Volume 149, Issue 1, Pages 75-87 (March 2012) COSA-1 Reveals Robust Homeostasis and Separable Licensing and Reinforcement Steps Governing Meiotic Crossovers  Rayka Yokoo, Karl A. Zawadzki, Kentaro Nabeshima, Melanie Drake, Swathi Arur, Anne M. Villeneuve  Cell  Volume 149, Issue 1, Pages 75-87 (March 2012) DOI: 10.1016/j.cell.2012.01.052 Copyright © 2012 Elsevier Inc. Terms and Conditions

Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 COSA-1 Is Required to Convert Meiotic DSBs into Interhomolog COs (A) Full karyotypes of individual diakinesis-stage oocytes. Six DAPI-stained bodies in the wild-type nucleus correspond to six pairs of homologs connected by chiasmata; 12 individual chromosomes (univalents) in the cosa-1mutant nucleus reflect a lack of chiasmata. (B) cosa-1mutant pachytene nuclei in which pairing was assessed either by FISH at the 5S rDNA locus (chromosome V) or by immunostaining for X chromosome pairing center (X-PC)-binding protein HIM-8. A single FISH or HIM-8 signal in each nucleus indicates successful pairing. (C) cosa-1 mutant pachytene nuclei, showing colocalization of SC lateral element protein HIM-3 and SC central region protein SYP-1 between parallel tracks of DAPI-stained chromatin. (D) Immunolocalization of RAD-51 in mid-to-late pachytene nuclei in the cosa-1 mutant. RAD-51 foci indicative of DSB formation are abundant in midpachytene and are greatly reduced or absent in most late-pachytene nuclei (asterisk indicates an apoptotic nucleus). (E) Early diplotene nuclei. In WT, SYP-1 and HTP-1/2 are localized to reciprocal domains on each chromosome pair; in cosa-1, this indicator of CO formation is not observed, as SYP-1 and HTP-1/2 remain extensively colocalized. Scale bar, 5 μm. See also Table S1. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 COSA-1 Is a Distant Member of the Cyclin Superfamily with Orthologs in Metazoa (A) (Top) Predicted gene structure of C. elegans cosa-l with mutant alleles indicated. Gray, UTR; magenta, coding exons. (Bottom) Construct used to express GFP::COSA-1. Green, GFP coding sequence; blue, extra tags and linker sequences. (B) Phylogenetic tree depicting a sampling of metazoan species. Green indicates lineages in which COSA-1, MSH-4, and MSH-5 orthologs are all present; red indicates the absence of all three from Drosophilid species. (C) Predicted structure of residues 56–360 of C. elegans COSA-1 (yellow and magenta) aligned with crystal structure of residues 167–426 of human cyclin B1 (cyan). N-terminal residues of COSA-1 and cyclin B1 were removed to aid visualization of the two core cyclin-fold motifs. In canonical cyclins, cyclin-fold motifs consist of five α helices, with well-conserved interhelical angles in the N-terminal cyclin box motif. In the predicted COSA-1 structure, the N-terminal cyclin box is interrupted by an insertion of 33 amino acids, modeled here as an extension of α helix 2 and an additional helix (magenta, α-2.5). Predicted α helices 3–5 of COSA-1 align well with the corresponding helices of cyclin B1 and cyclin A, which contribute to the cyclin/CDK interface in cyclin A/CDK2 (Jeffrey et al., 1995). See also Figures S1 and S6 and Extended Experimental Procedures. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 GFP::COSA-1 Localizes to Foci Corresponding to CO Sites (A and B) IF images of a portion of a gonad extending from midpachytene through diplotene and early diakinesis. GFP::COSA-1 foci are detected from late pachytene through early diakinesis. (A) (Left inset) Late-pachytene nuclei, each containing six bright foci. (Right insets) Diplotene nuclei, with one focus on each chromosome pair; bottom panel shows COSA-1 foci positioned at the site of the single emerging chiasma on each chromosome pair. (B) Relationship between localization of GFP::COSA-1 and ZHP-3. (Upper-left inset) Six COSA-1 foci in a midpachytene nucleus with ZHP-3 in long stretches along the chromosomes. (Upper-right inset) COSA-1 localized at one end of each comet-like stretch of ZHP-3. (Bottom inset) COSA-1 and ZHP-3 colocalization in a diplotene nucleus. (C) Representative images of GFP::COSA-1 localization in late-diplotene/early diakinesis nuclei, highlighting the location of GFP::COSA-1 at the site of the single emerging chiasma on each chromosome pair. (Large panels) Full projections of entire nuclei showing all six bivalents; asterisk indicates a bivalent depicted in smaller panels, which shows partial projections of individual bivalents. Scale bars, 5 μm except in the single bivalent panels in (C), in which scale bar is 1 μm. See also Figures S2 and S3. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 MSH-5 Colocalizes with and Depends on COSA-1 (A) IF images showing that MSH-5 foci are detected in midpachytene nuclei in excess of eventual COs (cyan inset) and then decline by late pachytene, when they colocalize with GFP::COSA-1 foci (yellow inset). (B) (Left) Late-pachytene nuclei from a wild-type germline, showing comet-like localization of ZHP-3 with COSA-1 foci at the comet heads. (Right) Late-pachytene nuclei from a cosa-1 mutant, showing persistence of ZHP-3 localization along the length of the chromosomes and a lack of MSH-5 foci. Scale bars, 5 μm; for insets, scale bars, 1 μm. See also Figure S4. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Time Course of Localization of CO Proteins at IR-Induced Recombination Sites Immunolocalization of CO proteins (GFP::COSA-1, MSH-5, and/or ZHP-3) in pachytene nuclei from gfp::cosa-1; spo-11 worms, either in the absence of IR (A, left) or at the indicated times following exposure to 1 kRad IR. Scale bars, 5 μm. (A) Localization of COSA-1 and ZHP-3 or MSH-5 in late-pachytene nuclei in the absence of IR (pre-IR) and 8 hr post-IR. In the unirradiated spo-11 control, ZHP-3 persists along the lengths of the chromosomes, and the majority of nuclei lack COSA-1 and MSH-5 foci; a subset of nuclei have one or two COSA-1/MSH-5 aggregates (indicated by asterisks). 8 hr post-IR: six bright COSA-1 foci localize at the heads of comet-like ZHP-3 signals. (B) Mid-to-late pachytene region of a 1 hr post-IR germline. Abundant IR-induced MSH-5 foci are detected specifically in midpachytene nuclei (left), whereas MSH-5 foci are not detected above baseline in late-pachytene nuclei (right; 0, 1, or 2 MSH-5 signals colocalize with COSA-1, as in unirradiated controls). (C) GFP::COSA-1 localization in nuclei within the late-pachytene region at 2.5 and 4 hr post-IR; fields also include a few midpachytene nuclei (at the left) and a few early diplotene nuclei (at the right). Circles indicate nuclei in which six COSA-1 foci are detected. At 2.5 hr post-IR, nuclei with six COSA-1 foci are limited to a narrow zone near the start of the late-pachytene region. At 4 hr post-IR, the zone of nuclei with six COSA-1 foci has expanded, presumably reflecting movement into and progression through late pachytene of nuclei that had been exposed to IR during midpachytene. (D) Localization of MSH-5 and COSA-1 at 8 hr post-IR in a region spanning the mid-to-late pachytene transition. (Left inset) Midpachytene nuclei, showing MSH-5-only foci, in excess of eventual COs. (Right) Late-pachytene nuclei, showing six MSH-5 foci that colocalize with six COSA-1 foci. See also Figure S5. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Dose-Response Analysis Reveals a Highly Nonlinear Relationship between IR-Induced DSBs and COSA-1 Foci (A) Paired IF images showing GFP::COSA-1 foci in late-pachytene nuclei from gfp::cosa-1; spo-11 germlines exposed to the indicated IR doses, fixed 8 hr post-IR, with numbers of foci in each nucleus indicated. Scale bar, 5 μm. (B) Stacked bar graph showing percentages of nuclei with the indicated numbers of COSA-1 foci at different IR doses. (C) Graph showing the highly nonlinear relationship between IR dose and the mean number of COSA-1 foci per nucleus. Experimental data points are plotted in red, with error bars indicating standard deviation. Our mathematical model (μ = 6(1 − e−cr)) is plotted in blue; see Results and Extended Experimental Procedures. (D) Graph depicting linear relationship between IR dose and inferred mean number of DSBs per chromosome pair, calculated from our empirical data based on the postulates of our model. Empirical data points are in red, with linear regression in blue. See also Extended Experimental Procedures. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Relationship of COSA-1 to COs in Conditions that Alter CO Number (A) Graph showing percentages of nuclei with the indicated numbers of COSA-1 foci in strains with altered numbers of COs. Numbers of foci in the rtel-1(tm1866) and dpy-28(s939) mutants did not differ significantly from the control (Mann-Whitney test). Worms homozygous for the mnT12(X;IV) fusion chromosome have only five chromosome pairs, and mnT12 undergoes only one CO in the majority of meioses; an average of 5.3 COSA-1 foci per nucleus was observed in mnT12 homozygotes. Numbers of COSA-1 foci in rtel-1; mnT12 worms did not differ significantly from mnT12 controls. ∗WT control contains the gfp::cosa-1 transgene in an otherwise wild-type background. (B) The rtel-1 mutation does not suppress the lack of chiasmata caused by loss of cosa-1 function. Graph shows percent of diakinesis nuclei with a given number of DAPI-stained bodies. As in cosa-1(tm3298) single mutants, 11–12 DAPI bodies were detected in rtel-1; cosa-1 double mutants, reflecting a lack of chiasmata. Numbers of oocyte nuclei scored: wild-type (n = 164), rtel-1 (n = 125), cosa-1 (n = 116), rtel-1; cosa-1 (n = 114). (C) Bar graph indicating genetic map distances (cM ± 95% C.I.) for the unc-60 dpy-11 interval measured for worms of the indicated genotypes (see Extended Experimental Procedures). ∗∗p < 0.001; ∗p = 0.01. The CO frequency in the rtel-1 mutant (19.6 cM) was significantly elevated over wild-type (13.4 cM, p = 0.0002; Fisher's exact test) and cosa-1/+ (13.6 cM, p = 0.0006) controls, which did not differ from each other. rtel-1 also differed significantly from rtel-1; cosa-1/+ (15.6 cM, p = 0.01), indicating that elevation in CO frequency was suppressed in rtel-1; cosa-1/+ worms. (D) Paired 3D volume renderings of a representative nucleus used to quantify X chromosome-associated COSA-1 foci in the dpy-28 mutant. Staining for chromosome axis protein HTP-3 reveals the paths of synapsed chromosome pairs; arrow indicates the X chromosome, marked by X-PC-associated protein HIM-8. Scale bar, 2 μm. Expected incidence of X chromosomes with two or more COSA-1 foci was estimated to be ≥ 36% based on frequencies of 2-CO and 3-CO products detected by genetic assay (Tsai et al., 2008). See also Table S1. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S1 Predicted Structure of Human COSA1/CNTD1 Shows Similarity to Bovine Cyclin A, Related to Figure 2 The predicted structure of residues 47-330 of human COSA1/CNTD1 (yellow, magenta) aligned with the crystal structure of residues 208-430 of bovine cyclin A (cyan). N-terminal protein segments of both COSA1/CNTD1 and cyclin A have been removed to enable visualization of the N- and C-terminal cyclin fold motifs. In the context of canonical cyclins, cyclin-fold motifs consist of 5 α helices, with well-conserved interhelical angles in the N-terminal cyclin box motif. In the predicted CNTD1 structure, the N-terminal cyclin box motif is interrupted by an insertion of approximately 26 amino acids, modeled here as an extension of α-helix 2 and an additional helix (magenta, designated α-2.5). Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S2 The gfp::cosa-1 Transgene Rescues the Diakinesis Phenotype of cosa-1(tm3298), Related to Figure 3 Bar graph depicting quantitation of the complementation experiment described in Supplemental Experimental Procedures. As in wild-type controls, 6 DAPI-stained bodies were detected in most diakinesis-stage oocytes in meIs8; cosa-1(tm3298) worms (which carry a transgene expressing GFP::COSA-1), reflecting successful chiasma formation. In contrast 11-12 DAPI stained bodies were detected in cosa-1 (tm3298) oocytes, reflecting a lack of chiasmata. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S3 RAD-51 and GFP::COSA-1 Foci Exhibit Distinct Temporal and Spatial Localization Patterns, Related to Figure 3 Immunofluorescence images showing a portion of a wild-type germline extending from the mid-to-late pachytene stage. RAD-51 foci are abundant in midpachytene nuclei and decline at the mid-to-late pachytene transition, when COSA-1 foci begin to appear. Insets show fields of nuclei containing only RAD-51 foci (left, midpachytene), only COSA-1 foci (right, late pachytene) or both (middle, transition). Even when RAD-51 and COSA-1 foci are detected in the same nucleus, they do not co-localize. Scale bar = 5 μm. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S4 COSA-1 Localization Is Dependent on MSH-5 and ZHP-3, Related to Figure 4 (A) Immunofluorescence images showing a portion of a germline extending from midpachytene to early diplotene. GFP::COSA-1 localizes to 6 bright foci in late-pachytene and diplotene nuclei in the heterozygous msh-5(me23)/+ control germline but is not detected above background in the homozygous msh-5(me23) mutant. Scale bar = 5 μm. (B) Bar graph indicating percent of worms exhibiting normal GFP::COSA-1 localization (6 bright foci per nucleus) or abnormal GFP::COSA-1 localization (0 or 1 focus/aggregate per nucleus) among zhp-3(me95) homozygotes (n = 100) and sibling controls (+/+ and zhp-3/+; n = 100). GFP::COSA-1 was visualized in live worms that also expressed mCherry::Histone H2B. mCherry::Histone H2B was used to visualize chromosomes in order to distinguish zhp-3(me95) homozygotes (which had 12 univalents in diakinesis oocytes) from controls (which had 6 bivalents). Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S5 Localization of Recombination Proteins in Irradiation Time Course Experiments, Related to Figure 5 Immunofluorescence images showing localization of recombination proteins in germ cell nuclei from spo-11(me44) mutant worms expressing GFP::COSA-1. Scale bars = 5 μm. (A) Nuclei from the mid-to-late pachytene transition region, fixed 2.5 hr post-IR and stained for RAD-51 and COSA-1 foci. Both RAD-51 and COSA-1 foci are detected, but they do not colocalize. (B) Late-pachytene nuclei from a germline fixed at 4 hr post-IR, stained for ZHP-3 and GFP::COSA-1. COSA-1 is localized in bright foci marking the presumptive CO sites, and ZHP-3 has begun to relocalize, exhibiting local peaks of high signal intensity that colocalize with the COSA-1 foci. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S6 Recombinant MSH-5 Is Phosphorylated In Vitro by Human CDK1/CyclinA2, Related to Figure 2 Left, autoradiograph showing P32 incorporated into 100 ng of Histone H1, His tagged MSH-5 and GFP during incubation with purified human CDK1/CyclinA2 kinase. In vitro kinase assays were performed for 15 min using 5U of recombinant CDK1/CyclinA2 kinase and 100 ng of target protein per reaction. Right, Coomassie blue-stained 10% SDS PAGE gel run in parallel with the assay gel, showing the corresponding purified target proteins used in these assays. Cell 2012 149, 75-87DOI: (10.1016/j.cell.2012.01.052) Copyright © 2012 Elsevier Inc. Terms and Conditions