Meiotic recombination and the crossover assurance checkpoint in Caenorhabditis elegans

Abstract

During meiotic prophase, chromosomes pair and synapse with their homologs and undergo programmed DNA double-strand break (DSB) formation to initiate meiotic recombination. These DSBs are processed to generate a limited number of crossover recombination products on each chromosome, which are essential to ensure faithful segregation of homologous chromosomes. The nematode Caenorhabditis elegans has served as an excellent model organism to investigate the mechanisms that drive and coordinate these chromosome dynamics during meiosis. Here we focus on our current understanding of the regulation of DSB induction in C. elegans. We also review evidence that feedback regulation of crossover formation prolongs the early stages of meiotic prophase, and discuss evidence that this can alter the recombination pattern, most likely by shifting the genome-wide distribution of DSBs.

Keywords: Cell cycle; Chromatin; Chromosome structure; Double-strand breaks; Feedback control; Meiosis; Recombination.

Figure 1. Programmed double-strand break formation during meiosis in C. elegans

A) Schematic of C. elegans germline anatomy and chromosome configurations during early meiotic prophase. Meiotic recombination initiates via programmed DSB formation during the leptotene/zygotene stages (transition zone), and crossover formation is completed during pachytene. A single recombination intermediate is designated to become a crossover, and excess breaks are repaired through non-crossover pathways. B) The distribution of meiotic DSBs, as determined by RAD-51 ChIP-seq, mirrors the distribution of crossover frequencies along the C. elegans chromosomes, except in subtelomeric regions, which show DSB enrichment but few or no crossovers. DSBs, like crossovers, are enriched on the chromosome arms relative to the centers. Locally they are associated with actively transcribed genes, and are negatively correlated with H3K9 di- and tri-methylation, although these marks are enriched on the arms. A newly discovered mark, trimethylated H3K23, may also antagonize DSBs.

Figure 2. Chromosome structure and recombination initiation machinery in C. elegans

Upon meiotic entry, chromosomes are reorganized around a central axis comprised of cohesins and meiosis-specific HORMA proteins. The formation of DSBs depends on this remodeling. The interplay between cohesins and condensins is thought to control axis length. Several C. elegans proteins, including HIM-17, XND-1, HIM-5, REC-1, DSB-1, and DSB-2, promote breaks or affect the distribution of breaks. ChIP-seq evidence, as well as mutant analysis, suggests that HIM-17 and XND-1 regulate the expression of downstream genes. HIM-5 is also associated with the autosomes and depleted on the transcriptionally-inactive X chromosome. DSB-1 and DSB-2 bind to meiotic chromosomes during the time of DSB induction and may contribute to recruiting and/or activating SPO-11. SPO-11-DNA adducts are subsequently removed by nucleolytic activity. This requires the MRE11-RAD-50 complex, which also is required for SPO-11-dependent DSB formation. The formation of DSBs also depends on the meiosis-specific CHK-2 kinase and is likely negatively regulated by the DNA damage kinases ATR (ATL-1) and/or ATM (ATM-1).

Figure 3. Feedback regulation of meiotic progression through CHK-2 impacts the timing and distribution of DSBs

A) CHK-2 acts as a master regulator of meiotic prophase. Its kinase activity is required for DSBs and also for homolog pairing and synapsis. CHK-2 activity normally declines during early pachytene, once all chromosomes synapse. If crossover intermediates fail to be established on all chromosomes, CHK-2 activity is prolonged. HORMA domain proteins at the chromosome axis are critical for this crossover assurance mechanism, but it is unknown how they sense or transduce crossover failures to prolong CHK-2 activity. When the checkpoint is activated, DSB-1 and DSB-2 persist along meiotic chromosomes, and (if breaks are made) the number of RAD-51 foci typically rises to higher levels than seen in wild-type animals. Biased crossover formation on chromosome arms is greatly reduced under these conditions, suggesting that the central chromosome regions experience more DSBs. B) When crossovers cannot be generated on all chromosomes, an extended zone of RAD-51 foci is observed, and more foci are detected per nucleus. Here, two gonad arms immunostained for RAD-51 are shown, one from a wild-type hermaphrodite (top) and one from a rad-54 mutant (bottom). Insets below each gonad show higher-magnification views of the boxed regions. In wild-type gonads RAD-51 foci peak in number at early pachytene and disappear by late pachytene, while in rad-54 mutants they progressively increase in number until nuclei reach the most proximal region of the gonad, and disappear as the chromosomes condense at diakinesis. These foci can be difficult to resolve and count reliably at such high densities, but frequently exceed 50 per nucleus. Other mutants with crossover defects have similar but less extreme effects than rad-54 mutants, which are defective in removal of RAD-51 from recombination intermediates.

Figure 3. Feedback regulation of meiotic progression through CHK-2 impacts the timing and distribution of DSBs

A) CHK-2 acts as a master regulator of meiotic prophase. Its kinase activity is required for DSBs and also for homolog pairing and synapsis. CHK-2 activity normally declines during early pachytene, once all chromosomes synapse. If crossover intermediates fail to be established on all chromosomes, CHK-2 activity is prolonged. HORMA domain proteins at the chromosome axis are critical for this crossover assurance mechanism, but it is unknown how they sense or transduce crossover failures to prolong CHK-2 activity. When the checkpoint is activated, DSB-1 and DSB-2 persist along meiotic chromosomes, and (if breaks are made) the number of RAD-51 foci typically rises to higher levels than seen in wild-type animals. Biased crossover formation on chromosome arms is greatly reduced under these conditions, suggesting that the central chromosome regions experience more DSBs. B) When crossovers cannot be generated on all chromosomes, an extended zone of RAD-51 foci is observed, and more foci are detected per nucleus. Here, two gonad arms immunostained for RAD-51 are shown, one from a wild-type hermaphrodite (top) and one from a rad-54 mutant (bottom). Insets below each gonad show higher-magnification views of the boxed regions. In wild-type gonads RAD-51 foci peak in number at early pachytene and disappear by late pachytene, while in rad-54 mutants they progressively increase in number until nuclei reach the most proximal region of the gonad, and disappear as the chromosomes condense at diakinesis. These foci can be difficult to resolve and count reliably at such high densities, but frequently exceed 50 per nucleus. Other mutants with crossover defects have similar but less extreme effects than rad-54 mutants, which are defective in removal of RAD-51 from recombination intermediates.