Kisspeptin 234 sale br Results br Discussion Here we establi

Results
Discussion Here we establish a key kinase cascade that acts at PCs to promote homolog pairing and synapsis. CHK-2 phosphorylates the zinc finger proteins that specify PCs, which, in turn, primes their recruitment of PLK-2 (Figure 7D). By recruiting these two kinases, PCs serve as signaling hubs that mediate early meiotic chromosome dynamics. Although most Chk2 family kinases function downstream of ATM/ATR in the DNA damage response (Matsuoka et al., 1998), C. elegans CHK-2 does not require DSBs for its initial activation and lacks clusters of SQ/TQ sites that define ATM/ATR targets. Instead, CHK-2 is essential for DSB formation and acts as a master regulator that governs pairing, synapsis, and recombination during meiotic prophase (MacQueen and Villeneuve, 2001). This rewiring of regulatory circuitry may have accompanied the emergence of homolog pairing mechanisms that function independently of meiotic recombination in C. elegans (Dernburg et al., 1998). We demonstrate that CHK-2′s kinase activity normally declines when all Kisspeptin 234 sale have accomplished synapsis and crossover, but is prolonged in mutants that disrupt synapsis or crossover formation. Adding to recent evidence for feedback regulation of crossover formation (Rosu et al., 2013, Stamper et al., 2013, Woglar et al., 2013), we now show that two distinct pathways controlled by CHK-2 (synapsis and meiotic recombination) both feed back to regulate CHK-2 activity (Figure 7D). This common circuitry delays meiotic progression and extends the temporal window for active pairing, synapsis, and DSB formation. This mechanism meets the original definition of a cell-cycle checkpoint (Hartwell and Weinert, 1989) in that it makes meiotic progression contingent on the formation of a crossover on each homolog pair. Our finding that mutations in the meiotic HORMA domain proteins fail to extend CHK-2 activity despite severe defects in pairing, synapsis, and crossover formation highlights the central and conserved role of these proteins in checkpoint control. Although CHK-2 is primarily detected at PCs, it clearly acts elsewhere within the nucleus. Our evidence demonstrates that the meiotic checkpoint is fully functional even in the absence of PC activity and that CHK-2 feedback regulation is, rather, a nucleus-wide response. This directly refutes the recent proposal that HIM-8 is required for feedback in early meiotic prophase (Silva et al., 2014). This confusion arose because the previous study did not directly monitor CHK-2 activity but, instead, the recruitment of PLK-2 to PCs, which requires not only CHK-2 activity but also the zinc finger proteins, specifically HIM-8, under conditions where the ZIMs do not remain phosphorylated. Our work explains the previously enigmatic observation that the polarized nuclear organization that defines the transition zone is extended greatly in synapsis-defective mutants (Colaiácovo et al., 2003, MacQueen et al., 2002, Phillips et al., 2005). Synapsis is initiated in the transition zone, and, therefore, a nucleus-wide signal generated from asynapsed chromosomes (e.g., in syp-2, him-8, and plk-2; plk-1(RNAi)) can maintain the phosphorylation of all PC proteins by CHK-2. On the other hand, a signal from chromosomes that have synapsed but lack crossovers (e.g., in zhp-3, msh-5, spo-11, and him-5) would be generated in early pachytene, when ZIMs are no longer detected at the autosomal PCs and transition zone morphology is no longer observed (Phillips and Dernburg, 2006). At this stage of meiosis, only the phosphoepitope on HIM-8 normally remains evident, and, therefore, only HIM-8 phosphorylation appears to be prolonged by crossover failures. Why HIM-8 phosphorylation and binding to the X chromosome PC persists longer than for the ZIMs is unknown. Nevertheless, feedback regulation maintains the status of the meiotic cell cycle at which errors are first detected. Different meiotic lesions extend the zone of CHK-2 activity to different degrees, indicating that feedback is graded rather than binary. Clear evidence for this comes from our analysis of a series of mutations in HTP-3 that disrupt individual HIM-3 recruitment. As the synapsis defects become progressively more severe, the duration of CHK-2 activation increases (Figure 5). This graded meiotic checkpoint resembles the rheostat-like behavior of the spindle assembly checkpoint (SAC) that monitors kinetochore-microtubule attachment during mitosis (Collin et al., 2013, Dick and Gerlich, 2013). The strength of the SAC correlates with the amount of Mad2 recruited to kinetochores, which, in turn, depends on a hierarchical assembly of other checkpoint proteins at the kinetochore (London and Biggins, 2014). We have now shown that both HTP-1 and HTP-2 are required to generate a maximal signal from asynapsed chromosomes and that both HIM-3 and HTP-1/2 are required for the feedback regulation. HTP-3 contains two binding sites for HTP-1/2 and four for HIM-3, which can recruit additional HTP-1/2 through its C-terminal motif (Kim et al., 2014). Therefore, HTP-3 acts as a scaffold for checkpoint activation, and the hierarchical network of HIM-3 and HTP-1/2 may allow flexibility in responding to diverse meiotic defects.