A compartmentalized signaling network mediates crossover control in meiosis
Meiosis gives rise to haploid gametes through two sequential rounds of nuclear division . In most eukaryotes , accurate meiotic chromosome segregation requires that every pair of homologous chromosomes attains at least one crossover ( CO ) recombination product , which creates a stable interhomolog connection known as a chiasma ( Page and Hawley , 2003 ) . However , the total number of COs per cell is typically far too low to assure CO formation on each chromosome by a Poisson process; indeed , in many species each homolog pair undergoes only a single CO ( Mercier et al . , 2015 ) . In cases where multiple COs occur per chromosome pair , they are nonrandomly far apart , a phenomenon known as ‘crossover interference’ ( Muller , 1916; Sturtevant , 1915 ) . Despite our longstanding awareness of crossover patterning , the mechanisms that ensure this highly nonrandom process remain poorly understood and controversial . Meiotic recombination is initiated by programmed DNA double-strand breaks ( DSBs ) catalyzed by the conserved topoisomerase-like enzyme Spo11 ( SPO-11 in C . elegans ) ( Dernburg et al . , 1998; Keeney , 2008; Keeney et al . , 1997 ) . A subset of DSBs is processed to become COs , while the rest are repaired through alternate pathways . In several model organisms , two CO pathways have been elucidated: The ‘class I’ pathway , which is subject to CO assurance and interference , requires two meiosis-specific homologs of the MutS bacterial mismatch repair protein , Msh4 and Msh5 , which form a complex known as MutSγ . Components of the synaptonemal complex ( SC ) , an ordered , periodic proteinaceous structure that assembles between homologous chromosomes during meiotic prophase , are also required for class I COs . An alternate enzymatic pathway can give rise to both noncrossovers and ‘class II’ COs , which do not show interference . In the nematode C . elegans , only class I COs normally occur , and only a single CO occurs per chromosome pair ( Martinez-Perez and Colaiácovo , 2009 ) . No COs occur in the absence of SC assembly ( MacQueen et al . , 2002 ) . Additional factors required for meiotic COs , but not for homolog pairing , synapsis , or other homologous recombination , include MSH-4 and MSH-5 ( Kelly et al . , 2000; Zalevsky et al . , 1999 ) , the cyclin-related protein COSA-1 ( Yokoo et al . , 2012 ) , and ZHP-3 , a RING finger protein ( Jantsch et al . , 2004 ) . COs act together with sister chromatid cohesion to orchestrate two successive rounds of chromosome segregation . In mammals and most other model organisms , cohesion between sister chromatids is maintained near centromeres during the first division ( MI ) , but arm cohesion must be released to allow homologs to separate ( Duro and Marston , 2015 ) . In C . elegans , which lacks defined centromeres , cohesion is spatially regulated downstream of CO designation . Late in meiotic prophase , the chromosome region on one side of the designated CO site becomes enriched for several proteins , including HTP-1/2 , LAB-1 , and REC-8 , which act together to maintain cohesion through the first division . Conversely , the reciprocal chromosome region becomes depleted of these proteins but retains a stretch of SC . This ‘short arm’ eventually recruits the Aurora B kinase AIR-2 , which is required to release cohesion and allow MI segregation ( de Carvalho et al . , 2008; Ferrandiz et al . , 2018; Kaitna et al . , 2002; Martinez-Perez et al . , 2008; Nabeshima et al . , 2005; Rogers et al . , 2002; Severson and Meyer , 2014 ) . It has been unclear whether the SC that remains along this arm contributes to regulating cohesion . The hierarchy of steps leading to this remodeling , and particularly the initial trigger for this asymmetry , are also poorly understood . Several recent studies have indicated that structural proteins within assembled SCs are highly mobile ( Nadarajan et al . , 2017; Pattabiraman et al . , 2017; Rog et al . , 2017; Voelkel-Meiman et al . , 2012 ) . We have also presented evidence that this material likely assembles through regulated phase separation ( Rog et al . , 2017 ) . When SCs cannot assemble between chromosomes , as in C . elegans mutants lacking the essential chromosome axis protein HTP-3 , or in budding yeast lacking DSBs , SC proteins self-assemble to form irregularly shaped , but internally structured , nuclear bodies known as polycomplexes ( Roth , 1966 ) , which do not appear to be associated with chromosomes . Observations of C . elegans polycomplexes in vivo revealed that they behave as liquid-like droplets , in that they rapidly change shape and fuse to form larger bodies ( Rog et al . , 2017 ) . Taken together , these observations indicate that SC proteins self-assemble through regulated coacervation to form a liquid crystalline material – defined as an ordered assembly of molecules that diffuse freely relative to each other . Normally this material preferentially assembles as a bilaterally symmetrical lamina between paired axes , but in the absence of the proper chromosomal substrate , it forms polycomplexes . Growing evidence has implicated the SC in CO regulation , particularly in C . elegans ( Hayashi et al . , 2010; Libuda et al . , 2013; MacQueen et al . , 2002; Rog et al . , 2017; Sym and Roeder , 1994 ) . The mobility of proteins within assembled SCs suggests that biochemical signals such as enzymes or posttranslational modifications could move through this material to regulate CO formation along paired chromosomes . In support of this idea , a number of crossover factors and DNA damage repair components have been found to associate with both SCs and polycomplexes . In budding yeast , these include Msh4 , Msh5 , Spo16 , Spo22 ( Zip4 ) , Zip2 , and Zip3 , and the 9-1-1 complex component Mec3 ( Shinohara et al . , 2015; Tsubouchi et al . , 2006 ) . Zip3 is unique among these proteins in that it localizes throughout the body of polycomplexes , suggesting that it has an intrinsic affinity for SC proteins , while all other proteins reported to date appear to localize to a ‘cap’ or focus at the surface of polycomplexes . We observed that ZHP-3 , a C . elegans homolog of yeast Zip3 ( Agarwal and Roeder , 2000 ) , similarly localizes throughout polycomplexes ( Rog et al . , 2017 ) . Interestingly , ZHP-3 and Zip3 share homology with Hei10 and RNF212 , which have been implicated in crossover control across diverse eukaryotes ( Chelysheva et al . , 2012; De Muyt et al . , 2014; Gray and Cohen , 2016; Qiao et al . , 2014; Reynolds et al . , 2013; Wang et al . , 2012; Ward et al . , 2007 ) . All of these proteins contain an N-terminal RING finger domain , a central coiled-coil domain , and a C-terminal domain that is predicted to be largely unstructured . Three paralogs of ZHP-3 are expressed in the C . elegans germline , but their functions have not previously been described . Here we define essential meiotic roles for these proteins . We report that this family of four paralogs , which we designate as ZHP-1–4 , likely act as two heterodimeric complexes to regulate CO formation . Like ZHP-3 , the other 3 proteins localize throughout SCs in early prophase , and are also recruited to polycomplexes . Together with other known and unknown factors , these ZHP proteins appear to form a signaling network that acts within the SC to ensure CO formation while limiting the number of CO-designated sites , and may thus mediate both CO assurance and CO interference . In addition , the ZHP proteins in C . elegans act upstream of other regulators to direct chromosome remodeling in response to crossover formation , which enables the stepwise removal of cohesion that enables two successive rounds of chromosome segregation during meiosis . Homology between the ZHP proteins and meiotic regulators from other phyla indicate that similar mechanisms likely underlie meiotic CO control in most eukaryotes , and may also play previously-unrecognized roles in regulating sister chromatid cohesion during meiosis .
As described in the Introduction , the RING finger protein ZHP-3 was initially identified as a candidate meiotic factor in C . elegans based on its homology to Zip3 , a component of the ‘synapsis initiation complex’ in yeast ( Agarwal and Roeder , 2000 ) . Targeted disruption of zhp-3 revealed that it is required for CO formation , but dispensable for homolog pairing and synapsis ( Jantsch et al . , 2004 ) . The C . elegans genome includes three additional predicted genes with the same domain structure and significant homology to ZHP-3 ( Figure 1—figure supplement 1A–B ) . Several independent analyses indicate that all four genes are expressed in the germline , consistent with a role in meiosis ( WormBase; ( WormBase , 2017 ) ; S . K . and A . F . D . unpublished ) . All four predicted proteins have similar N-terminal C3HC4-type RING-finger domains ( Figure 1—figure supplement 1A–B ) , which are usually associated with ubiquitin E3 ligase activity ( Deshaies and Joazeiro , 2009 ) . The C-terminal regions of these proteins are divergent and lack obvious structural domains , but contain a number of potential post-translational modification sites ( Figure 1—figure supplement 1A ) . Based on their similarity to each other , and on evidence presented here that they function together to form a regulatory circuit , we have named these genes zhp-1 ( F55A12 . 10 ) , zhp-2 ( D1081 . 9 ) and zhp-4 ( Y39B6A . 16 ) ( zip3 homologous protein ) . This numbering reflects their physical order in the C . elegans genome: zhp-1 , -2 , and -3 are clustered on Chromosome I , while zhp-4 is on Chromosome V . To gain insight into the evolution of these proteins , we searched for homologs in other sequenced nematode genomes ( Figure 1—figure supplement 1C ) . Orthologs of each protein can be identified in C . briggsae and C . remanei , as well as in other nematode genera , indicating that these proteins diversified at least tens to hundreds of millions of years ago ( Figure 1—figure supplement 1C–D and data not shown ) . Their N-terminal regions show homology to the mammalian CO regulator RNF212 , and ZHP-1 and ZHP-2 are also recognized by BLAST as homologs of HEI10 , which is involved in CO regulation in mammals , plants , and fungi ( Chelysheva et al . , 2012; De Muyt et al . , 2014; Qiao et al . , 2014; Rao et al . , 2017; Reynolds et al . , 2013 , Wang et al . , 2012; Ward et al . , 2007 ) ( data not shown ) . To investigate the ZHP proteins , we inserted epitope tags at the C-terminus of each coding sequence; these tagged proteins supported normal meiosis ( Supplementary file 1 , Table S1 ) and were readily localized by immunofluorescence with epitope-specific antibodies ( Figure 1—figure supplement 2 and Figure 2—figure supplement 2A–B ) . At meiotic entry the ZHP proteins localized to small polycomplexes , which appear prior to synapsis and contain the four known SC structural proteins , SYP-1 , -2 , -3 , and -4 ( Goldstein , 2013; Rog et al . , 2017; Roth , 1966 ) . Once SC assembly initiated , they localized to SCs ( Figure 1 ) . As meiosis progressed , the four proteins showed two distinct patterns of localization ( Figure 1 ) : From early prophase to mid-pachynema , ZHP-1 and -2 became brighter and more contiguous along the length of SCs , although the intensity of staining was not homogeneous . Upon the appearance of GFP-COSA-1 foci , which mark designated CO sites from mid-pachynema through diplonema ( Yokoo et al . , 2012 ) , ZHP-1 and -2 became confined to the SC on one side of each CO . Intriguingly , this restriction was observed in pachytene nuclei that retained SC ( Figure 1A–B ) and the HORMA domain proteins HTP-1/2 ( data not shown ) on both sides of the CO , and represents the earliest known molecular differentiation of the two chromosome domains that will become the long and short arms of the bivalent . ZHP-1 and -2 remained associated with SC proteins along the short arm of each bivalent as the SC disappeared from the long arm , and persisted as long as SC proteins were present along the short arms , through late diakinesis . ZHP-3 and -4 exhibited a similar distribution to ZHP-1 and -2 during early prophase , but showed a distinct pattern starting at mid-pachytene , when crossover designation ( the selection of one recombination intermediate as the eventual crossover site for each pair of chromosomes ) is thought to occur in C . elegans ( Figure 1C–D ) ( Yokoo et al . , 2012 ) . Upon the detection of GFP-COSA-1 foci , which mark the subset of recombination intermediates that will become COs , ZHP-3 and -4 both accumulated at these sites and gradually disappeared from the SC along both arms of the chromosomes . These observations are largely consistent with previous characterization of ZHP-3 and a partially functional GFP-ZHP-3 fusion protein ( Bhalla et al . , 2008; Jantsch et al . , 2004 ) . The SC in C . elegans comprises at least four proteins , known as SYP-1–4 , which are mutually dependent for assembly between chromosomes or as polycomplexes . We found that none of the four ZHP proteins associated along chromosomes in syp-1 or other syp mutants ( Figure 1—figure supplement 3A–C , and data not shown ) . Intriguingly , in the region of the germline that normally corresponds to late pachynema , the four ZHP proteins colocalized within nuclear puncta devoid of SC proteins , indicating that the ZHPs can interact with each other in the absence of SCs or polycomplexes ( Figure 1—figure supplement 3C , lower panel ) . These puncta do not coincide with known DSB repair proteins ( Figure 1—figure supplement 3D ) . The ZHP proteins were also less abundant in syp-1 mutants ( Figure 1—figure supplement 3E–K ) , suggesting that their stability is enhanced by association with SCs , particularly that of ZHP-1 and -2 ( Figure 1—figure supplement 3F and J ) . Because the ZHP proteins relocalized upon CO designation ( Figure 1 ) , we examined their distribution under conditions where COs fail to occur . In mutants lacking SPO-11 , MSH-5 , or COSA-1 , ZHPs were detected along the length of SCs through diplonema ( data not shown ) . Thus , relocalization of the ZHPs in late prophase depends on DSBs and pro-CO factors . To investigate the meiotic functions of the ZHP proteins , we first exploited the auxin-inducible degradation system . Each of the ZHP genes was tagged with a 3xFLAG epitope or GFP and a 44-aa degron sequence ( hereafter ‘AID’ ) in a strain that expresses the F-box protein AtTIR1 throughout the germline ( Zhang et al . , 2015 ) . When adult animals were transferred to plates containing 1 mM auxin , all AID-tagged proteins were robustly depleted within 1–2 hr ( Figure 2—figure supplement 1A–B ) . To assess the efficacy of the knockdown , we compared meiotic chromosome segregation in hermaphrodites carrying the previously characterized zhp-3 ( jf61 ) null allele ( Jantsch et al . , 2004 ) to auxin-treated worms expressing ZHP-3-AID-GFP ( Figure 2—figure supplement 1C ) . AID-mediated depletion of ZHP-3 quantitatively phenocopied the null allele with respect to both embryonic viability ( ∆zhp-3: 2 . 06 ± 0 . 62%; zhp-3::AID: 1 . 83 ± 0 . 82% ) and the incidence of males among the self-progeny of hermaphrodites , which reflects X-chromosome nondisjunction ( Hodgkin et al . , 1979 ) ( ∆zhp-3: 43 . 75 ± 27 . 59%; zhp-3::AID: 37 . 02 ± 38 . 12% ) ( Figure 2—figure supplement 1C ) , indicating that auxin-induced degradation effectively eliminates the function of ZHP-3 . To determine the dependence of the ZHP proteins on each other for their localization and stability , we exposed worms to auxin for 24 hr , so that a pool of nuclei had entered and progressed through meiotic prophase in the absence of AID-tagged protein . We found that ZHP-1 and -2 depend on each other for both their chromosome association and stability ( Figure 2A–D ) . ZHP-3 and -4 were also interdependent for their localization to chromosomes ( Figure 2E and G and Figure 2—figure supplement 2C ) . ZHP-3 was unstable in the absence of ZHP-4 ( Figure 2F ) and ZHP-4 levels were somewhat lower when ZHP-3 was depleted ( Figure 2H and Figure 2—figure supplement 2D ) . Other RING finger proteins such as BRCA1/BARD1 are known to act as heterodimers ( Brzovic et al . , 2001; Metzger et al . , 2014; Wu et al . , 1996 ) . The similarity and interdependence of localization of ZHP-1 and -2 , and of ZHP-3 and -4 , suggested that these four proteins might function as two pairs . Using yeast two-hybrid analysis , we found that ZHP-1 and -2 indeed interact with each other , and that ZHP-3 and -4 also physically interact ( Figure 2I and Figure 2—figure supplement 2E ) . This evidence for specific pairwise physical interactions , together with their physical distributions ( above ) and functional analysis ( below ) , indicate that ZHP-1 and -2 form a complex , and that ZHP-3 and -4 similarly act as a pair . Hereafter we refer to these two dimeric activities as ZHP-1/2 and ZHP-3/4 for simplicity . Using a number of established assays , we investigated the meiotic functions of these RING finger proteins . At late diakinesis , association between homologs is normally maintained by chiasmata , and six condensed bivalents can be detected in each wild-type oocyte nucleus . We exposed zhp-AID strains to auxin for 24 hr and then analyzed chiasma formation . Depletion of ZHP-1 or ZHP-2 yielded 8–12 DAPI-staining bodies at diakinesis , representing 4–12 univalents and 0–4 bivalents ( Figure 3A–B ) . In contrast , depletion of ZHP-3 or ZHP-4 resulted in a complete failure of chiasma formation , indicated by the appearance of 12 achiasmate chromosomes in each nucleus ( Figure 3C–D ) , consistent with previous characterization of ZHP-3 ( Bhalla et al . , 2008; Jantsch et al . , 2004 ) . Because a few chiasmata were observed when ZHP-1-AID or ZHP-2-AID were depleted , we were concerned that these proteins might not be efficiently degraded . We therefore introduced null mutations in zhp-1 and zhp-2 ( see Materials and methods ) . Homozygous mutants produced few viable progeny and a high incidence of males among the self-progeny of hermaphrodites ( Figure 3E ) , both of which are indicative of meiotic chromosome missegregation . They also showed identical distributions of DAPI-staining bodies at diakinesis to that observed in AID-depleted animals ( Figure 3—figure supplement 1A–C and data not shown ) . Additionally , both zhp-1 and -2 null mutants produced more viable progeny than zhp-3 null mutants ( ***p<0 . 001 , two-sided Student’s t-test ) ( Figure 3E ) , consistent with a less absolute dependence of chiasma formation on ZHP-1/2 than on ZHP-3/4 . We wondered whether the few chiasmata in hermaphrodites lacking ZHP-1/2 might preferentially occur on the X chromosome , which shows some differences from autosomes in its genetic requirements for DSB induction and CO formation ( Yu et al . , 2016 ) . To investigate this , we used fluorescence in situ hybridization ( FISH ) to mark Chromosome V and the X chromosome in zhp-1 mutants . By examining nuclei at diakinesis , we found that chiasma formation was impaired to a similar degree on both chromosomes . Our observations are quantitatively consistent with a low number of chiasmata distributed evenly among all six chromosome pairs ( Figure 3F–G ) . ZHP-3 was previously found to be dispensable for homologous chromosome pairing , synapsis , and DSB induction ( Jantsch et al . , 2004 ) . Pairing and synapsis also occurred normally in the absence of ZHP-1 , -2 , or -4 ( Figure 3—figure supplement 2 ) . RAD-51 foci , which mark recombination intermediates and require DSB induction ( Colaiácovo et al . , 2003 ) , were also abundant in all zhp mutants ( Figure 3—figure supplement 3A–F ) . Thus , the high frequency of achiasmate chromosomes in the absence of ZHPs reflects defects in CO recombination downstream of pairing , synapsis , and DSBs . Prior work has established the existence of a ‘crossover assurance checkpoint’ in C . elegans that delays meiotic progression when recombination intermediates or pro-CO factors are absent on one or more chromosomes due to defects in homolog pairing , synapsis , DSB induction , or recombination ( Carlton et al . , 2006; Kim et al . , 2015; Yu et al . , 2016 ) . Activation of this checkpoint prolongs the activity of the CHK-2 kinase , resulting in an extended region and elevated number of RAD-51 foci , and a shift in the pattern of meiotic recombination , but does not perturb CO interference . We observed that phosphorylation of HIM-8 and its paralogs , a marker for CHK-2 activity , was extended when any of the ZHPs was depleted ( Figure 3—figure supplement 3G–I ) , and RAD-51 foci were more abundant ( Figure 3—figure supplement 3A–F ) . Thus , the crossover assurance checkpoint does not require ZHP activity . We noted differences in the dynamics of DNA repair when ZHP-1/2 were depleted as compared to ZHP-3/4 ( Figure 3—figure supplement 3A–F ) . In the absence of ZHP-3/4 , RAD-51 foci reached higher peak numbers than in wild-type animals or in the absence of ZHP-1/2 , but depletion of ZHP-1/2 caused a greater delay in both the accumulation and disappearance of RAD-51 foci than depletion of ZHP-3/4 . Thus , the two ZHP complexes likely play distinct roles in the processing of recombination intermediates , similar to observations of HEI10 and RNF212 in mouse spermatocytes ( Qiao et al . , 2014 ) . To probe the role of ZHPs in recombination , we examined the cytological distributions of MSH-5 , which can be detected at sites of early recombination intermediates , and COSA-1 , which forms bright foci at sites designated for CO formation ( Yokoo et al . , 2012 ) . In wild-type and control ( non-auxin-treated ) worms , ~10–20 MSH-5 foci per nucleus could be detected by mid-pachytene ( Figure 4—figure supplement 1A ) , after which six GFP-COSA-1 foci – one per pair of chromosomes – were observed ( Figure 4A ) , and MSH-5 became restricted to these sites ( Figure 4—figure supplement 1 ) , as previously described ( Yokoo et al . , 2012 ) . In worms depleted of ZHP-3/4 , neither MSH-5 nor COSA-1 foci were detected during meiotic prophase ( Figure 4B , Figure 4—figure supplement 1B and data not shown ) . This indicates that ZHP-3/4 are likely required for stable association of MutSγ with early recombination intermediates . In striking contrast , depletion of ZHP-1/2 resulted in appearance of up to ~50 MSH-5 foci and up to ~30 dim but detectable GFP-COSA-1 foci per oocyte nucleus ( Figure 4C–D , and Figure 4—figure supplement 1C–D ) . Notably , the number of detectable foci increased in number as nuclei advanced from mid- to late pachytene , and then rapidly declined ( Figure 4D and Figure 4—figure supplement 1D ) . The dim GFP-COSA-1 foci in ZHP-1/2-depleted worms showed extensive colocalization with MSH-5 foci ( Figure 4—figure supplement 1E ) . Together with prior evidence , this suggested that MSH-5 and COSA-1 colocalize at many recombination intermediates in the absence of ZHP-1/2 . Indeed , it is likely that these pro-CO factors initially colocalize at all strand exchange intermediates , and that the difference in the number of detected foci of MSH-5 and COSA-1 is due to the relative sensitivity of the available detection reagents . To validate the idea that these foci correspond to recombination intermediates , we eliminated recombination by disrupting DSB formation . We AID-tagged SPO-11 and either ZHP-1 or ZHP-2 in the same strain , and found that both proteins could be effectively co-depleted by auxin treatment ( Figure 4—figure supplement 2A and data not shown ) . This abolished all MSH-5 and GFP-COSA-1 foci ( Figure 4E and data not shown ) , supporting the idea that the dim foci observed in the absence of ZHP-1/2 are indeed recombination intermediates . Co-depletion of ZHP-1 and ZHP-4 also eliminated GFP-COSA-1 foci ( Figure 4F and Figure 4—figure supplement 2B ) and bivalent formation ( Figure 4G ) , supporting the conclusion that ZHP-3/4 are required to stabilize MutSγ at all recombination intermediates . Depletion of ZHP-1/2 also resulted in persistence of ZHP-3 and -4 throughout SCs in late pachytene nuclei ( Figure 4—figure supplement 2C and data not shown ) . These observations indicate that ZHP-1/2 are required to restrict pro-CO proteins to a single recombination intermediate along each pair of homologs during late prophase . We next sought to understand why oocytes lacking ZHP-1/2 have abundant , dim COSA-1 foci but few bivalent chromosomes . This discrepancy could indicate that the dim foci do not mature as COs , or that they do become COs but fail to give rise to stable chiasmata . To address this , we mapped COs in zhp-1 null mutants by whole genome sequencing ( see Materials and methods ) . Using this genetic recombination assay , an average of 2 . 8 ± 1 . 8 ( SD ) COs per oocyte were detected in zhp-1 mutants , compared to 7 . 3 ± 3 . 0 ( SD ) per oocyte ( slightly more than the expected number of 6 ) in wild-type animals ( Figure 4H ) . These data are consistent with the observed number of bivalents ( Figure 3 and Figure 3—figure supplement 1 ) , and indicate that only a small fraction of COSA-1-positive foci in zhp-1/2 mutants mature to become COs and chiasmata . The excess number but weak immunofluorescence of GFP-COSA-1 foci in the absence of ZHP-1/2 suggested that one or more limiting components might be distributed to an excess of recombination intermediates . We therefore wondered whether accumulation of pro-CO factors at specific recombination intermediates could be rescued by restricting the number of these intermediates . Mutations in dsb-2 cause an age-dependent reduction in meiotic DSBs: very few RAD-51 foci and chiasmata are seen in older ( 48 hr post-L4 ) dsb-2 hermaphrodites , while somewhat more RAD-51 foci and chiasmata are seen in younger adults ( Rosu et al . , 2013 ) . We co-depleted DSB-2 and ZHP-2 using the AID system ( Figure 5—figure supplement 1A ) , fixed worms at 24 and 48 hr post-L4 , and stained for RAD-51 and GFP-COSA-1 . Fewer COSA-1 foci and bivalents were observed in animals depleted for both DSB-2 and ZHP-2 than DSB-2 alone , although the number of RAD-51 foci was similar ( Figure 5A–C , Figure 5—figure supplement 1B and data not shown ) . Intriguingly , while younger dsb-2 mutant animals have more meiotic DSBs ( Rosu et al . , 2013 and data not shown ) , we observed fewer bright GFP-COSA-1 foci in young ZHP-2 depleted animals: only 11% of nuclei contained a bright GFP-COSA-1 focus , compared to 49% in older animals ( Figure 5B–C ) . The number of bivalents in these animals corresponded well with bright GFP-COSA-1 foci , and only chromosomes with bright COSA-1 foci displayed the normal hallmarks of chromosome remodeling ( see below ) , indicating that only bright COSA-1 foci reliably mature as COs . As another approach to examine how the number of intermediates impacts CO maturation in the absence of ZHP-1/2 , we co-depleted ZHP-2 and SPO-11 to abolish programmed meiotic DSBs , and exposed these animals to varying doses of ionizing radiation . When SPO-11 alone was depleted by the AID system , GFP-COSA-1 foci and bivalents were eliminated . Following 10 Gy of radiation , a dose sufficient to ensure CO formation on most chromosomes in the absence of SPO-11 , six bright GFP-COSA-1 foci were observed in each nucleus , as previously described ( Libuda et al . , 2013; Yokoo et al . , 2012 ) ( Figure 5—figure supplement 1C ) . When SPO-11 and ZHP-2 were co-depleted , bright GFP-COSA-1 foci were observed following low doses of radiation , but these never exceeded two per nucleus ( Figure 5D ) . At 10 Gy , only extremely dim GFP-COSA-1 foci were detected ( Figure 5D ) . Taken together , these observations indicate that ZHP-1/2 are dispensable for a single break to become a functional interhomolog CO , but act to limit or focus the activity of ZHP-3/4 and/or other pro-CO factors to select a single recombination intermediate on each chromosome and to ensure CO maturation in the context of excess DSBs . A higher fraction of recombination intermediates become crossovers when breaks are severely reduced , but even when very few DSBs are present , ZHP-1/2 contribute to the accumulation of pro-CO factors , which in turn promotes CO maturation . These results indicate that ZHP-1/2 play dual roles: they restrict the accumulation of pro-CO factors to a subset of recombination intermediates and promote their accumulation at a subset of sites . We tested whether these two functions could be separated by reducing the amount of either protein . When strains carrying AID-tagged ZHP-1 or -2 were exposed to low concentrations of auxin , these proteins could be partially depleted ( Figure 5—figure supplement 2A–B and data not shown ) . At very low protein levels , we saw a reduction in the number of GFP-COSA-1 foci , and never observed more than six bright COSA-1 foci per nucleus . Thus , concentrations of ZHP-1/2 required to promote accumulation of pro-CO factors are also sufficient to limit their association with recombination sites ( Figure 5—figure supplement 2C and data not shown ) , indicating that these functions are tightly coupled . As described in the Introduction , stepwise chromosome segregation in C . elegans meiosis requires functional differentiation of the chromosome regions on either side of the crossover . Here we report that even prior to any apparent reorganization of HTP-1/2 or SC proteins , ZHP-1/2 became restricted to the SC on one side of the CO , the presumptive short arm ( Figure 1 and data not shown ) . In addition , depletion of ZHP-1 or -2 resulted in aberrant chromosome remodeling: HTP-1/2 and SYP-1 persisted along most chromosomes , both recombinant and achiasmate ( Figure 6—figure supplement 1A–C ) . These observations suggested that ZHP-1/2 might regulate chromosome remodeling , in addition to their roles in CO control . To further probe the contribution of ZHP-1/2 to remodeling , we examined dsb-2 mutants , in which some recombination intermediates accumulate pro-CO factors and undergo CO maturation even in the absence of ZHP-1/2 . The low number of COs in dsb-2 mutants also allows direct comparison between recombinant and nonexchange chromosomes in the same nucleus , and thus at the same cell cycle stage . As described above , at 48 h-post L4 , most late pachytene nuclei in dsb-2 mutants had either 0 or 1 COSA-1 focus . These foci also accumulated ZHP-3 and ZHP-4 , which disappeared from the corresponding SCs on both sides of these sites ( Figure 6A and data not shown ) . SC proteins and the Polo kinase PLK-2 also became enriched along these chromosomes relative to nonexchange chromosomes ( Figure 6A and D ) , as previously described ( Machovina et al . , 2016; Pattabiraman et al . , 2017 ) . We also observed that PLK-2 , which plays a poorly characterized role in remodeling ( Harper et al . , 2011 ) , became co-enriched with ZHP-1/2 on one side of each CO ( Figure 6E , upper panel ) . Similar enrichment of PLK-2 along the short arm was observed in wild-type animals ( Figure 6—figure supplement 2A–B ) . This had not been apparent using PLK-2 antibodies , but was readily detected with epitope-tagged PLK-2 , as recently reported ( Harper et al . , 2011; Pattabiraman et al . , 2017 ) . When we co-depleted DSB-2 and ZHP-1/2 , ZHP-3 still accumulated at the few bright COSA-1 foci and was depleted from the corresponding SCs on both sides of these sites ( Figure 6B ) . This indicates that accumulation of pro-CO factors , rather than ZHP-1/2 activity per se , leads to depletion of ZHP-3/4 from the SC . However , none of the other hallmarks of CO-induced remodeling were observed in these animals , including accumulation of SC proteins and PLK-2 , and asymmetrical localization of PLK-2 , HTP-1/2 , and LAB-1 ( Figure 6B–E and data not shown ) . Instead , a small focus of PLK-2 was detected at CO sites ( Figure 6E , lower panel ) . During diplotene-diakinesis , SC proteins also became restricted to this focus , while HTP-1/2 persisted along all chromosome arms ( Figure 6C , lower panel ) . We also depleted PLK-2 with the AID system ( Figure 6F ) . Under these conditions , both the accumulation of pro-CO factors at a subset of recombination intermediates and the asymmetric localization of ZHP-1/2 were delayed , but still occurred ( Figure 6F , lower panel ) . Thus , ZHP-1/2 are required for the asymmetric localization of PLK-2 , but not vice versa , placing ZHP-1/2 upstream of PLK-2 in the remodeling pathway . Because ZHP-3/4 are strictly required for CO formation , we could not analyze their roles in chromosome remodeling . However , prior work has found that a GFP-tagged ZHP-3 transgene acts as a separation-of-function allele that is proficient for CO formation but defective for remodeling ( Bhalla et al . , 2008 ) . This remodeling process ultimately leads to the spatial partitioning of cohesin complexes containing distinct kleisin subunits to reciprocal chromosome domains ( Severson et al . , 2009; Severson and Meyer , 2014 ) . The most mature oocyte in each gonad arm , referred to as the ‘−1 oocyte , ’ normally shows enrichment of REC-8 along the long arms of each bivalent , while COH-3/4 are retained along the short arms . In ZHP-1/2 depleted worms , REC-8 persisted along all arms , while COH-3/4 virtually disappeared by the −1 oocyte ( Figure 6—figure supplement 2C ) , consistent with the absence of other ‘short arm’ hallmarks . Taken together , our analysis indicates that the ZHP circuit acts at or near the top of the hierarchy that mediates chromosome remodeling in response to CO designation , and directly couples these two key mechanisms to promote proper chromosome segregation . Our observations also imply that the persistence of SC along the short arm following CO designation plays an important role in remodeling by maintaining ZHP-1/2 and PLK-2 activity along this region . Together with previous observations , our findings indicate that a biochemical signaling cascade acts in cis within individual SC compartments to mediate and respond to CO designation ( Libuda et al . , 2013; Machovina et al . , 2016 ) . In C . elegans lacking the chromosome axis protein HTP-3 , large polycomplexes appear shortly after meiotic entry and persist throughout meiotic prophase ( Goodyer et al . , 2008; Rog et al . , 2017; Severson and Meyer , 2014 ) . These polycomplexes recapitulate both the internal order and the liquid-like behavior of SCs , and ZHP-3 concentrates within these bodies ( Rog et al . , 2017 ) . At mid-prophase , a focus of COSA-1 appears at the surface of each polycomplex . ZHP-3 also accumulates at these foci and gradually disappears from the interior of polycomplexes . Thus , ZHP-3 and COSA-1 recapitulate the dynamic relocalization and interdependence that they show at CO sites on the surface of polycomplexes , even in the absence of DSBs or recombination intermediates ( Rog et al . , 2017 ) . To investigate this circuitry further , we disrupted htp-3 in various tagged strains . All 4 ZHP proteins localized throughout polycomplexes during early prophase ( Figure 7A–B , and data not shown ) , consistent with the idea that they associate with SC proteins . In contrast to ZHP-3 ( Rog et al . , 2017 ) , ZHP-1/2 did not accumulate at COSA-1 foci , but instead remained diffusely localized throughout polycomplexes during late prophase ( Figure 7A–B and data not shown ) . COSA-1 foci appeared during late prophase even when ZHP-1/2 were depleted , but under these conditions , ZHP-3 remained distributed throughout polycomplexes in addition to concentrating with COSA-1 ( Figure 7C ) , further indicating that ZHP-1/2 promote the accumulation of ZHP-3/4 . We also found that MSH-5 colocalized with ZHP-3/4 and COSA-1 foci on polycomplexes ( Figure 7D ) . PLK-2 localized throughout polycomplexes even prior to the detection of COSA-1 foci , but became noticeably brighter afterwards , in the presence or absence of ZHP-1/2 ( Figure 7E–F ) . Together with the observations of dsb-2 mutants described above , these findings demonstrate that key aspects of the regulatory circuit that designates a subset of recombination intermediates for CO formation act autonomously within each contiguous SC or polycomplex . Remarkably , our study indicates that this circuit is triggered within polycomplexes in response to progression of nuclei into late prophase , despite the absence of any DSBs or CO intermediates in htp-3 mutants . These observations indicate that the ‘designation’ of one and only one site per compartment , which mirrors the logic of crossover assurance and interference , is an intrinsic property of this compartmentalized circuitry .
Evidence that the SC behaves as a phase-separated , liquid crystalline compartment suggested how this material might act as a conduit for diffusion of biochemical signals along the interface between paired chromosomes ( Rog et al . , 2017 ) . Here we provide evidence that CO assurance , CO interference , and CO maturation are coordinately regulated by the ZHP RING finger proteins ( Figure 8 ) , all of which are concentrated within the SC . Spatial confinement of the ZHPs within this interface between homologs can explain how each pair of chromosomes in the same nucleus is regulated independently , although CO designation is normally coupled to global cell cycle signals . These results add further support to the idea that CO regulation is mediated through this liquid crystalline medium . CO interference in C . elegans normally limits COs to one per homolog pair . This ‘winner takes all’ logic is reminiscent of other well-studied biological symmetry-breaking circuits , such as the selection of a single bud site by growing S . cerevisiae cells ( Goryachev and Pokhilko , 2008; Howell et al . , 2012; Ozbudak et al . , 2005 ) . Our evidence indicates that ZHP-1/2 are crucial for both positive feedback that promotes accumulation of pro-CO factors , including ZHP-3/4 , at a single recombination intermediate , and negative feedback that depletes these factors from other candidate sites ( Figure 8 ) . Similar feedback loops have been proposed in previous work on crossover regulation ( Qiao et al . , 2014; Rao et al . , 2017; Reynolds et al . , 2013; Yokoo et al . , 2012 ) . Indeed , coupled positive and negative feedback mechanisms are likely essential for any robust , switch-like decision executed by biological circuits ( Pfeuty and Kaneko , 2009 ) . A full understanding of CO control will clearly require identification of the substrates of the ZHP ligase family and the effects of their modification . A single crossover site could be selected , and other sites deselected , via a ‘trigger wave’ that moves rapidly through the SC from an initial site of stochastic CO designation and inhibits selection of nearby recombination intermediates ( Figure 8A ) . However , such a mechanism cannot easily be reconciled with observations that pro-crossover factors , including Zip3 , accumulate at recombination intermediates even prior to completion of synapsis in budding yeast , and likely in other organisms . Alternatively , coupled positive and negative regulation may give rise to a periodic standing wave , or Turing pattern ( Turing , 1952 ) , of CO designation potential along each chromosome . This type of patterning requires interaction between two diffusible activities: an activating activity – or ‘morphogen , ’ to use Turing’s term – that acts locally and shows autocatalytic or cooperative behavior , and an inhibitory activity that acts over a greater distance . This type of pattern has intrinsic periodicity that depends on the activities and diffusion rates of the activator and inhibitor . Such a pattern could thus be established prior to full SC assembly , in which case designation of the first CO site would spatially fix one node of the pattern ( Figure 8A ) . A Turing patterning mechanism is also consistent with evidence presented here and in previous studies that stabilization of recombination intermediates and CO patterning are tightly coupled . Evidence that mammalian RNF212 and Arabidopsis HEI10 act as dosage-sensitive modulators of recombination rates ( Qiao et al . , 2014; Reynolds et al . , 2013; Ziolkowski et al . , 2017 ) is compatible with either a temporally-dependent or standing wave model , since an increase or decrease in these activities could alter either the rate of signal propagation or the frequency of a waveform along paired chromosomes . While the SC is an unusual , liquid crystalline material , liquid-liquid phase separation ( LLPS ) has been implicated in subcellular compartmentalization of diverse signaling mechanisms ( Banani et al . , 2017 ) . By diffusing and interacting within a spatially confined liquid compartment , biochemical signals could potentially establish a variety of periodic , Turing-type patterns within cells . Analogously , diffusion of signaling molecules within cell membranes can create two-dimensional patterns , which have been proposed to orchestrate polarity decisions ( Goryachev and Pokhilko , 2008 ) , as well as more elaborate patterning such as hexagonal arrays of stereocilia ( Jacobo and Hudspeth , 2014 ) . Intriguingly , the ZHP proteins and their homologs are very similar to TRIM E3 ligases ( tripartite motif: RING , B-box , coiled-coil domain ) , a very large family of enzymes with diverse cellular roles . The functions of most TRIM ligases are unknown , but like the ZHP proteins , they often form homo- or heterodimers , and many of these proteins appear to localize to cellular compartments , including globular nuclear and cytoplasmic bodies , or to filamentous structures such as microtubules ( Rajsbaum et al . , 2014; Reymond et al . , 2001 ) . Thus , we speculate that compartmentalized regulatory mechanisms with similarities to the circuitry illuminated here may control other spatially-patterned activities within cells . RING finger proteins homologous to the ZHP family play important roles in CO formation in diverse eukaryotes ( Agarwal and Roeder , 2000; Ahuja et al . , 2017; Chelysheva et al . , 2012; Gray and Cohen , 2016; Lake et al . , 2015; Qiao et al . , 2014; Rao et al . , 2017; Reynolds et al . , 2013; Wang et al . , 2012; Ward et al . , 2007; Zhang et al . , 2014a ) . Interestingly , the number of these proteins varies among organisms . The cytological distributions and apparent functions of ZHP-3/4 and ZHP-1/2 are very similar to those reported for the mammalian recombination regulators RNF212 and HEI10 , respectively . Like ZHP-3/4 , mouse RNF212 is required to stabilize recombination intermediates ( Reynolds et al . , 2013 ) , while HEI10 , like ZHP-1/2 , is required to limit the number of RNF212/MutSγ-positive foci along chromosomes and for robust CO maturation ( Qiao et al . , 2014 ) . Most ZHP homologs also contain predicted coiled-coil domains and localize to the central region of the SC , including Zip3 in budding yeast , which forms foci along chromosomes but localizes throughout polycomplexes . A notable exception is a recently identified homolog required for crossovers in the ciliate Tetrahymena thermophila . This protein lacks a coiled-coil domain , likely related to the fact that this organism lacks SCs ( Shodhan et al . , 2017 ) . HEI10 in Sordaria and Arabidopsis , and Zip3 in budding yeast , may be bifunctional proteins with roles similar to both ZHP-1/2 and ZHP-3/4 , as suggested by their localization to CO sites and the apparent absence of RNF212 in these clades ( Agarwal and Roeder , 2000; Chelysheva et al . , 2012; De Muyt et al . , 2014 ) . We speculate that they could play dual roles by partnering with multiple E2 conjugating enzymes , or perhaps with distinct substrate-recognition factors . In mammals , RNF212 and HEI10 are thought to mediate opposing pro- and anti-crossover activities through SUMOylation and SUMO-dependent ubiquitylation , respectively ( Qiao et al . , 2014; Rao et al . , 2017; Reynolds et al . , 2013 ) ; a similar ‘SUMO-ubiquitin switch’ has been proposed based on findings in Sordaria ( De Muyt et al . , 2014 ) . In budding yeast , Sordaria , and mice , SUMOylation is essential for SC assembly , and thus for CO regulation ( De Muyt et al . , 2014; Hooker and Roeder , 2006; Klug et al . , 2013; Leung et al . , 2015; Lin et al . , 2010; Rao et al . , 2017; Voelkel-Meiman et al . , 2013 ) . In C . elegans we do not detect SUMO cytologically either along the SC or at CO sites , even with highly sensitive tools such as epitope-tagged SUMO transgenes ( Pelisch and Hay , 2016; data not shown ) . Further , genetic analysis has indicated that SMO-1 ( SUMO ) and its E2 conjugating enzyme UBC-9 are dispensable for synapsis and CO control in C . elegans , although they play key roles downstream of COs to mediate meiotic chromosome segregation in oocytes ( Bhalla et al . , 2008; Pelisch et al . , 2017 ) . Taken together , we favor the idea that each pair of ZHP proteins acts as a ubiquitin ligase , like the ‘lion’s share’ of RING finger proteins ( Deshaies and Joazeiro , 2009 ) . Our analysis also reveals that the C . elegans ZHP proteins likely function as heterodimers , or perhaps as higher order oligomers . It will be interesting to determine whether ZHP homologs in other organisms also have obligate partners , or perhaps homooligomerize , since this knowledge may help to illuminate their in vivo activities and substrates . In addition to their roles in CO control , the ZHP family also link COs to chromosome remodeling , which enables the stepwise release of cohesion during the MI and MII divisions ( Figure 8 ) . Specifically , we find that CO designation results in enrichment of ZHP-1/2 along one chromosome arm , which in turn promotes concentration of PLK-2 within this domain , asymmetric disassembly of the SC , and spatial regulation of cohesin function . It is not clear whether sister chromatid cohesion during meiosis is regulated in the same fashion in other organisms . In budding and fission yeasts , Drosophila , diverse plants , and mammals , cohesion near centromeres is specifically retained during the first division ( Duro and Marston , 2015 ) . Cohesin complexes containing the meiosis-specific kleisin Rec8 are enriched in pericentromeric heterochromatin and sometimes at centromere cores , and Shugoshin/MEI-S332 , a Rec8-specific protective factor , is also recruited to these regions . In principle , programmed release of cohesion along arms and its retention near centromeres could suffice to allow homologs to segregate in MI while maintaining a link between sisters until MII . However , experimental evidence from several organisms indicates that CO formation leads to a local disruption of cohesion , which can predispose chromosomes to missegregate when COs occur close to centromeres ( Brar and Amon , 2008 ) . In light of other similarities between ZHP-1/2 and HEI10 , this raises the possibility that HEI10 may also influence sister chromatid cohesion , perhaps via spatial control of Polo-like kinases , which promote release of cohesion in mitosis and meiosis ( Archambault and Glover , 2009 ) . If so , HEI10 activity may directly impact chromosome missegregation in human oocytes , which has been attributed to compromised cohesion , and also – very recently – to inefficient CO maturation ( Wang et al . , 2017 ) , another process that involves HEI10 .
All C . elegans strains were maintained on nematode growth medium ( NGM ) plates seeded with OP50 bacteria at 20°C . All epitope-tagged genes used in this study were fully functional , as indicated by their ability to support normal meiosis ( Supplementary file 1 , Table S1 ) . New alleles and their detailed information are listed in Supplementary file 1 , Tables S2 and S3 . Unless otherwise indicated , new alleles in this study were generated by genome editing , by injection of Cas9-gRNA RNP complexes , using dpy-10 Co-CRISPR to enrich for edited progeny ( Arribere et al . , 2014; Paix et al . , 2015 ) . Cas9-NLS protein was produced by the MacroLab at UC Berkeley . The protein was complexed in vitro with equimolar quantities of duplexed tracrRNA and target-specific crRNAs , purchased from Integrated DNA Technologies ( IDT ) . Repair templates were single- or double-stranded amplicons generated by asymmetric or standard PCR , respectively , or Ultramer oligonucleotides purchased from IDT . Each 10 µl of injection mixture contained: 16 µM [Cas9 protein + gRNA] ( target gene + dpy-10 ) , plus 0 . 16–2 µM long and/or 6 µM oligonucleotide repair templates . In cases where Unc worms were injected ( e . g . , to tag zhp genes in a syp-1 ( me17 ) /nT1 strain ) , plasmids encoding green fluorescent reporter proteins were co-injected to enrich for edited progeny . Injected animals were transferred to individual plates and incubated at 20 ˚C . After 4 days , F1 Rol or Dpy progeny ( dpy-10 heterozygotes or homozygotes , respectively ) from ‘jackpot’ broods containing multiple Rol/Dpy animals were picked to individual plates , allowed to produce self-progeny , then lysed and screened by PCR . Disruption of the zhp-1 gene was accomplished by inserting the sequence TAAGCTCGAG after the 6th codon , introducing a stop codon , a frameshift , and an Xho I restriction site . Three independent alleles of the same sequence were generated: ie43 and ie44 in the N2 strain background , and ie46 in the CB4856 strain . The zhp-2 gene was similarly disrupted by insertion of TAATAATTAATTAG after the 7th codon , resulting in multiple stop codons and a frameshift . See Supplementary file 1 , Table S4 for a full list of strains used . Unless otherwise indicated , young adults were used for both immunofluorescence and Western blotting assays . Synchronized young adults were obtained by picking L4 larvae and culturing them for 20–24 hr at 20°C . To deplete AID-tagged proteins in a strain expressing the AtTIR1 transgene , animals were treated with auxin ( indole acetic acid , IAA ) as previously described ( Zhang et al . , 2015 ) . Briefly , NGM agar was supplemented with 1 mM IAA just before pouring plates . E . coli OP50 bacteria cultures were concentrated and spread on plates , which were allowed to dry before transferring C . elegans onto the plates . We note that several major advantages of the AID system have facilitated our study of these proteins in meiosis: ( i ) this approach enables temporal control and germline-specific degradation of proteins; ( ii ) complex strains that combine various mutations and epitope-tagged alleles can be constructed far more easily , since balancer chromosomes are not required to maintain conditional alleles of genes essential for reproduction . Additionally , control experiments can be performed using the same strains without auxin treatment , and partial protein depletion can be achieved by titrating auxin concentrations . To quantify brood size and males among self-progeny , L4 hermaphrodites were picked onto individual plates and transferred to new plates daily over 4 days . Eggs were counted every day . Viable progeny and males were counted when the F1 reached the L4 or adult stages . To analyze viability and fertility in the presence of auxin , L1s were transferred onto auxin plates and grown to the L4 stage . Hermaphrodites were then transferred onto fresh auxin plates , transferred daily over 4 days , and progeny were scored as described above . The zhp-1 gene was disrupted by CRISPR/Cas9-based genome editing in both the N2 Bristol and CB4856 Hawaiian strain backgrounds , as described above ( allele designations: ie43 and ie46 , respectively ) . By crossing animals carrying these mutations , we generated N2/CB4856 hybrids lacking zhp-1 . To map meiotic COs that occurred during oogenesis in these animals , hybrid hermaphrodites were backcrossed to ( zhp-1+ ) CB4856 males . Hermaphrodite progeny from this cross were picked to individual 60 mm plates and allowed to produce self-progeny until the plates were starved . Genomic DNA was extracted from these pools of progeny using Gentra Puregene Tissue Kit ( QIAGEN , Hilden , Germany ) , and quantified by Qubit ( Invitrogen ) . Sequencing libraries were constructed in the Functional Genomics Lab ( FGL ) , a QB3-Berkeley Core Research Facility at UC Berkeley . DNA was Fragmented with an S220 Focused-Ultrasonicator ( Covaris , Woburn , MA ) , then cleaned and concentrated with the MinElute PCR Purification kit ( QIAGEN ) . Library preparation was done on an Apollo 324 with PrepX ILM 32i DNA Library Kits ( WaferGen Biosystems , Fremont , CA ) . 9 cycles of PCR amplification were used . The average insert size of libraries was 380 bp . 150PE sequencing at approximately 9x coverage was carried out on an Illumina Hiseq 2500 by the Vincent J . Coates Genomics Sequencing Laboratory at UC Berkeley . Two libraries for each of the parental strains ( N2 and CB4856 ) were prepared in parallel , sequenced at 30x coverage , and aligned to the reference genome sequences available through Wormbase or ( Thompson et al . , 2015 ) . To map COs , the data were analyzed using the MSG software package ( https://github . com/JaneliaSciComp/msg ) ( Andolfatto et al . , 2011 ) . To test for interactions among the ZHP proteins using the yeast two-hybrid system , coding sequences for ZHP-1-V5 , ZHP-2-3xFLAG , ZHP-3-3xFLAG and ZHP-4-HA were synthesized as gBlocks by Integrated DNA Technologies ( IDT ) . Epitope tags were appended to the protein sequences so that expression in yeast could be monitored . The coding sequence for ZHP-4 was codon-optimized by IDT to facilitate gBlock synthesis and expression in yeast . Each of these sequences was fused to both the GAL4 DNA binding domain and the GAL4 activation domain by insertion into pDEST32 and pDEST22 , respectively , using the Gateway cloning system , following the manufacturer’s instructions ( Invitrogen ) . All possible pairwise combinations of the four ZHP proteins , including potential homodimers , were tested using the ProQuestTM Two-Hybrid System ( Invitrogen ) . Briefly , each pair of bait and prey plasmids was co-transformed into yeast strain MaV203 using standard LiAc-mediated transformation . To test for specific interactions , 2–4 independent clones of each strain were assayed for HIS3 , URA3 and LacZ induction . Negative and positive controls provided with the ProQuestTM kit ( Cat . Series 10835 ) were run in parallel . Immunofluorescence experiments were performed as previously described ( Zhang et al . , 2015 ) , except that samples were mounted in Prolong Diamond mounting medium with DAPI ( Millipore Sigma ) . Primary antibodies were obtained from commercial sources or have been previously described , and were diluted as follows: Rabbit anti-SYP-1 [1:500 , ( MacQueen et al . , 2002 ) , Rabbit anti-SYP-2 [1:500 , ( Colaiácovo et al . , 2003 ) , Rabbit anti-HTP-1 [1:500 , ( Martinez-Perez et al . , 2008 ) , Rabbit anti-RAD-51 ( 1:5 , 000 , Novus Biologicals , #29480002 ) , Rabbit anti-pHIM-8/ZIMs [1:500 , ( Kim et al . , 2015 ) , Rabbit MSH-5 [1:5 , 000 , modENCODE/SDIX , #SDQ2376 , ( Yokoo et al . , 2012 ) , Rabbit anti-REC-8 ( 1:5 , 000 , modENCODE/SDIX , #SDQ0802 ) , Rabbit anti-COH-3 ( 1:5 , 000 , modENCODE/SDIX , #SDQ3972 ) , Guinea pig anti-PLK-2 [1:100 , ( Harper et al . , 2011 ) , Goat anti-SYP-1 [1:300 , ( Harper et al . , 2011 ) , Chicken anti-HTP-3 [1:500 , ( MacQueen et al . , 2005 ) , Rabbit anti-ZHP-3 [1:5 , 000 , modENCODE/SDIX , #SDQ3956 , ( Rog et al . , 2017 ) , Rat anti-HIM-8 [1:500 , ( Phillips et al . , 2005 ) , Mouse anti-FLAG ( 1:500 , Sigma , #F1804 ) , Mouse anti-HA ( 1:400 , Thermo Fisher , #26183 ) , Mouse anti-V5 ( 1:500 , Thermo Fisher , #R960-25 ) , Rabbit anti-V5 ( 1:250 , Millipore Sigma , #V8137 ) , Mouse anti-GFP ( 1:500 , Millipore Sigma , #11814460001 ) . Secondary antibodies labeled with Alexa 488 , Cy3 or Cy5 were purchased from Jackson ImmunoResearch ( West Grove , PA ) and used at 1:500 . Chromosomal in situ hybridization was performed as previously described ( Dernburg et al . , 1998 ) with modifications . Briefly , worms were dissected and fixed essentially according to the immunofluorescence protocol described above . Fixed worm gonads were then washed with 2xSSCT and incubated in 50% formamide/2xSSCT overnight at 37°C . Tissue and probes were denatured at 91°C for 2 min , then hybridized overnight at 37 ˚C . Hybridized gonads were then washed and stained with DAPI . Two chromosome-specific probes were used: an oligonucleotide targeting a short repetitive sequence on the X chromosome ( Lieb et al . , 2000; Phillips et al . , 2005 ) and the 5S rDNA repeat on the right arm of chromosome V ( Dernburg et al . , 1998 ) . FISH probes were labeled with aminoallyl-dUTP ( Millipore Sigma ) by terminal transferase-mediated 3’ end-labeling , followed by conjugation to Alexa 488-NHS-ester ( Thermo Fisher Scientific ) or Cy3-NHS-ester ( Themo Fisher Scientific ) , as previously described ( Dernburg , 2011 ) All images were acquired as z-stacks through 8 µm depth at intervals of 0 . 2 µm using a DeltaVision Elite microscope ( GE ) with a 100x , 1 . 4 N . A . oil-immersion objective . Iterative 3D deconvolution , image projection and colorization were carried out using the SoftWoRx package and Adobe Photoshop CC 2014 . Adult worms were picked into SDS sample buffer and lysed by boiling for 30 min , a single freeze/thaw cycle in liquid nitrogen , and occasionally vortexing . Whole worm lysates were separated on 4–12% polyacrylamide gradient gels and blotted with indicated antibodies: Mouse anti-GFP ( 1:1 , 000 , Millipore Sigma , #11814460001 ) , Mouse anti-FLAG ( 1:1 , 000 , Millipore Sigma , #F1804 ) , Mouse anti-HA ( 1:1 , 000 , Thermo Fisher , #26183 ) , Mouse anti-V5 ( 1:1 , 000 , Thermo Fisher , #R960-25 ) , Mouse anti-α-tubulin ( 1:5 , 000 , Millipore Sigma , #05–829 ) , Guinea pig anti-HTP-3 [1:1 , 500 , ( MacQueen et al . , 2005 ) . HRP-conjugated secondary antibodies ( Jackson Immunoresearch #115-035-068 ) and SuperSignal West Femto Maximum Sensitivity Substrate ( Thermo Fisher , #34095 ) were used for detection . To quantify Western blots , TIF images were collected for each blot using a Chemidoc system ( Bio-Rad , Hercules , CA ) . Integrated intensities for relevant bands were then calculated using ImageJ . To normalize for sample loading , the indicated band intensity was divided by the corresponding α-tubulin or HTP-3 band intensity . Each normalized band intensity is expressed as the percentage of the intensity at t = 0 or in a control sample . Quantification methods and statistical parameters are indicated in the legend of each figure , including error calculations ( SD or SEM ) , n values , statistical tests , and p-values . p<0 . 05 was considered to be significant .
During meiosis , each pair of homologous chromosomes typically undergoes at least one crossover ( crossover assurance ) , but these exchanges are strictly limited in number and widely spaced along chromosomes ( crossover interference ) . The molecular basis for this chromosome-wide regulation remains mysterious . A family of meiotic RING finger proteins has been implicated in crossover regulation across eukaryotes . Caenorhabditis elegans expresses four such proteins , of which one ( ZHP-3 ) is known to be required for crossovers . Here we investigate the functions of ZHP-1 , ZHP-2 , and ZHP-4 . We find that all four ZHP proteins , like their homologs in other species , localize to the synaptonemal complex , an unusual , liquid crystalline compartment that assembles between paired homologs . Together they promote accumulation of pro-crossover factors , including ZHP-3 and ZHP-4 , at a single recombination intermediate , thereby patterning exchanges along paired chromosomes . These proteins also act at the top of a hierarchical , symmetry-breaking process that enables crossovers to direct accurate chromosome segregation .
Most human cells contain 23 pairs of chromosomes , giving 46 chromosomes in total . When a cell divides , it typically copies all its chromosomes and distributes the copies equally between the two new cells , so that they also have 46 chromosomes . Cells in the reproductive organs undergo a special division process called meiosis , which halves the number of chromosomes . As a result , sperm and eggs have just 23 chromosomes , and so when they combine , the fertilized egg receives a complete set of 46 chromosomes . A similar process happens in all species that use sexual reproduction . As the chromosomes prepare to separate , they line up side by side in matching pairs . During this period , DNA from one chromosome will swap with DNA from its partner . These events are called “crossovers , ” and because such exchanges can happen at many locations along the chromosomes , no two sperm or eggs are the same . Historic studies in fruit flies revealed that chromosomes do not mix their DNA at random . After one crossover occurs , it is less likely that another will happen , and if there are two crossovers , the second one tends be far away from the first . This suggests that there must be a signal that tells the chromosomes about the exchange . However , the nature of the signals and how they are communicated along pairs of chromosomes remain mysterious . A structure called the “synaptonemal complex” holds chromosomes together while they mix their DNA . In 2017 , researchers found that this structure behaves like a liquid crystal: its molecules are organized into a regular , repeating pattern , but they move freely , like a fluid . If signals could move through this material , this might explain how information spreads along paired chromosomes . Now , Zhang et al . – including two researchers involved in the 2017 work – identify some of the signals in the small roundworm , Caenorhabditis elegans , as four related proteins named ZHPs . When tagged with fluorescent markers and followed under a microscope , all four ZHP proteins moved to the liquid crystal-like synaptonemal complex during meiosis . Depleting the proteins at this crucial time revealed their roles . Two of the proteins are needed for chromosomes to mix their DNA , while the other two control the number of exchanges between each pair of chromosomes . Successful meiosis depended upon all four ZHPs , and so too did the fertility of the worms . The next step is to find the other molecules that interact with the ZHP proteins during meiosis . Since similar proteins appear in other species , including humans , this could help to reveal more about how genetic traits from our parents mix and match . In the future , studies that build on these findings could also help scientists to understand how errors in these processes give rise to birth defects and infertility .