosomes locate and recognize their appropriate pairing partners? How is recognition coordinated with assembly of the synaptonemal complex (SC), a highly ordered protein scaffold that stabilizes homolog association, so that synapsis occurs only between correct partners?
How do cells sense a chromosome pair that has not yet undergone a crossover? How does a crossover trigger global changes in structure and function along a whole chromosome pair? How do crossover-triggered changes inhibit other crossovers?
COORDINATING CHROMOSOME STRUCTURE WITH RECOMBINATION:
Double-strand DNA breaks (DSBs) are dangerous to genomic integrity. How is their formation and repair coordinated with other features of the meiotic program? How does chromatin state affect competence for DSB formation?
How does chromosome organization established during prophase lead to orderly segregation? How does the oocyte assemble a bipolar spindle in the absence of centrosomes? What special mechanisms ensure inheritance of sex chromosomes?
EVOLUTION OF MEIOTIC MACHINERY
What mechanisms are responsible for the rapid divergence of meiotic structural proteins?
- Advanced Genetics
GENE 205 (Win)
- Frontiers in Biological Research
BIOC 215, DBIO 215, GENE 215 (Aut, Win, Spr)
Independent Studies (10)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr)
- Directed Reading in Genetics
GENE 299 (Aut, Win, Spr)
- Graduate Research
DBIO 399 (Aut, Win, Spr)
- Graduate Research
GENE 399 (Aut, Win, Spr)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr)
- Medical Scholars Research
GENE 370 (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Supervised Study
GENE 260 (Aut, Win, Spr)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr)
- Undergraduate Research
GENE 199 (Aut, Win, Spr)
- Directed Reading in Developmental Biology
Prior Year Courses
- Advanced Genetics
GENE 205 (Win)
- Frontiers in Biological Research
BIOC 215, DBIO 215, GENE 215 (Aut, Win, Spr)
- Advanced Genetics
GENE 205 (Win)
- Frontiers in Biological Research
BIOC 215, DBIO 215, GENE 215 (Aut, Win)
- Advanced Genetics
Mammalian CNTD1 is critical for meiotic crossover maturation and deselection of excess precrossover sites.
journal of cell biology
2014; 205 (5): 633-641
Meiotic crossovers (COs) are crucial for ensuring accurate homologous chromosome segregation during meiosis I. Because the double-strand breaks (DSBs) that initiate meiotic recombination greatly outnumber eventual COs, this process requires exquisite regulation to narrow down the pool of DSB intermediates that may form COs. In this paper, we identify a cyclin-related protein, CNTD1, as a critical mediator of this process. Disruption of Cntd1 results in failure to localize CO-specific factors MutLγ and HEI10 at designated CO sites and also leads to prolonged high levels of pre-CO intermediates marked by MutSγ and RNF212. These data show that maturation of COs is intimately coupled to deselection of excess pre-CO sites to yield a limited number of COs and that CNTD1 coordinates these processes by regulating the association between the RING finger proteins HEI10 and RNF212 and components of the CO machinery.
View details for DOI 10.1083/jcb.201401122
View details for PubMedID 24891606
Meiotic chromosome structures constrain and respond to designation of crossover sites.
2013; 502 (7473): 703-706
Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologues that ensure their proper segregation at meiosis I. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are the initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair. Moreover, crossovers tend to inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known as crossover interference. Here we show that the synaptonemal complex, a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by crossover recombination events. Using a cytological marker of crossover sites in Caenorhabditis elegans, we show that partial depletion of the synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the effective distance over which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover, we show that crossovers are associated with a local 0.4-0.5-micrometre increase in chromosome axis length. We propose that meiotic crossover regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome structure to inhibit other crossovers at additional sites.
View details for DOI 10.1038/nature12577
View details for PubMedID 24107990
- The C-elegans DSB-2 Protein Reveals a Regulatory Network that Controls Competence for Meiotic DSB Formation and Promotes Crossover Assurance PLOS GENETICS 2013; 9 (8)
Meiotic HORMA domain proteins prevent untimely centriole disengagement during Caenorhabditis elegans spermatocyte meiosis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (10): E898-E907
In many species where oocytes lack centrosomes, sperm contribute both genetic material and centriole(s) to the zygote. Correct centriole organization during male meiosis is critical to guarantee a normal bipolar mitotic spindle in the zygote. During Caenorhabditis elegans male meiosis, centrioles normally undergo two rounds of duplication, resulting in haploid sperm each containing a single tightly engaged centriole pair. Here we identify an unanticipated role for C. elegans HORMA (Hop1/Rev7/Mad2) domain proteins HTP-1/2 and HIM-3 in regulating centriole disengagement during spermatocyte meiosis. In him-3 and htp-1 htp-2 mutants, centrioles separate inappropriately during meiosis II, resulting in spermatids with disengaged centrioles. Moreover, extra centrosomes are detected in a subset of zygotes. Together, these data implicate HIM-3 and HTP-1/2 in preventing centriole disengagement during meiosis II. We showed previously that HTP-1/2 prevents premature loss of sister chromatid cohesion during the meiotic divisions by inhibiting removal of meiotic cohesin complexes containing the REC-8 subunit. Worms lacking REC-8, or expressing a mutant separase protein with elevated local concentration at centrosomes and in sperm, likewise exhibit inappropriate centriole separation during spermatocyte meiosis. These observations are consistent with HIM-3 and HTP-1/2 preventing centriole disengagement by inhibiting separase-dependent cohesin removal. Our data suggest that the same specialized meiotic mechanisms that function to prevent premature release of sister chromatid cohesion during meiosis I in C. elegans also function to inhibit centriole separation at meiosis II, thereby ensuring that the zygote inherits the appropriate complement of chromosomes and centrioles.
View details for DOI 10.1073/pnas.1213888110
View details for Web of Science ID 000316377400007
View details for PubMedID 23401519
COSA-1 Reveals Robust Homeostasis and Separable Licensing and Reinforcement Steps Governing Meiotic Crossovers
2012; 149 (1): 75-87
Crossovers (COs) between homologous chromosomes ensure their faithful segregation during meiosis. We identify C. elegans COSA-1, a cyclin-related protein conserved in metazoa, as a key component required to convert meiotic double-strand breaks (DSBs) into COs. During late meiotic prophase, COSA-1 localizes to foci that correspond to the single CO site on each homolog pair and indicate sites of eventual concentration of other conserved CO proteins. Chromosomes gain and lose competence to load CO proteins during meiotic progression, with competence to load COSA-1 requiring prior licensing. Our data further suggest a self-reinforcing mechanism maintaining CO designation. Modeling of a nonlinear dose-response relationship between IR-induced DSBs and COSA-1 foci reveals efficient conversion of DSBs into COs when DSBs are limiting and a robust capacity to limit cytologically differentiated CO sites when DSBs are in excess. COSA-1 foci serve as a unique live cell readout for investigating CO formation and CO interference.
View details for DOI 10.1016/j.cell.2012.01.052
View details for Web of Science ID 000302235400011
View details for PubMedID 22464324
Robust Crossover Assurance and Regulated Interhomolog Access Maintain Meiotic Crossover Number
2011; 334 (6060): 1286-1289
Most organisms rely on interhomolog crossovers (COs) to ensure proper meiotic chromosome segregation but make few COs per chromosome pair. By monitoring repair events at a defined double-strand break (DSB) site during Caenorhabditis elegans meiosis, we reveal mechanisms that ensure formation of the obligate CO while limiting CO number. We find that CO is the preferred DSB repair outcome in the absence of inhibitory effects of other (nascent) recombination events. Thus, a single DSB per chromosome pair is largely sufficient to ensure CO formation. Further, we show that access to the homolog as a repair template is regulated, shutting down simultaneously for both CO and noncrossover (NCO) pathways. We propose that regulation of interhomolog access limits CO number and contributes to CO interference.
View details for DOI 10.1126/science.1212424
View details for Web of Science ID 000297553600053
View details for PubMedID 22144627
- DNA Helicase HIM-6/BLM Both Promotes MutS gamma-Dependent Crossovers and Antagonizes MutS gamma-Independent Interhomolog Associations During Caenorhabditis elegans Meiosis GENETICS 2014; 198 (1): 193-?
- Evidence That Masking of Synapsis Imperfections Counterbalances Quality Control to Promote Efficient Meiosis PLOS GENETICS 2013; 9 (12)
Identification of DSB-1, a Protein Required for Initiation of Meiotic Recombination in Caenorhabditis elegans, Illuminates a Crossover Assurance Checkpoint
2013; 9 (8)
Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.
View details for DOI 10.1371/journal.pgen.1003679
View details for Web of Science ID 000323830300030
View details for PubMedID 23990794
Chromosome movements promoted by the mitochondrial protein SPD-3 are required for homology search during Caenorhabditis elegans meiosis.
2013; 9 (5)
Pairing of homologous chromosomes during early meiosis is essential to prevent the formation of aneuploid gametes. Chromosome pairing includes a step of homology search followed by the stabilization of homolog interactions by the synaptonemal complex (SC). These events coincide with dramatic changes in nuclear organization and rapid chromosome movements that depend on cytoskeletal motors and are mediated by SUN-domain proteins on the nuclear envelope, but how chromosome mobility contributes to the pairing process remains poorly understood. We show that defects in the mitochondria-localizing protein SPD-3 cause a defect in homolog pairing without impairing nuclear reorganization or SC assembly, which results in promiscuous installation of the SC between non-homologous chromosomes. Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis. Pairing center regions localize to SUN-1 aggregates at meiosis onset in spd-3 mutants; and pairing-promoting proteins, including cytoskeletal motors and polo-like kinase 2, are normally recruited to the nuclear envelope. However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing. SUN-1 aggregate movement is also impaired following inhibition of mitochondrial respiration or dynein knockdown, suggesting that mitochondrial function is required for motor-driven SUN-1 movement. The reduced chromosome-end mobility of spd-3 mutants impairs coupling of SC assembly to homology recognition and causes a delay in meiotic progression mediated by HORMA-domain protein HTP-1. Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.
View details for DOI 10.1371/journal.pgen.1003497
View details for PubMedID 23671424
- Assembly of the Synaptonemal Complex Is a Highly Temperature-Sensitive Process That Is Supported by PGL-1 During Caenorhabditis elegans Meiosis G3-GENES GENOMES GENETICS 2013; 3 (4): 585-595
Assembly of the Synaptonemal Complex is a Highly Temperature-Sensitive Process that is Supported by PGL-1 during Caenorhabditis elegans Meiosis.
G3 (Bethesda, Md.)
Successful chromosome segregation during meiosis depends on the synaptonemal complex (SC), a structure that stabilizes pairing between aligned homologous chromosomes. Here we show that SC assembly is a temperature sensitive process during C. elegans meiosis. Temperature sensitivity of SC assembly was initially revealed through identification of the germline-specific P-granule component PGL-1 as a factor promoting stable homolog pairing. Using an assay system that monitors homolog pairing in vivo, we showed that depletion of PGL-1 at 25°C disrupts homolog pairing. Analysis of homolog pairing at other chromosomal loci in a pgl-1 null mutant revealed a pairing defect similar to that observed in mutants lacking SC central region components. Further, loss of pgl-1 function at temperatures ?25° results in severe impairment in loading of SC central region component SYP-1 onto chromosomes, resulting in formation of SYP-1 aggregates. SC assembly is also temperature sensitive in wild-type worms, which exhibit similar SYP-1 loading defects and formation of SYP-1 aggregates at temperatures ?26.5°. Temperature shift analyses suggest that assembly of the SC is temperature sensitive, but maintenance of the SC is not. We suggest that the ts nature of SC assembly may contribute to fitness and adaptation capacity in C. elegans by enabling meiotic disruption in response to environmental change, thereby increasing the production of male progeny available for outcrossing.
View details for PubMedID 23550120
Full-Length Synaptonemal Complex Grows Continuously during Meiotic Prophase in Budding Yeast
2012; 8 (10)
the synaptonemal complex (SC) links two meiotic prophase chromosomal events: homolog pairing and crossover recombination. SC formation involves the multimeric assembly of coiled-coil proteins (Zip1 in budding yeast) at the interface of aligned homologous chromosomes. However, SC assembly is indifferent to homology and thus is normally regulated such that it occurs only subsequent to homology recognition. Assembled SC structurally interfaces with and influences the level and distribution of interhomolog crossover recombination events. Despite its involvement in dynamic chromosome behaviors such as homolog pairing and recombination, the extent to which SC, once installed, acts as an irreversible tether or maintains the capacity to remodel is not clear. Experiments presented here reveal insight into the dynamics of the full-length SC in budding yeast meiotic cells. We demonstrate that Zip1 continually incorporates into previously assembled synaptonemal complex during meiotic prophase. Moreover, post-synapsis Zip1 incorporation is sufficient to rescue the sporulation defect triggered by SCs built with a mutant version of Zip1, Zip1-4LA. Post-synapsis Zip1 incorporation occurs initially with a non-uniform spatial distribution, predominantly associated with Zip3, a component of the synapsis initiation complex that is presumed to mark a subset of crossover sites. A non-uniform dynamic architecture of the SC is observed independently of (i) synapsis initiation components, (ii) the Pch2 and Pph3 proteins that have been linked to Zip1 regulation, and (iii) the presence of a homolog. Finally, the rate of SC assembly and SC central region size increase in proportion to Zip1 copy number; this and other observations suggest that Zip1 does not exit the SC structure to the same extent that it enters. Our observations suggest that, after full-length assembly, SC central region exhibits little global turnover but maintains differential assembly dynamics at sites whose distribution is patterned by a recombination landscape.
View details for DOI 10.1371/journal.pgen.1002993
View details for Web of Science ID 000310528400020
View details for PubMedID 23071451
HAL-2 Promotes Homologous Pairing during Caenorhabditis elegans Meiosis by Antagonizing Inhibitory Effects of Synaptonemal Complex Precursors
2012; 8 (8)
During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification.
View details for DOI 10.1371/journal.pgen.1002880
View details for Web of Science ID 000308529300038
View details for PubMedID 22912597
Chromosome Painting Reveals Asynaptic Full Alignment of Homologs and HIM-8-Dependent Remodeling of X Chromosome Territories during Caenorhabditis elegans Meiosis
2011; 7 (8)
During early meiotic prophase, a nucleus-wide reorganization leads to sorting of chromosomes into homologous pairs and to establishing associations between homologous chromosomes along their entire lengths. Here, we investigate global features of chromosome organization during this process, using a chromosome painting method in whole-mount Caenorhabditis elegans gonads that enables visualization of whole chromosomes along their entire lengths in the context of preserved 3D nuclear architecture. First, we show that neither spatial proximity of premeiotic chromosome territories nor chromosome-specific timing is a major factor driving homolog pairing. Second, we show that synaptonemal complex-independent associations can support full lengthwise juxtaposition of homologous chromosomes. Third, we reveal a prominent elongation of chromosome territories during meiotic prophase that initiates prior to homolog association and alignment. Mutant analysis indicates that chromosome movement mediated by association of chromosome pairing centers (PCs) with mobile patches of the nuclear envelope (NE)-spanning SUN-1/ZYG-12 protein complexes is not the primary driver of territory elongation. Moreover, we identify new roles for the X chromosome PC (X-PC) and X-PC binding protein HIM-8 in promoting elongation of X chromosome territories, separable from their role(s) in mediating local stabilization of pairing and association of X chromosomes with mobile SUN-1/ZYG-12 patches. Further, we present evidence that HIM-8 functions both at and outside of PCs to mediate chromosome territory elongation. These and other data support a model in which synapsis-independent elongation of chromosome territories, driven by PC binding proteins, enables lengthwise juxtaposition of chromosomes, thereby facilitating assessment of their suitability as potential pairing partners.
View details for DOI 10.1371/journal.pgen.1002231
View details for Web of Science ID 000294297000027
View details for PubMedID 21876678
An Asymmetric Chromosome Pair Undergoes Synaptic Adjustment and Crossover Redistribution During Caenorhabditis elegans Meiosis: Implications for Sex Chromosome Evolution
2011; 187 (3): 685-699
Heteromorphic sex chromosomes, such as the X/Y pair in mammals, differ in size and DNA sequence yet function as homologs during meiosis; this bivalent asymmetry presents special challenges for meiotic completion. In Caenorhabditis elegans males carrying mnT12, an X;IV fusion chromosome, mnT12 and IV form an asymmetric bivalent: chromosome IV sequences are capable of pairing and synapsis, while the contiguous X portion of mnT12 lacks a homologous pairing partner. Here, we investigate the meiotic behavior of this asymmetric neo-X/Y chromosome pair in C. elegans. Through immunolocalization of the axis component HIM-3, we demonstrate that the unpaired X axis has a distinct, coiled morphology while synapsed axes are linear and extended. By showing that loci at the fusion-proximal end of IV become unpaired while remaining synapsed as pachytene progresses, we directly demonstrate the occurrence of synaptic adjustment in this organism. We further demonstrate that meiotic crossover distribution is markedly altered in males with the asymmetric mnT12/+ bivalent relative to controls, resulting in greatly reduced crossover formation near the X;IV fusion point and elevated crossovers at the distal end of the bivalent. In effect, the distal end of the bivalent acts as a neo-pseudoautosomal region in these males. We discuss implications of these findings for mechanisms that ensure crossover formation during meiosis. Furthermore, we propose that redistribution of crossovers triggered by bivalent asymmetry may be an important driving force in sex chromosome evolution.
View details for DOI 10.1534/genetics.110.124958
View details for Web of Science ID 000288457800005
View details for PubMedID 21212235
Meiotic Errors Activate Checkpoints that Improve Gamete Quality without Triggering Apoptosis in Male Germ Cells
2010; 20 (23): 2078-2089
Meiotic checkpoints ensure the production of gametes with the correct complement and integrity of DNA; in metazoans, these pathways sense errors and transduce signals to trigger apoptosis to eliminate damaged germ cells. The extent to which checkpoints monitor and safeguard the genome differs between sexes and may contribute to the high frequency of human female meiotic errors. In the C. elegans female germline, DNA damage, chromosome asynapsis, and/or unrepaired meiotic double-strand breaks (DSBs) activate checkpoints that induce apoptosis; conversely, male germ cells do not undergo apoptosis.Here we show that the recombination checkpoint is in fact activated in male germ cells despite the lack of apoptosis. The 9-1-1 complex and the phosphatidylinositol 3-kinase-related protein kinase ATR, sensors of DNA damage, are recruited to chromatin in the presence of unrepaired meiotic DSBs in both female and male germlines. Furthermore, the checkpoint kinase CHK-1 is phosphorylated and the p53 ortholog CEP-1 induces expression of BH3-only proapoptotic proteins in germlines of both sexes under activating conditions. The core cell death machinery is expressed in female and male germlines; however, CED-3 caspase is not activated in the male germline. Although apoptosis is not triggered, checkpoint activation in males has functional consequences for gamete quality, because there is reduced viability of progeny sired by males with a checkpoint-activating defect in the absence of checkpoint function.We propose that the recombination checkpoint functions in male germ cells to promote repair of meiotic recombination intermediates, thereby improving the fidelity of chromosome transmission in the absence of apoptosis.
View details for DOI 10.1016/j.cub.2010.10.008
View details for Web of Science ID 000285213500021
View details for PubMedID 20970339
The Synaptonemal Complex Shapes the Crossover Landscape Through Cooperative Assembly, Crossover Promotion and Crossover Inhibition During Caenorhabditis elegans Meiosis
2010; 186 (1): 45-U101
The synaptonemal complex (SC) is a highly ordered proteinaceous structure that assembles at the interface between aligned homologous chromosomes during meiotic prophase. The SC has been demonstrated to function both in stabilization of homolog pairing and in promoting the formation of interhomolog crossovers (COs). How the SC provides these functions and whether it also plays a role in inhibiting CO formation has been a matter of debate. Here we provide new insight into assembly and function of the SC by investigating the consequences of reducing (but not eliminating) SYP-1, a major structural component of the SC central region, during meiosis in Caenorhabditis elegans. First, we find an increased incidence of double CO (DCO) meiotic products following partial depletion of SYP-1 by RNAi, indicating a role for SYP-1 in mechanisms that normally limit crossovers to one per homolog pair per meiosis. Second, syp-1 RNAi worms exhibit both a strong preference for COs to occur on the left half of the X chromosome and a significant bias for SYP-1 protein to be associated with the left half of the chromosome, implying that the SC functions locally in promoting COs. Distribution of SYP-1 on chromosomes in syp-1 RNAi germ cells provides strong corroboration for cooperative assembly of the SC central region and indicates that SYP-1 preferentially associates with X chromosomes when it is present in limiting quantities. Further, the observed biases in the distribution of both COs and SYP-1 protein support models in which synapsis initiates predominantly in the vicinity of pairing centers (PCs). However, discontinuities in SC structure and clear gaps between localized foci of PC-binding protein HIM-8 and X chromosome-associated SYP-1 stretches allow refinement of models for the role of PCs in promoting synapsis. Our data suggest that the CO landscape is shaped by a combination of three attributes of the SC central region: a CO-promoting activity that functions locally at CO sites, a cooperative assembly process that enables CO formation in regions distant from prominent sites of synapsis initiation, and CO-inhibitory role(s) that limit CO number.
View details for DOI 10.1534/genetics.110.115501
View details for Web of Science ID 000281950600005
View details for PubMedID 20592266
Coordinating cohesion, co-orientation, and congression during meiosis: lessons from holocentric chromosomes
GENES & DEVELOPMENT
2010; 24 (3): 219-228
Organisms that reproduce sexually must reduce their chromosome number by half during meiosis to generate haploid gametes. To achieve this reduction in ploidy, organisms must devise strategies to couple sister chromatids so that they stay together during the first meiotic division (when homologous chromosomes separate) and then segregate away from one another during the second division. Here we review recent findings that shed light on how Caenorhabditis elegans, an organism with holocentric chromosomes, deals with these challenges of meiosis by differentiating distinct chromosomal subdomains and remodeling chromosome structure during prophase. Furthermore, we discuss how features of chromosome organization established during prophase affect later chromosome behavior during the meiotic divisions. Finally, we illustrate how analysis of holocentric meiosis can inform our thinking about mechanisms that operate on monocentric chromosomes.
View details for DOI 10.1101/gad.1863610
View details for Web of Science ID 000274157300001
View details for PubMedID 20123904
Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line
2010; 6 (1)
Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.
View details for DOI 10.1371/journal.pgen.1000830
View details for Web of Science ID 000274194300036
View details for PubMedID 20107519
A Caenorhabditis elegans RNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference
2009; 183 (4): 1297-1314
Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing through formation of RNA duplexes. Although progress has been made in identifying the capabilities of siRNAs in silencing foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a Caenorhabditis elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. In a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role of the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis.
View details for DOI 10.1534/genetics.109.109686
View details for Web of Science ID 000272435000010
View details for PubMedID 19805814
Lateral microtubule bundles promote chromosome alignment during acentrosomal oocyte meiosis
NATURE CELL BIOLOGY
2009; 11 (7): 839-U135
Although centrosomes serve to organize microtubules in most cell types, oocyte spindles form and mediate meiotic chromosome segregation in their absence. Here, we used high-resolution imaging of both bipolar and experimentally generated monopolar spindles in Caenorhabditis elegans to reveal a surprising organization of microtubules and chromosomes within acentrosomal structures. We found that homologous chromosome pairs (bivalents) are surrounded by microtubule bundles running along their sides, whereas microtubule density is extremely low at chromosome ends despite a high concentration of kinetochore proteins at those regions. Furthermore, we found that the chromokinesin KLP-19 (kinesin-like protein 19) is targeted to a ring around the centre of each bivalent and provides a polar ejection force that is required for congression. Together, these observations create a new picture of chromosome-microtubule association in acentrosomal spindles and reveal a mechanism by which metaphase alignment can be achieved using this organization. Specifically, we propose that ensheathment by lateral microtubule bundles places spatial constraints on the chromosomes, thereby promoting biorientation, and that localized motors mediate movement along these bundles, thereby promoting alignment.
View details for DOI 10.1038/ncb1891
View details for Web of Science ID 000267603100012
View details for PubMedID 19525937
Analysis of meiotic recombination in Caenorhabditis elegans.
Methods in molecular biology (Clifton, N.J.)
2009; 557: 77-97
Caenorhabditis elegans is an important experimental organism for the study of recombination during meiosis. A variety of techniques have been developed for the measurement of meiotic recombination in C. elegans, ranging from traditional genetic measures to direct cytological determination of chiasma frequency. Here, we provide methods for some of the varied approaches used for the study of meiotic recombination in these tiny but powerful worms.
View details for DOI 10.1007/978-1-59745-527-5_7
View details for PubMedID 19799178
Ensuring an exit strategy: RTeL1 Restricts Rogue Recombination
2008; 135 (2): 213-215
Success of homologous recombination-based DNA repair depends not only on recombinases, which promote invasion of the homologous DNA duplex that serves as a template for repair, but also on antirecombinases, which dismantle recombination intermediates to allow completion of repair. In this issue, Barber et al. (2008) identify a previously elusive antirecombinase activity important for maintaining genome stability in animals.
View details for DOI 10.1016/j.cell.2008.10.003
View details for Web of Science ID 000260130400011
View details for PubMedID 18957197
Crossovers trigger a remodeling of meiotic chromosome axis composition that is linked to two-step loss of sister chromatid cohesion
GENES & DEVELOPMENT
2008; 22 (20): 2886-2901
Segregation of homologous chromosomes during meiosis depends on linkages (chiasmata) created by crossovers and on selective release of a subset of sister chromatid cohesion at anaphase I. During Caenorhabditis elegans meiosis, each chromosome pair forms a single crossover, and the position of this event determines which chromosomal regions will undergo cohesion release at anaphase I. Here we provide insight into the basis of this coupling by uncovering a large-scale regional change in chromosome axis composition that is triggered by crossovers. We show that axial element components HTP-1 and HTP-2 are removed during late pachytene, in a crossover-dependent manner, from the regions that will later be targeted for anaphase I cohesion release. We demonstrate correspondence in position and number between chiasmata and HTP-1/2-depleted regions and provide evidence that HTP-1/2 depletion boundaries mark crossover sites. In htp-1 mutants, diakinesis bivalents lack normal asymmetrical features, and sister chromatid cohesion is prematurely lost during the meiotic divisions. We conclude that HTP-1 is central to the mechanism linking crossovers with late-prophase bivalent differentiation and defines the domains where cohesion will be protected until meiosis II. Further, we discuss parallels between the pattern of HTP-1/2 removal in response to crossovers and the phenomenon of crossover interference.
View details for DOI 10.1101/gad.1694108
View details for Web of Science ID 000260073200015
View details for PubMedID 18923085
C. elegans germ cells switch between distinct modes of double-strand break repair during meiotic prophase progression
2007; 3 (11): 2068-2084
Chromosome inheritance during sexual reproduction relies on deliberate induction of double-strand DNA breaks (DSBs) and repair of a subset of these breaks as interhomolog crossovers (COs). Here we provide a direct demonstration, based on our analysis of rad-50 mutants, that the meiotic program in Caenorhabditis elegans involves both acquisition and loss of a specialized mode of double-strand break repair (DSBR). In premeiotic germ cells, RAD-50 is not required to load strand-exchange protein RAD-51 at sites of spontaneous or ionizing radiation (IR)-induced DSBs. A specialized meiotic DSBR mode is engaged at the onset of meiotic prophase, coincident with assembly of meiotic chromosome axis structures. This meiotic DSBR mode is characterized both by dependence on RAD-50 for rapid accumulation of RAD-51 at DSB sites and by competence for converting DSBs into interhomolog COs. At the mid-pachytene to late pachytene transition, germ cells undergo an abrupt release from the meiotic DSBR mode, characterized by reversion to RAD-50-independent loading of RAD-51 and loss of competence to convert DSBs into interhomolog COs. This transition in DSBR mode is dependent on MAP kinase-triggered prophase progression and coincides temporally with a major remodeling of chromosome architecture. We propose that at least two developmentally programmed switches in DSBR mode, likely conferred by changes in chromosome architecture, operate in the C. elegans germ line to allow formation of meiotic crossovers without jeopardizing genomic integrity. Our data further suggest that meiotic cohesin component REC-8 may play a role in limiting the activity of SPO-11 in generating meiotic DSBs and that RAD-50 may function in counteracting this inhibition.
View details for DOI 10.1371/journal.pgen.0030191
View details for Web of Science ID 000251310200003
View details for PubMedID 17983271
Differential timing of S phases, X chromosome replication, and meiotic prophase in the C-elegans germ line
2007; 308 (1): 206-221
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.
View details for DOI 10.1016/j.ydbio.2007.05.019
View details for Web of Science ID 000248589700017
View details for PubMedID 17599823
Synapsis-Defective mutants reveal a correlation between chromosome conformation and the mode of double-strand break repair during Caenorhabditis elegans meiosis
2007; 176 (4): 2027-2033
SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspring accompanied by the disappearance of RAD-51 foci suggests that DSBs are being repaired in these synapsis-defective mutants. Our studies indicate that once interhomolog recombination is impaired, both intersister recombination and nonhomologous end-joining pathways may contribute to repair during germline meiosis. Moreover, our studies suggest that the conformation of chromosomes may influence the mode of DSB repair employed during meiosis.
View details for DOI 10.1534/genetics.107.076968
View details for Web of Science ID 000249530000009
View details for PubMedID 17565963
SYP-3 restricts synaptonemal complex assembly to bridge paired. chromosome axes during meiosis in Caenorhabditis elegans
2007; 176 (4): 2015-2025
Synaptonemal complex (SC) formation must be regulated to occur only between aligned pairs of homologous chromosomes, ultimately ensuring proper chromosome segregation in meiosis. Here we identify SYP-3, a coiled-coil protein that is required for assembly of the central region of the SC and for restricting its loading to occur only in an appropriate context, forming structures that bridge the axes of paired meiotic chromosomes in Caenorhabditis elegans. We find that inappropriate loading of central region proteins interferes with homolog pairing, likely by triggering a premature change in chromosome configuration during early prophase that terminates the search for homologs. As a result, syp-3 mutants lack chiasmata and exhibit increased chromosome mis-segregation. Altogether, our studies lead us to propose that SYP-3 regulates synapsis along chromosomes, contributing to meiotic progression in early prophase.
View details for DOI 10.1534/genetics.107.072413
View details for Web of Science ID 000249530000008
View details for PubMedID 17565948
A role for Caenorhabditis elegans chromatin-associated protein HIM-17 in the proliferation vs. meiotic entry decision
2007; 175 (4): 2029-2037
Chromatin-associated protein HIM-17 was previously shown to function in the chromosomal events of meiotic prophase. Here we report an additional role for HIM-17 in regulating the balance between germ cell proliferation and meiotic development. A cryptic function for HIM-17 in promoting meiotic entry and/or inhibiting proliferation was revealed by defects in germline organization in him-17 mutants grown at high temperature (25 degrees) and by a synthetic tumorous germline phenotype in glp-1(ar202); him-17 mutants at 15 degrees.
View details for DOI 10.1534/genetics.107.070987
View details for Web of Science ID 000246448800042
View details for PubMedID 17237503
Regulation of sperm activation by SWM-1 is required for reproductive success of C-elegans males
2006; 16 (3): 252-263
Sexual reproduction in animals requires the production of highly specialized motile sperm cells that can navigate to and fertilize ova. During sperm differentiation, nonmotile spermatids are remodeled into motile spermatozoa through a process known as spermiogenesis. In nematodes, spermiogenesis, or sperm activation, involves a rapid cellular morphogenesis that converts unpolarized round spermatids into polarized amoeboid spermatozoa capable of both motility and fertilization.Here we demonstrate, by genetic analysis and in vivo and in vitro cell-based assays, that the temporal and spatial localization of spermiogenesis are critical determinants of male fertility in C. elegans, a male/hermaphrodite species. We identify swm-1 as a factor important for male but not hermaphrodite fertility. We show that whereas in wild-type males, activation occurs after spermatids are transferred to the hermaphrodite, swm-1 mutants exhibit ectopic activation of sperm within the male reproductive tract. This ectopic activation leads to infertility by impeding sperm transfer. The SWM-1 protein is composed of a signal sequence and two trypsin inhibitor-like domains and likely functions as a secreted serine protease inhibitor that targets two distinct proteases.These findings support a model in which (1) proteolysis acts as an important in vivo trigger for sperm activation and (2) regulating the timing of proteolysis-triggered activation is crucial for male reproductive success. Furthermore, our data provide insight into how a common program of gamete differentiation can be modulated to allow males to participate in reproduction in the context of a male/hermaphrodite species where the capacity for hermaphrodite self-fertilization has rendered them nonessential for progeny production.
View details for DOI 10.1016/j.cub.2005.12.041
View details for Web of Science ID 000235347400025
View details for PubMedID 16461278
Chromosome sites play dual roles to establish homologous synapsis during meiosis in C-elegans
2005; 123 (6): 1037-1050
We have investigated the role of pairing centers (PCs), cis-acting sites required for accurate segregation of homologous chromosomes during meiosis in C. elegans. We find that these sites play two distinct roles that contribute to proper segregation. Chromosomes lacking PCs usually fail to synapse and also lack a synapsis-independent stabilization activity. The presence of a PC on just one copy of a chromosome pair promotes synapsis but does not support synapsis-independent pairing stabilization, indicating that these functions are separable. Once initiated, synapsis is highly processive, even between nonhomologous chromosomes of disparate lengths, elucidating how translocations suppress meiotic recombination in C. elegans. These findings suggest a multistep pathway for chromosome synapsis in which PCs impart selectivity and efficiency through a "kinetic proofreading" mechanism. We speculate that concentration of these activities at one region per chromosome may have coevolved with the loss of a point centromere to safeguard karyotype stability.
View details for DOI 10.1016/j.cell.2005.09.034
View details for Web of Science ID 000234177200013
View details for PubMedID 16360034
HTP-1-dependent constraints coordinate homolog pairing and synapsis and promote chiasma formation during C-elegans meiosis
GENES & DEVELOPMENT
2005; 19 (22): 2727-2743
Synaptonemal complex (SC) assembly must occur between correctly paired homologous chromosomes to promote formation of chiasmata. Here, we identify the Caenorhabditis elegans HORMA-domain protein HTP-1 as a key player in coordinating establishment of homolog pairing and synapsis in C. elegans and provide evidence that checkpoint-like mechanisms couple these early meiotic prophase events. htp-1 mutants are defective in the establishment of pairing, but in contrast with the pairing-defective chk-2 mutant, SC assembly is not inhibited and generalized nonhomologous synapsis occurs. Extensive nonhomologous synapsis in htp-1; chk-2 double mutants indicates that HTP-1 is required for the inhibition of SC assembly observed in chk-2 gonads. htp-1 mutants show a decreased abundance of nuclei exhibiting a polarized organization that normally accompanies establishment of pairing; analysis of htp-1; syp-2 double mutants suggests that HTP-1 is needed to prevent premature exit from this polarized nuclear organization and that this exit stops homology search. Further, based on experiments monitoring the formation of recombination intermediates and crossover products, we suggest that htp-1 mutants are defective in preventing the use of sister chromatids as recombination partners. We propose a model in which HTP-1 functions to establish or maintain multiple constraints that operate to ensure coordination of events leading to chiasma formation.
View details for DOI 10.1101/gad.1338505
View details for Web of Science ID 000233366600008
View details for PubMedID 16291646
Crossing over is coupled to late meiotic prophase bivalent differentiation through asymmetric disassembly of the SC
JOURNAL OF CELL BIOLOGY
2005; 168 (5): 683-689
Homologous chromosome pairs (bivalents) undergo restructuring during meiotic prophase to convert a configuration that promotes crossover recombination into one that promotes bipolar spindle attachment and localized cohesion loss. We have imaged remodeling of meiotic chromosome structures after pachytene exit in Caenorhabditis elegans. Chromosome shortening during diplonema is accompanied by coiling of chromosome axes and highly asymmetric departure of synaptonemal complex (SC) central region proteins SYP-1 and SYP-2, which diminish over most of the length of each desynapsing bivalent while becoming concentrated on axis segments distal to the single emerging chiasma. This and other manifestations of asymmetry along chromosomes are lost in synapsis-proficient crossover-defective mutants, which often retain SYP-1,2 along the full lengths of coiled diplotene axes. Moreover, a gamma-irradiation treatment that restores crossovers in the spo-11 mutant also restores asymmetry of SYP-1 localization. We propose that crossovers or crossover precursors serve as symmetry-breaking events that promote differentiation of subregions of the bivalent by triggering asymmetric disassembly of the SC.
View details for DOI 10.1083/jcb.200410144
View details for Web of Science ID 000227461800005
View details for PubMedID 15738262
Chromosome-wide regulation of meiotic crossover formation in Caenorhabditis elegans requires properly assembled chromosome axes
2004; 168 (3): 1275-1292
Most sexually reproducing organisms depend on the regulated formation of crossovers, and the consequent chiasmata, to accomplish successful segregation of homologous chromosomes at the meiosis I division. A robust, chromosome-wide crossover control system limits chromosome pairs to one crossover in most meioses in the nematode Caenorhabditis elegans; this system has been proposed to rely on structural integrity of meiotic chromosome axes. Here, we test this hypothesis using a mutant, him-3(me80), that assembles reduced levels of meiosis-specific axis component HIM-3 along cohesin-containing chromosome axes. Whereas pairing, synapsis, and crossing over are eliminated when HIM-3 is absent, the him-3(me80) mutant supports assembly of synaptonemal complex protein SYP-1 along some paired chromosomes, resulting in partial competence for chiasma formation. We present both genetic and cytological evidence indicating that the him-3(me80) mutation leads to an increased incidence of meiotic products with two crossovers. These results indicate that limiting the amount of a major axis component results in a reduced capacity to communicate the presence of a (nascent) crossover and/or to discourage others in response.
View details for Web of Science ID 000225767400015
View details for PubMedID 15579685
C-elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination
2004; 118 (4): 439-452
Initiation of meiotic recombination by double-strand breaks (DSBs) must occur in a controlled fashion to avoid jeopardizing genome integrity. Here, we identify chromatin-associated protein HIM-17 as a link between chromatin state and DSB formation during C. elegans meiosis. Dependencies of several meiotic prophase events on HIM-17 parallel those seen for DSB-generating enzyme SPO-11: HIM-17 is essential for DSB formation but dispensable for homolog synapsis. Crossovers and chiasmata are eliminated in him-17 null mutants but are restored by artificially induced DSBs, indicating that all components required to convert DSBs into chiasmata are present. Unlike SPO-11, HIM-17 is also required for proper accumulation of histone H3 methylation at lysine 9 on meiotic prophase chromosomes. HIM-17 shares structural features with three proteins that interact genetically with LIN-35/Rb, a known component of chromatin-modifying complexes. Furthermore, DSB levels and incidence of chiasmata can be modulated by loss of LIN-35/Rb. These and other data suggest that chromatin state governs the timing of DSB competence.
View details for Web of Science ID 000223650900007
View details for PubMedID 15315757
A component of C-elegans meiotic chromosome axes at the interface of homolog alignment, synapsis, nuclear reorganization, and recombination
2004; 14 (7): 585-592
A universal feature of meiotic prophase is the pairing of homologous chromosomes, a fundamental prerequisite for the successful completion of all subsequent meiotic events. HIM-3 is a Caenorhabditis elegans meiosis-specific non-cohesin component of chromosome axes that is required for synapsis. Our characterization of new him-3 alleles reveals previously unknown functions for the protein. HIM-3 is required for the establishment of initial contacts between homologs, for the nuclear reorganization characteristic of early meiotic prophase, and for the coordination of these events with synaptonemal complex (SC) assembly. Despite the absence of homolog alignment, we find that recombination is initiated efficiently, indicating that initial pairing is not a prerequisite for early steps of the recombination pathway. Surprisingly, RAD-51-marked recombination intermediates disappear with apparent wild-type kinetics in him-3 null mutants in which homologs are spatially unavailable for recombination, raising the possibility that HIM-3's presence at chromosome axes inhibits the use of sister chromatids as templates for repair. We propose that HIM-3 is a molecular link between multiple landmark events of meiotic prophase; it is critical for establishing chromosome identity by configuring homologs to facilitate their recognition while simultaneously imposing structural constraints that later promote the formation of the crossover essential for proper segregation.
View details for DOI 10.1016/j.cub.2004.03.033
View details for Web of Science ID 000220809900024
View details for PubMedID 15062099
Methods for analyzing checkpoint responses in Caenorhabditis elegans.
Methods in molecular biology (Clifton, N.J.)
2004; 280: 257-274
In response to genotoxic insults, cells activate DNA damage checkpoint pathways that stimulate DNA repair, lead to a transient cell cycle arrest, and/or elicit programmed cell death (apoptosis) of affected cells. The Caenorhabditis elegans germ line was recently established as a model system to study these processes in a genetically tractable, multicellular organism. The utility of this system was revealed by the finding that upon treatment with genotoxic agents, premeiotic C. elegans germ cells transiently halt cell cycle progression, whereas meiotic prophase germ cells in the late pachytene stage readily undergo apoptosis. Further, accumulation of unrepaired meiotic recombination intermediates can also lead to the apoptotic demise of affected pachytene cells. DNA damage-induced cell death requires key components of the evolutionarily conserved apoptosis machinery. Moreover, both cell cycle arrest and pachytene apoptosis responses depend on conserved DNA damage checkpoint proteins. Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways. In this chapter, we will briefly review the C. elegans DNA damage-response field, and we will discuss in detail the methods that are being used to assay DNA damage responses in C. elegans.
View details for PubMedID 15187259
Chromosome-wide control of meiotic crossing over in C-elegans
2003; 13 (18): 1641-1647
A central event in sexual reproduction is the reduction in chromosome number that occurs at the meiosis I division. Most eukaryotes rely on crossing over between homologs, and the resulting chiasmata, to direct meiosis I chromosome segregation, yet make very few crossovers per chromosome pair. This indicates that meiotic recombination must be tightly regulated to ensure that each chromosome pair enjoys the crossover necessary to ensure correct segregation. Here, we investigate control of meiotic crossing over in Caenorhabditis elegans, which averages only one crossover per chromosome pair per meiosis, by constructing genetic maps of end-to-end fusions of whole chromosomes. Fusion of chromosomes removes the requirement for a crossover in each component chromosome segment and thereby reveals a propensity to restrict the number of crossovers such that pairs of fusion chromosomes composed of two or even three whole chromosomes enjoy but a single crossover in the majority of meioses. This regulation can operate over physical distances encompassing half the genome. The meiotic behavior of heterozygous fusion chromosomes further suggests that continuous meiotic chromosome axes, or structures that depend on properly assembled axes, may be important for crossover regulation.
View details for DOI 10.1016/j.cub.2003.08.026
View details for Web of Science ID 000185404800026
View details for PubMedID 13678597
Synaptonemal complex assembly in C-elegans is dispensable for loading strand-exchange proteins but critical for proper completion of recombination
2003; 5 (3): 463-474
Here we probe the relationships between assembly of the synaptonemal complex (SC) and progression of recombination between homologous chromosomes during Caenorhabditis elegans meiosis. We identify SYP-2 as a structural component of the SC central region and show that central region assembly depends on proper morphogenesis of chromosome axes. We find that the SC central region is dispensable for initiation of recombination and for loading of DNA strand-exchange protein RAD-51, despite the fact that extensive RAD-51 loading normally occurs in the context of assembled SC. Further, persistence of RAD-51 foci and absence of crossover products in meiotic mutants suggests that SC central region components and recombination proteins MSH-4 and MSH-5 are required to promote conversion of resected double-strand breaks into stable post-strand exchange intermediates. Our data also suggest that early prophase barriers to utilization of sister chromatids as repair templates do not depend on central region assembly.
View details for Web of Science ID 000185309600014
View details for PubMedID 12967565
A gene recommender algorithm to identify coexpressed genes in C-elegans
2003; 13 (8): 1828-1837
One of the most important uses of whole-genome expression data is for the discovery of new genes with similar function to a given list of genes (the query) already known to have closely related function. We have developed an algorithm, called the gene recommender, that ranks genes according to how strongly they correlate with a set of query genes in those experiments for which the query genes are most strongly coregulated. We used the gene recommender to find other genes coexpressed with several sets of query genes, including genes known to function in the retinoblastoma complex. Genetic experiments confirmed that one gene (JC8.6) identified by the gene recommender acts with lin-35 Rb to regulate vulval cell fates, and that another gene (wrm-1) acts antagonistically. We find that the gene recommender returns lists of genes with better precision, for fixed levels of recall, than lists generated using the C. elegans expression topomap.
View details for DOI 10.1101/gr.1125403
View details for Web of Science ID 000184530900005
View details for PubMedID 12902378
Synapsis-dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C-elegans
GENES & DEVELOPMENT
2002; 16 (18): 2428-2442
Analysis of Caenorhabditis elegans syp-1 mutants reveals that both synapsis-dependent and -independent mechanisms contribute to stable, productive alignment of homologous chromosomes during meiotic prophase. Early prophase nuclei undergo normal reorganization in syp-1 mutants, and chromosomes initially pair. However, the polarized nuclear organization characteristic of early prophase persists for a prolonged period, and homologs dissociate prematurely; furthermore, the synaptonemal complex (SC) is absent. The predicted structure of SYP-1, its localization at the interface between intimately paired, lengthwise-aligned pachytene homologs, and its kinetics of localization with chromosomes indicate that SYP-1 is an SC structural component. A severe reduction in crossing over together with evidence for accumulated recombination intermediates in syp-1 mutants indicate that initial pairing is not sufficient for completion of exchange and implicates the SC in promoting crossover recombination. Persistence of polarized nuclear organization in syp-1 mutants suggests that SC polymerization may provide a motive force or signal that drives redispersal of chromosomes. Whereas our analysis suggests that the SC is required to stabilize pairing along the entire lengths of chromosomes, striking differences in peak pairing levels for opposite ends of chromosomes in syp-1 mutants reveal the existence of an additional mechanism that can promote local stabilization of pairing, independent of synapsis.
View details for DOI 10.1101/gad.1011602
View details for Web of Science ID 000178129600013
View details for PubMedID 12231631
A targeted RNAi screen for genes involved in chromosome morphogenesis and nuclear organization in the Caenorhabditis elegans germline
2002; 162 (1): 113-128
We have implemented a functional genomics strategy to identify genes involved in chromosome morphogenesis and nuclear organization during meiotic prophase in the Caenorhabditis elegans germline. This approach took advantage of a gene-expression survey that used DNA microarray technology to identify genes preferentially expressed in the germline. We defined a subset of 192 germline-enriched genes whose expression profiles were similar to those of previously identified meiosis genes and designed a screen to identify genes for which inhibition by RNA interference (RNAi) elicited defects in function or development of the germline. We obtained strong germline phenotypes for 27% of the genes tested, indicating that this targeted approach greatly enriched for genes that function in the germline. In addition to genes involved in key meiotic prophase events, we identified genes involved in meiotic progression, germline proliferation, and chromosome organization and/or segregation during mitotic growth.
View details for Web of Science ID 000178363400010
View details for PubMedID 12242227
2001; 106 (6): 647-650
Sexual reproduction predominates among eukaryotic organisms on our planet. While debate continues over why this should be so, burgeoning genomic and functional information now allows us to begin to think reasonably about some of the events that may have occurred to make sex possible in the first place.
View details for Web of Science ID 000171213000001
View details for PubMedID 11572770
Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C-elegans chk-2
GENES & DEVELOPMENT
2001; 15 (13): 1674-1687
Analysis of mutants defective in meiotic chromosome pairing has uncovered a role for Caenorhabditis elegans chk-2 in initial establishment of pairing between homologous chromosomes during early meiotic prophase. chk-2 is also required for the major spatial reorganization of nuclei that normally accompanies the onset of pairing, suggesting a mechanistic coupling of these two events. Despite failures in pairing, nuclear reorganization, and crossover recombination, chk-2 mutants undergo many other aspects of meiotic chromosome morphogenesis and complete gametogenesis. Although chk-2 encodes a C. elegans ortholog of the Cds1/Chk2 checkpoint protein kinases, germ-line nuclei in chk-2 mutants are competent to arrest proliferation in response to replication inhibition and to trigger DNA damage checkpoint responses to ionizing radiation. However, chk-2 mutants are defective in triggering the pachytene DNA damage checkpoint in response to an intermediate block in the meiotic recombination pathway, suggesting that chk-2 is required either for initiation of meiotic recombination or for monitoring a specific subset of DNA damage lesions. We propose that chk-2 functions during premeiotic S phase to enable chromosomes to become competent for subsequent meiotic prophase events and/or to coordinate replication with entry into prophase.
View details for Web of Science ID 000169824600009
View details for PubMedID 11445542
- Development - How to stimulate your partner SCIENCE 2001; 291 (5511): 2099-?
C-elegans mre-11 is required for meiotic recombination and DNA repair but is dispensable for the meiotic G(2) DNA damage checkpoint
GENES & DEVELOPMENT
2001; 15 (5): 522-534
We investigated the roles of Caenorhabditis elegans MRE-11 in multiple cellular processes required to maintain genome integrity. Although yeast Mre11 is known to promote genome stability through several diverse pathways, inviability of vertebrate cells that lack Mre11 has hindered elucidation of the in vivo roles of this conserved protein in metazoan biology. Worms homozygous for an mre-11 null mutation are viable, allowing us to demonstrate in vivo requirements for MRE-11 in meiotic recombination and DNA repair. In mre-11 mutants, meiotic crossovers are not detected, and oocyte chromosomes lack chiasmata but appear otherwise intact. gamma-irradiation of mre-11 mutant germ cells during meiotic prophase eliminates progeny survivorship and induces chromosome fragmentation and other cytologically visible abnormalities, indicating a defect in repair of radiation-induced chromosome damage. Whereas mre-11 mutant germ cells are repair-deficient, they retain function of the meiotic G(2) DNA damage checkpoint that triggers germ cell apoptosis in response to ionizing radiation. Although mre-11/mre-11 animals derived from heterozygous parents are viable and produce many embryos, there is a marked drop both in the number and survivorship of embryos produced by succeeding generations. This progressive loss of fecundity and viability indicates that MRE-11 performs a function essential for maintaining reproductive capacity in the species.
View details for Web of Science ID 000167419000004
View details for PubMedID 11238374
Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis
2000; 156 (2): 617-630
Crossing over and chiasma formation during Caenorhabditis elegans meiosis require msh-5, which encodes a conserved germline-specific MutS family member. msh-5 mutant oocytes lack chiasmata between homologous chromosomes, and crossover frequencies are severely reduced in both oocyte and spermatocyte meiosis. Artificially induced DNA breaks do not bypass the requirement for msh-5, suggesting that msh-5 functions after the initiation step of meiotic recombination. msh-5 mutants are apparently competent to repair breaks induced during meiosis, but accomplish repair in a way that does not lead to crossovers between homologs. These results combine with data from budding yeast to establish a conserved role for Msh5 proteins in promoting the crossover outcome of meiotic recombination events. Apart from the crossover deficit, progression through meiotic prophase is largely unperturbed in msh-5 mutants. Homologous chromosomes are fully aligned at the pachytene stage, and germ cells survive to complete meiosis and gametogenesis with high efficiency. Our demonstration that artificially induced breaks generate crossovers and chiasmata using the normal meiotic recombination machinery suggests (1) that association of breaks with a preinitiation complex is not a prerequisite for entering the meiotic recombination pathway and (2) that the decision for a subset of recombination events to become crossovers is made after the initiation step.
View details for Web of Science ID 000089766800014
View details for PubMedID 11014811
Transgene-mediated cosuppression in the C-elegans germ line
GENES & DEVELOPMENT
2000; 14 (13): 1578-1583
Functional silencing of chromosomal loci can be induced by transgenes (cosuppression) or by introduction of double-stranded RNA (RNAi). Here, we demonstrate the generality of and define rules for a transgene-mediated cosuppression phenomenon in the Caenorhabditis elegans germ line. Functional repression is not a consequence of persistent physical association between transgenes and endogenous genes or of mutations in affected genes. The cosuppression mechanism likely involves an RNA mediator that defines its target specificity, reminiscent of RNAi. Cosuppression is strongly abrogated in rde-2 and mut-7 mutants, but is not blocked in an rde-1 mutant, indicating that cosuppression and RNAi have overlapping but distinct genetic requirements.
View details for Web of Science ID 000088146000002
View details for PubMedID 10887151
Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast
1999; 153 (3): 1271-1283
Formation of crossovers between homologous chromosomes during Caenorhabditis elegans meiosis requires the him-14 gene. Loss of him-14 function severely reduces crossing over, resulting in lack of chiasmata between homologs and consequent missegregation. Cytological analysis showing that homologs are paired and aligned in him-14 pachytene nuclei, together with temperature-shift experiments showing that him-14 functions during the pachytene stage, indicate that him-14 is not needed to establish pairing or synapsis and likely has a more direct role in crossover formation. him-14 encodes a germline-specific member of the MutS family of DNA mismatch repair (MMR) proteins. him-14 has no apparent role in MMR, but like its Saccharomyces cerevisiae ortholog MSH4, has a specialized role in promoting crossing over during meiosis. Despite this conservation, worms and yeast differ significantly in their reliance on this pathway: whereas worms use this pathway to generate most, if not all, crossovers, yeast still form 30-50% of their normal number of crossovers when this pathway is absent. This differential reliance may reflect differential stability of crossover-competent recombination intermediates, or alternatively, the presence of two different pathways for crossover formation in yeast, only one of which predominates during nematode meiosis. We discuss a model in which HIM-14 promotes crossing over by interfering with Holliday junction branch migration.
View details for Web of Science ID 000083539500017
View details for PubMedID 10545458
Meiotic recombination in C-elegans initiates by a conserved mechanism and is dispensable for homologous chromosome synapsis
1998; 94 (3): 387-398
Chromosome segregation at meiosis I depends on pairing and crossing-over between homologs. In most eukaryotes, pairing culminates with formation of the proteinaceous synaptonemal complex (SC). In budding yeast, recombination initiates through double-strand DNA breaks (DSBs) and is thought to be essential for SC formation. Here, we examine whether this mechanism for initiating meiotic recombination is conserved, and we test the dependence of homologous chromosome synapsis on recombination in C. elegans. We find that a homolog of the yeast DSB-generating enzyme, Spo11p, is required for meiotic exchange in this metazoan, and that radiation-induced breaks partially alleviate this dependence. Thus, initiation of recombination by DSBs is apparently conserved. However, homologous synapsis is independent of recombination in the nematode, since it occurs normally in a C. elegans spo-11 null mutant.
View details for Web of Science ID 000075308400014
View details for PubMedID 9708740
- ?Chromosome Organization, Meiosis and Mitosis?. in C. elegans, II. (ed. D. Riddle, B. Meyer, Blumenthal, T. and Priess, J.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1997
Telomeric repeats (TTAGGC)(n) are sufficient for chromosome capping function in Caenorhabditis elegans
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1996; 93 (17): 8983-8988
Telomeres are specialized structures located at the ends of linear eukaryotic chromosomes that ensure their complete replication and protect them from fusion and degradation. We report here the characterization of the telomeres of the nematode Caenorhabditis elegans. We show that the chromosomes terminate in 4-9 kb of tandem repeats of the sequence TTAGGC. Furthermore, we have isolated clones corresponding to 11 of the 12 C. elegans telomeres. Their subtelomeric sequences are all different from each other, demonstrating that the terminal TTAGGC repeats are sufficient for general chromosomal capping functions. Finally, we demonstrate that the me8 meiotic mutant, which is defective in X chromosome crossing over and segregation, bears a terminal deficiency, that was healed by the addition of telomeric repeats, presumably by the activity of a telomerase enzyme. The 11 cloned telomeres represent an important advance for the completion of the physical map and for the determination of the entire sequence of the C. elegans genome.
View details for Web of Science ID A1996VD43400037
View details for PubMedID 8799140
CIS-ACTING LOCUS THAT PROMOTES CROSSING-OVER BETWEEN X-CHROMOSOMES IN CAENORHABDITIS-ELEGANS
1994; 136 (3): 887-902
This study reports the characterization of a cis-acting locus on the Caenorhabditis elegans X chromosome that is crucial for promoting normal levels of crossing over specifically between the X homologs and for ensuring their proper disjunction at meiosis I. The function of this locus is disrupted by the mutation me8, which maps to the extreme left end of the X chromosome within the region previously implicated by studies of X; A translocations and X duplications to contain a meiotic pairing site. Hermaphrodites homozygous for a deletion of the locus (Df/Df) or heterozygous for a deletion and the me8 mutation (me8/Df) exhibit extremely high level of X chromosome nondisjunction at the reductional division; this is correlated with a sharp decrease in crossing over between the X homologs as evidenced both by reductions in genetic map distances and by the presence of achiasmate chromosomes in cytological preparations of oocyte nuclei. Duplications of the wild-type region that are unlinked to the X chromosome cannot complement the recombination and disjunction defects in trans, indicating that this region must be present in cis to the X chromosome to ensure normal levels of crossing over and proper homolog disjunction. me8 homozygotes exhibit an altered distribution of crossovers along the X chromosome that suggests a defect in processivity along the X chromosome of an event that initiates at the chromosome end. Models are discussed in which the cis-acting locus deleted by the Dfs functions as a meiotic pairing center that recruits trans-acting factors onto the chromosomes to nucleate assembly of a crossover-competent complex between the X homologs. This pairing center might function in the process of homolog recognition, or in the initiation of homologous synapsis.
View details for Web of Science ID A1994MZ47400018
View details for PubMedID 8005443