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[Andreas Mbili]

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Chromosome movements and programmed DNA double-strand breaks (DSBs) promote homologue pairing and initiate recombination at meiosis onset. Meiotic progression involves checkpoint-controlled termination of these events when all homologue pairs achieve synapsis and form crossover precursors. Exploiting the temporo-spatial organisation of the C. elegans germline and time-resolved methods of protein removal, we show that surveillance of the synaptonemal complex (SC) controls meiotic progression. In nuclei with fully synapsed homologues and crossover precursors, removing different meiosis-specific cohesin complexes, which are individually required for SC stability, or a SC central region component causes functional redeployment of the chromosome movement and DSB machinery, triggering whole-nucleus reorganisation. This apparent reversal of the meiotic programme requires CHK-2 kinase reactivation via signalling from chromosome axes containing HORMA proteins, but occurs in the absence of transcriptional changes. Our results uncover an unexpected plasticity of the meiotic programme and show how chromosome signalling orchestrates nuclear organisation and meiotic progression.

The formation of haploid gametes from diploid germ cells during meiosis constitutes a cornerstone of sexual reproduction. Defects in this process cause sterility and aneuploid gametes that impair fitness of the resulting progeny. Accurate transmission of chromosomes into the gametes depends critically on the establishment of crossover (CO) events between paternal and maternal homologous chromosomes (homologues) during the long prophase preceding the first meiotic division, when COs, together with sister chromatid cohesion (SCC), ensure correct chromosome orientation on the spindle1. Thus, meiotic cells have evolved meiosis-specific chromosome structures and surveillance mechanisms to ensure that every pair of homologues is connected by COs before nuclei proceed to the first meiotic division.

The formation and repair of DSBs into COs is mechanistically coupled to early meiotic progression by a network of surveillance mechanisms that monitor specific, but incompletely understood, pairing and recombination intermediates7. The presence (or absence) of these meiotic chromosome metabolism intermediates results in signals that feedback to regulate checkpoint kinases, which then target components of the pairing, recombination, and cell cycle machineries8. These quality control mechanisms fulfil two important roles. Firstly, they act to limit the temporal window during which nuclei remain competent for DSB formation, ensuring timely cessation of this activity once CO precursors are formed on all chromosomes, thus preventing the genotoxic effects of excess DSBs. Secondly, they regulate meiotic progression in a checkpoint manner, inducing arrest of nuclei at the stage when defects in specific CO-promoting events are first detected. For example, mutations that impair DSB processing into CO precursors cause extension of DSB-permissive stages in yeast and C. elegans9,10,11. The regulated exit from DSB-permissive stages represents a fundamental transition of the meiotic programme, but how nucleus-wide loss in competence for DSB formation is sustained remains unclear.

The C. elegans germline provides a powerful system to investigate how feedback mechanisms integrate pairing and recombination with early meiotic progression12. CHK-2 promotes DSB formation, chromosome movement, and SC assembly during early prophase and the temporal window of CHK-2 activity is controlled by feedback from the progression of these events mediated by HORMADs13,14,15. Despite sharing some components, these feedback mechanisms are mechanistically different from checkpoints that induce apoptosis of nuclei with persistent DNA damage or asynapsed chromosomes at late pachytene16,17. Importantly, SC assembly is independent of recombination in worms18 and both processes are under surveillance to feedback on CHK-219. In wild-type germlines synapsis induces termination of CHK-2-dependent chromosome movements by pachytene entrance, while the formation of CO precursors triggers loss of CHK-2-dependent markers of DSB formation by mid pachytene. Once achieved, nucleus-wide loss of CHK-2 activity is thought of as a unidirectional transition of the meiotic programme that leads to the completion of recombination and progression towards chromosome segregation.

In the current work, we combine the experimental advantages of the C. elegans germline with temporally resolved protein removal methods to investigate how cohesin and the SC contribute to meiotic chromosome function once full synapsis and early recombination steps are completed. We uncover a role for REC-8 and COH-3/4 cohesin in promoting SC stability and demonstrate that direct or indirect, via cohesin removal, SC disassembly triggers rapid and nucleus-wide redeployment of the pairing and recombination machinery, inducing drastic changes in nuclear organisation. This apparent reversal in meiotic progression requires CHK-2 reactivation mediated by HORMA protein HTP-1, but occurs in the absence of transcriptional changes. Our findings have important implications for understanding the quality control mechanisms that ensure fertility in higher eukaryotes.

In most germlines, REC-83XTEV::GFP cleavage caused concentration of SYP-1 in a few aggregates per nucleus, where it colocalized with remaining GFP (REC-8) signals (Fig. 2a). These aggregates were more prominent in late pachytene nuclei, where they typically appeared as 6 chromosome-associated short stretches (Fig. 2a). Since late pachytene nuclei in worms contain six CO-designated sites, one per homologue pair, that are visible as 6 COSA-1 foci30, we tagged the cosa-1 gene with HA using CRISPR and crossed this allele into the REC-83XTEV::GFP strain. Following TEV injection, we observed that 92% of persistent REC-83XTEV::GFP stretches in late pachytene were associated with a COSA-1 focus and that most nuclei contained 6 COSA-1 foci (Fig. 2b and Supplementary Figs. 2c, d). These observations suggest that REC-8 cohesin is locally regulated around CO-designated sites.

These results demonstrate that pachytene nuclei with full SC and a normal complement of CO precursors retain the potential to reactivate early, CHK-2-dependent, prophase events and suggest that chromosome-bound cohesin plays an important role in regulating CHK-2 activity during pachytene.

SC depletion also triggered de novo association of DSB-2 with pachytene chromosomes (Supplementary Fig. 4d), and increased RAD-51 foci (Fig. 4e), consistent with de novo DSB formation. Interestingly, COSA-1 foci in late pachytene nuclei remained intact following complete SC disassembly (Supplementary Fig. 4e), consistent with previous reports that used 1,6-hexanediol to dissolve the SC in dissected germlines43. Therefore, SC removal is sufficient to induce CHK-2 reactivation in pachytene nuclei, suggesting that SC surveillance remains active once the structure is fully assembled.

HORMAD HTP-1 is a key component of the feedback mechanisms that couple SC assembly with meiotic progression during early prophase14,36. HTP-1 promotes persistence of CHK-2-dependent chromosome clustering in mutant backgrounds in which SC assembly fails in one or more chromosomes14,47, presumably by participating in the creation and transmission of a signal that sustains CHK-2 activity in the presence of unsynapsed chromosomes. To investigate if HTP-1 is required for the reactivation of CHK-2-dependent events observed when cohesin is removed from pachytene chromosomes, we induced TEV-mediated removal of COH-3 in germlines of htp-1 mutants. TEV injection induced efficient COH-3 removal in htp-1 mutant germlines, but failed to induce reappearance of PLK-2 aggregates or chromosome clustering in pachytene nuclei (Fig. 5f), as observed when COH-3 was removed in control germlines (Fig. 5g). This suggests that the structural changes that result when cohesin is removed from pachytene chromosomes are sensed and transmitted by the same, HTP-1-dependent, surveillance mechanisms that monitor SC assembly to regulate CHK-2 activity during early prophase.

Our findings reveal that nuclei with fully synapsed chromosomes and a normal complement of CO precursors undergo rapid, nucleus-wide, reestablishment of chromosome movement and DSB formation when SC stability is compromised. This process requires de novo CHK-2 activation and causes a dramatic reorganisation of the nucleus that appears to functionally revert pachytene nuclei back to earlier prophase stages, revealing a striking plasticity of the meiotic programme. We identify SC surveillance as the mechanism that regulates CHK-2 activity during pachytene. We also uncover a role for REC-8 and COH-3/4 cohesin in promoting SC stability in pachytene nuclei, as removal of either type of cohesin causes rapid SC disassembly and CHK-2 reactivation. This role of REC-8 and COH-3/4 (Rad21L) cohesin is different from their redundant role in promoting axis assembly at the onset of meiosis3,49, evidencing that the contribution of cohesin to landmark meiotic chromosome structures (axial elements and the SC) is more complex than previously thought. Moreover, we also provide evidence that cohesin may be locally regulated around CO sites, reminiscent of the local regulation of SC structure at CO sites50. Below, we discuss our findings in the context of a model in which SC surveillance orchestrates meiotic progression by continuously regulating CHK-2 activity (Fig. 8).

In C. elegans CHK-2 promotes chromosome movement, DSB formation, and SC assembly at meiotic entrance13 and CHK-2 activity is lost gradually; markers of chromosome movement disappear at pachytene entrance, coinciding with full synapsis, while markers of DSB formation disappear at mid pachytene, presumably once all chromosomes have formed CO precursors9,10,19. Analysis of mutants with defects in synapsis or CO formation suggest that the termination of chromosome movement at early pachytene and of DSB formation at mid pachytene, which are controlled by checkpoint mechanisms9,10,14,19, represent two key functional transitions of meiotic progression that culminate with a nucleus-wide loss in competency for CHK-2-dependent events. However, we now show that nuclei that have progressed normally to mid pachytene undergo rapid reactivation of CHK-2-dependent events when SC stability is compromised. This finding provides important insights into the surveillance mechanisms that regulate meiotic progression.

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