ZTF-8 Interacts with the 9-1-1 Complex and Is Required for DNA Damage Response and Double-Strand Break Repair in the C. elegans Germline

Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline

Crossover recombination mediated by HIM-18/SLX4-associated nucleases

Meiosis is a specialized cell division program that results in the formation of haploid gametes (i.e., sperm and eggs) from diploid parental cells, and is essential for all sexually reproducing organisms. Crossover formation, the reciprocal exchange of genetic information during recombination, is critical for accurate meiotic chromosome segregation. Misregulation of crossover formation leads to genomic instability and aneuploidy (cells with the incorrect number of chromosomes), resulting in tumorigenesis, birth defects, miscarriages, and infertility in humans. Recently, a shuriken/Swiss army knife-like multi-nuclease complex has been implicated in processing various types of DNA repair intermediates. However, how these nucleases coordinate their functions during repair remained unclear. Our studies in C. elegans revealed genetic redundancies between these nucleases for meiotic crossover formation and that they promote distinct crossover control at different chromosome regions. Specifically, XPF-1 acts redundantly with both MUS-81 and SLX-1 to resolve Holliday junction recombination intermediates into crossover products at designated future crossover sites on chromosome arms. In contrast, SLX-1 is required for suppression of crossovers at the center region of chromosomes. Altogether, our studies have shed light on the interplay between structure-specific endonucleases and uncovered their ability to exert either positive or negative meiotic crossover control on a chromosome region-specific basis

Crossing over is coupled to late meiotic prophase bivalent differentiation through asymmetric disassembly of the SC

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 γ-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

A C. elegans Screening Platform for the Rapid Assessment of Chemical Disruption of Germline Function

Background: Despite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals. Objectives: We developed an assay in Caenorhabditis elegans that fluorescently marks aneuploid embryos after chemical exposure. Methods: We qualified the predictive value of the assay against chemotherapeutic agents as well as environmental compounds from the ToxCast Phase I library by comparing results from the C. elegans assay with the comprehensive mammalian in vivo end point data from the ToxRef database. Results: The assay was highly predictive of mammalian reproductive toxicities, with a 69% maximum balanced accuracy. We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression. Conclusions: This C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function

SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4–dependent endonuclease activity toward replication forks, 5′-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs

Interplay between Structure-Specific Endonucleases for Crossover Control during Caenorhabditis elegans Meiosis

The number and distribution of crossover events are tightly regulated at prophase of meiosis I. The resolution of Holliday junctions by structure-specific endonucleases, including MUS-81, SLX-1, XPF-1 and GEN-1, is one of the main mechanisms proposed for crossover formation. However, how these nucleases coordinately resolve Holliday junctions is still unclear. Here we identify both the functional overlap and differences between these four nucleases regarding their roles in crossover formation and control in the Caenorhabditis elegans germline. We show that MUS-81, XPF-1 and SLX-1, but not GEN-1, can bind to HIM-18/SLX4, a key scaffold for nucleases. Analysis of synthetic mitotic defects revealed that MUS-81 and SLX-1, but not XPF-1 and GEN-1, have overlapping roles with the Bloom syndrome helicase ortholog, HIM-6, supporting their in vivo roles in processing recombination intermediates. Taking advantage of the ease of genetic analysis and high-resolution imaging afforded by C. elegans, we examined crossover designation, frequency, distribution and chromosomal morphology in single, double, triple and quadruple mutants of the structure-specific endonucleases. This revealed that XPF-1 functions redundantly with MUS-81 and SLX-1 in executing crossover formation during meiotic double-strand break repair. Analysis of crossover distribution revealed that SLX-1 is required for crossover suppression at the center region of the autosomes. Finally, analysis of chromosome morphology in oocytes at late meiosis I stages uncovered that SLX-1 and XPF-1 promote meiotic chromosomal stability by preventing formation of chromosomal abnormalities. We propose a model in which coordinate action between structure-specific nucleases at different chromosome domains, namely MUS-81, SLX-1 and XPF-1 at the arms and SLX-1 at the center region, exerts positive and negative regulatory roles, respectively, for crossover control during C. elegans meiosis

Heritable genome editing in C. elegans via a CRISPR-Cas9 system

CRISPR-Cas systems have been used with single-guide RNAs for accurate gene disruption and conversion in multiple biological systems. Here we report the use of the endonuclease Cas9 to target genomic sequences in the C. elegans germline, utilizing single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants

A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes

The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. Gcna gene expression is enriched in reproductive cells across eukarya – either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells of diverse multicellular animals. Studies of Gcna-mutant C. elegans and mice indicate that GCNA has functioned in reproduction for at least 600 million years. Homology to IDR-containing proteins implicated in DNA damage repair suggests that GCNA proteins may protect the genomic integrity of cells carrying a heritable genome.Life Sciences Research FoundationHoward Hughes Medical Institut

Leptotene/Zygotene Chromosome Movement Via the SUN/KASH Protein Bridge in Caenorhabditis elegans

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2–dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates