PCH-2 controls the number and distribution of crossovers in similar patterns on multiple chromosomes.

Genetic analysis of meiotic recombination in wildtype and pch-2 mutants. DCO indicates double crossovers. Physical and genetic maps of Chromosome I, III, IV and the X chromosome are depicted to scale. Genetic distance is shown in centimorgans. A * indicates a p-value < 0.05, a ** indicates a p value < 0.01 and a *** indicates p-value < 0.001.

PCH-2 prevents exogenous double strand breaks from becoming crossovers in early meiotic prophase.

A. Illustration of the irradiation experiments in control and pch-2 mutants. Box indicates late pachytene, the area where GFP::COSA-1 foci are analyzed. B. Fraction of meiotic nuclei with less than 6, 6, or greater than 6 GFP::COSA-1 foci in control animals (yellow, n = 446) and pch-2 mutants (blue, n = 552). C. Meiotic nuclei in control animals and pch-2 mutants 8 hours post irradiation stained for DAPI (magenta) and GFP::COSA-1 (green). Scale bar is 4 um. D. Fraction of meiotic nuclei with less than 6, 6, or greater than 6 GFP::COSA-1 foci in control animals (yellow, n = 143) and pch-2 mutants (blue, n = 125) 8 hours post irradiation. E. Meiotic nuclei in control animals and pch-2 mutants 24 hours post irradiation with DAPI (magenta) and GFP::COSA-1 (green). F. Fraction of meiotic nuclei with less than 6, 6, or greater than 6 GFP::COSA-1 foci in control animals (n = 179) and pch-2 mutants (n = 378) 24 hours post irradiation. A *** indicates p-value < 0.001, and a **** indicates p-value < 0.0001.

PCH-2 prevents SPO-11-induced double strand breaks from becoming crossovers in early meiotic prophase.

A. Illustration of the SPO-11 depletion experiment to assay GFP::COSA-1 in control animals and pch-2 mutants at different timepoints of auxin treatment. Each timepoint indicates when SPO-11 is depleted in the germline with auxin induced degradation. Box indicates late pachytene, the area where GFP::COSA-1 foci are analyzed. B. Representative images of meiotic nuclei in control animals and pch-2 mutants treated with auxin for 20 hours, stained for DAPI (magenta) and GFP::COSA-1 (green). Scale bar is 5 um. C. Number of GFP::COSA-1 foci in meiotic nuclei at different timepoints of auxin treatment in control (blue) and pch-2 mutants (yellow). Error bars represent standard error of the mean (SEM). N values are as follows: 36 hours on auxin, control (78 nuclei) and pch-2 (83 nuclei); 24 hours on auxin, control (132 nuclei) and pch-2 (155 nuclei); 22 hours on auxin, control (152 nuclei) and pch-2 (168 nuclei); 20 hours on auxin, control (139 nuclei) and pch-2 (157 nuclei); 18 hours on auxin, control (154 nuclei) and pch-2 (154 nuclei); and 24 hours on ethanol, control (86 nuclei) and pch-2 (143 nuclei). D. Illustration of the SPO-11 depletion experiment to assay bivalents in control animals and pch-2 mutants at different timepoints of auxin treatment. Each timepoint indicates when SPO-11 is depleted in the germline with auxin induced degradation. Box indicates diakinesis, where DAPI stained bodies are analyzed. E. Oocytes from control animals and pch-2 mutants stained for DAPI (magenta). Scale bar is 4um. F. Number of DAPI stained bodies in meiotic nuclei at different timepoints of auxin treatment in control animals and pch-2 mutants. N values are as follows: 48 hours on auxin, control (47 nuclei) and pch-2 (43 nuclei); 36 hours on auxin, control (46 nuclei) and pch-2 (50 nuclei); 34 hours on auxin, control (41 nuclei) and pch-2 (41 nuclei); 32 hours on auxin, control (51 nuclei) and pch-2 (47 nuclei); 30 hours on auxin, control (46 nuclei) and pch-2 (21 nuclei); and 36 hours on ethanol, control (52 nuclei) and pch-2 (48 nuclei). Error bars represent SEM and a * indicates p-value < 0.05 and a *** indicates p-value < 0.001.

PCH-2 is required for timely loading and removal of MSH-5 on meiotic chromosomes through its regulation of HIM-3.

A. Representative images of nuclei in different stages of meiotic prophase in control animals and pch-2 mutants stained for DAPI (magenta) and GFP::MSH-5 (green) Scale bar is 5 um. B. Scatter plot showing average GFP::MSH-5 foci per row of germline nuclei in control animals (yellow, 163 nuclei) and pch-2 mutants (blue, 195 nuclei) from the transition zone (TZ) to late pachytene (LP), normalized to 100. The line represents a rolling average of four rows. C. Representative images of nuclei in different stages of meiotic prophase in him-3R93Y mutants (left) and pch-2;him-3R93Ydouble mutants (right), stained for DAPI (magenta) and GFP::MSH-5 (green). D. Scatter plot showing average GFP::MSH-5 foci per row in him-3R93Y(brown, 183 nuclei) and pch-2;him-3R93Y mutants (pink, 163 nuclei) from the transition zone (TZ) to late pachytene (LP), normalized to 100. The line represents a rolling average of four rows. Similar data is provided for a control germline (opaque yellow, 236 nuclei) for comparison. E. Representative images of meiotic nuclei in control animals and pch-2 mutants stained for DAPI (blue), GFP::MSH-5 (green), and OLLAS::COSA-1 (red). Yellow circles indicate GFP::MSH-5 without OLLAS::COSA-1. Scale bar is 4um. F. Scatter plot showing average GFP::MSH-5 (green) and OLLAS::COSA-1 (red) foci per row in the last five rows of the germline in control animals (36 nuclei) and pch-2 mutants (45 nuclei). The line represents a rolling average of 2 rows. G. Swarm plot showing number of GFP::MSH-5 foci in control (yellow, 14 nuclei) and pch-2 mutant (blue, 29 nuclei) nuclei with less than 6 OLLAS::COSA-1 foci (left) and 6 OLLAS::COSA-1 foci (right). Error bars represent SEM. A * indicates a p-value less than 0.05 and a ** indicates a p-value < 0.01.

PCH-2 is removed when crossovers are designated.

A. Representative images of meiotic nuclei in dsb-2 animals 24 hours post L4 and 48 hours post L4 stained for DAPI (magenta), PCH-2 (red), and GFP::COSA-1 (green). Scale bar is 4 um. B. Stacked histograms showing percentage of PCH-2 positive (n = 43 at 24 hours, n = 42 at 48 hours) and negative (n = 194 at 24 hours, n = 130 at 48 hours) nuclei with (lime) and without (dark green) GFP::COSA-1 foci in dsb-2 mutants at 24 hours post L4 and 48 hours post L4. C. Representative images of meiotic nuclei in dsb-2::AID and dsb-2::AID;pch-2 mutants treated with auxin and stained for DAPI (magenta) and GFP::COSA-1 (green). Scale bar is 5 um. D. Swarm plot showing the number of GFP::COSA-1 foci in dsb-2::AID (gray) and dsb-2::AID;pch-2 (lemon) mutants when treated with ethanol or auxin. N values are as follows: dsb-2::AID (101 nuclei) and dsb-2::AID;pch-2 (154 nuclei) on ethanol, dsb-2::AID (136 nuclei) and dsb-2::AID;pch-2 (131 nuclei) on auxin. Error bars represent the SEM. A ** indicates a p-value less than 0.01 and a **** indicates p-value < 0.0001.

PCH-2 and high CHK-2 activity control the fate of early double strand breaks.

A. Illustration of CHK-2 activity in wildtype and syp-1T452A germlines. B. Representative images of meiotic nuclei late pachytene in syp-1T452A and pch-2;syp-1T452Amutants stained for DAPI (magenta) and GFP::COSA-1 (green). Scale bar is 5 um. C. Swarm plot showing number of GFP::COSA-1 foci in control animals (blue), pch-2 (yellow), syp-1T452A (maroon), and pch-2;syp-1T452A(light blue) mutants. Error bars represent SEM. D. Oocytes from syp-1T452Aand pch-2;syp-1T452A mutant worms stained for DAPI (magenta). Scale bar is 4um. D. Swarm plot showing number of DAPI stained bodies in control animals (blue, n = 154), pch-2 (yellow, n = 89), syp-1T452A (maroon, n = 247)), and pch-2;syp-1T452A(light blue, n = 242) mutants. Error bars represent SEM. A **** indicates p-value < 0.0001.

PCH-2 remodels HIM-3 to disassemble crossover-eligible intermediates, controlling crossover distribution and number.

A. Model for how pch-2 and him-3R93Ymutations genetically interact to affect the progression of meiotic recombination. HIM-3 adopts the closed conformation upon binding an interacting protein with a closure motif and its conversion to the extended conformation is facilitated by PCH-2’s remodeling of its HORMA domain. B. Model for how PCH-2 and HIM-3 progressively implement meiotic recombination during different stages of meiotic prophase.

Single nucleotide polymorphisms used for recombination assay

PCH-2 does not regulate GFP::MSH-5 loading and removal through HTP-3.

A. Representative images of nuclei in different stages of meiotic prophase in htp-3H96Y and pch-2; htp-3H96Y mutants stained for DAPI (magenta) and GFP::MSH-5 (green) Scale bar in all images is 5 um. B. Scatter plot showing average GFP::MSH-5 foci per row of germline nuclei in htp-3H96Y(blue, 132 nuclei) and pch-2; htp-3H96Y (green, 161 nuclei) mutants from the transition zone to late pachytene, normalized to 100. The line represents a rolling average of four rows. Similar data is provided for a control germline (opaque yellow, 163 nuclei) for comparison

pch-2 meiotic nuclei with elevated numbers of GFP::MSH-5 foci show defects in crossover assurance.

Gray scale images of control and pch-2 mutant nuclei stained for DAPI, GFP::MSH-5 and OLLAS::COSA-1. Scale bar in image is 4 um.

syp-1T452Aand pch-2;syp-1T452A mutants display a similar defect in meiotic progression.

A. Representative images of syp-1T452A and pch-2;syp-1T452Amutant germlines. Encircled nuclei indicate transition zone nuclei. Scale bar indicates 10 um. B. Quantification of fraction of rows of transition zone nuclei in 3 germlines in syp-1T452A (maroon) and pch-2;syp-1T452A(light blue) mutants.