The N-Terminal Region of the Polo Kinase Cdc5 is Required for Downregulation of the Meiotic Recombination Checkpoint

During meiosis, the budding yeast polo-like kinase Cdc5 is a crucial driver of the prophase I to meiosis I (G2/M) transition. The meiotic recombination checkpoint restrains cell cycle progression in response to defective recombination to ensure proper distribution of intact chromosomes to the gametes. This checkpoint detects unrepaired DSBs and initiates a signaling cascade that ultimately inhibits Ndt80, a transcription factor required for CDC5 gene expression. Previous work revealed that overexpression of CDC5 partially alleviates the checkpoint-imposed meiotic delay in the synaptonemal complex-defective zip1Δ mutant. Here, we show that overproduction of a Cdc5 version (Cdc5-ΔN70), lacking the N-terminal region required for targeted degradation of the protein by the APC/C complex, fails to relieve the zip1Δ-induced meiotic delay, despite being more stable and reaching increased protein levels. However, precise mutation of the consensus motifs for APC/C recognition (D-boxes and KEN) has no effect on Cdc5 stability or function during meiosis. Compared to the zip1Δ single mutant, the zip1Δ cdc5-ΔN70 double mutant exhibits an exacerbated meiotic block and reduced levels of Ndt80 consistent with persistent checkpoint activity. Finally, using a CDC5-inducible system, we demonstrate that the N-terminal region of Cdc5 is essential for its checkpoint erasing function. Thus, our results unveil an additional layer of regulation of polo-like kinase function in meiotic cell cycle control.

Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.

Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic NES or NLS sequences or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.

NAD+-dependent deacetylase SIRT1 is essential for meiotic progression and controls repair-recombination efficiency

Meiotic components and their functions have been extensively studied. Yet, the interplay between molecular factors and regulation of their functions that is brought about by post-translational modifications, specifically (de)-acetylation, is not well characterized. SIRT1, a NAD+-dependent deacetylase has been previously shown to be necessary for spermatogenesis. However, whether it has any role to play in mammalian meiosis remains to be uncovered. Our findings identify SIRT1 as a key determinant of meiotic progression. Knocking out SIRT1 specifically in meiocytes (SIRT1Δmeio) led to a delay in progression through pachytene and repair of double strand breaks. Interestingly, despite these deficits, meiotic loss of SIRT1 did not affect synapsis nor did it lead to pachytene arrest or apoptosis. Moreover, our results demonstrate that SIRT1 is required for regulating crossover frequency and its absence results in higher crossover events. Therefore, our study brings to the fore a novel regulatory factor/mechanism that is necessary for coupling of synapsis and recombination. This is noteworthy since mutations in core meiotic components result in gross defects in synapsis, repair and recombination, and very few studies have reported the differential regulation of these processes. Further, exposing SIRT1Δmeio to low/moderate doses of ©-irradiation indicated that SIRT1 might be involved in eliciting recombination checkpoint arrest and in its absence pachytene cells progress to diplotene stage, unlike in the SIRT1WT mice. Importantly, exogenous damage resulted in enhanced retention of ©H2AX in SIRT1Δmeio diplotene cells, reiterating the critical role that SIRT1 plays in regulating repair efficiency/kinetics. Molecularly, we find that SIRT1 interacts with MRN complex and lack of SIRT1 causes hyperacetylation of several non-histone proteins including the MRN components. Given that SIRT1Δmeio mice mimic MRN hypomorphs, we propose that SIRT1-dependent deacetylation of these proteins is crucial for normal meiotic progression. Taken together, our study uncovers a previously unappreciated role of SIRT1 in meiotic progression.Author SummaryMeiosis is a key process in germ cell development that is essential for generating genetic diversity via recombination. It involves precise spatio-temporal orchestration of various molecular events such as chromosomal synapsis, repair and recombination. Whereas the core meiotic components are well known, upstream factors that might be important for regulating their functions and also couple the downstream processes are less explored. In this paper, we report that SIRT1, a NAD+-dependent deacetylase, is necessary for meiotic progression by identifying its role in coupling of synapsis and recombination. By generating a meiosis specific knockout of SIRT1, we show that its absence in spermatocytes leads to inefficient/delayed repair and progression through pachytene. We have also uncovered that SIRT1 exerts control over recombination (cross over) frequency. Interestingly, our findings demonstrate that SIRT1 provides protection against exogenous genotoxic stress possibly by eliciting meiotic checkpoints. Thus, this study provides both cellular and molecular insights into the importance of SIRT1 mediated protein deacetylation in governing meiosis in mammals.

Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint

ABSTRACTThe meiotic recombination checkpoint blocks meiotic cell cycle progression in response to synapsis and/or recombination defects to prevent aberrant chromosome segregation. The evolutionarily-conserved budding yeast Pch2TRIP13 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1HORMAD adaptor at T318. In the wild type, Pch2 localizes to synapsed chromosomes and to the unsynapsed rDNA region (nucleolus), excluding Hop1. In contrast, in synaptonemal complex (SC)-defective zip1Δ mutants, which undergo checkpoint activation, Pch2 is detected only on the nucleolus. Alterations in some epigenetic marks that lead to Pch2 dispersion from the nucleolus suppress zip1Δ-induced checkpoint arrest. These observations have led to the notion that Pch2 nucleolar localization could be important for the meiotic recombination checkpoint. Here we investigate how Pch2 chromosomal distribution impacts on checkpoint function. We have generated and characterized several mutations that alter Pch2 localization pattern resulting in aberrant Hop1 distribution and compromised meiotic checkpoint response. Besides the AAA+ signature, we have identified a basic motif in the extended N-terminal domain critical for Pch2’s checkpoint function and localization. We have also examined the functional relevance of the described Orc1-Pch2 interaction. Both proteins colocalize in the rDNA, and Orc1 depletion during meiotic prophase prevents Pch2 targeting to the rDNA allowing unwanted Hop1 accumulation on this region. However, Pch2 association with SC components remains intact in the absence of Orc1. We finally show that checkpoint activation is not affected by the lack of Orc1 demonstrating that, in contrast to previous hypotheses, nucleolar localization of Pch2 is actually dispensable for the meiotic checkpoint.

Functional impact of the H2A.Z histone variant during meiosis in Saccharomyces cerevisiae

Among the collection of chromatin modifications that influence its function and structure, the substitution of canonical histones by the so-called histone variants is one of the most prominent actions. Since crucial meiotic transactions are modulated by chromatin, here we investigate the functional contribution of the H2A.Z histone variant during both unperturbed meiosis and upon challenging conditions where the meiotic recombination checkpoint is triggered in budding yeast by the absence of the synaptonemal complex component Zip1. We have found that H2A.Z localizes to meiotic chromosomes in an SWR1-dependent manner. Although meiotic recombination is not substantially altered, the htz1 mutant (lacking H2A.Z) shows inefficient meiotic progression, impaired sporulation and reduced spore viability. These phenotypes are likely accounted for by the misregulation of meiotic gene expression landscape observed in htz1. In the zip1 mutant, the absence of H2A.Z results in a tighter meiotic arrest imposed by the meiotic recombination checkpoint. We have found that Mec1-dependent Hop1-T318 phosphorylation and the ensuing Mek1 activation are not significantly altered in zip1 htz1; however, downstream checkpoint targets, such as the meiosis I-promoting factors Ndt80, Cdc5 and Clb1, are drastically down-regulated. The study of the checkpoint response in zip1 htz1 has also allowed us to reveal the existence of an additional function of the Swe1 kinase, independent of CDK inhibitory phosphorylation, which is relevant to restrain meiotic cell cycle progression. In summary, our study shows that the H2A.Z histone variant impacts various aspects of meiotic development adding further insight into the relevance of chromatin dynamics for accurate gametogenesis.

Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control.