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. 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. C. Fraction of meiotic nuclei with less than 6, 6, or greater than 6 GFP::COSA-1 foci in control animals(yellow) and pch-2 mutants (blue) mutants 8 hours post irradiation. D. Meiotic nuclei in control animals and pch-2 mutants 24 hours post irradiation with DAPI (magenta) and GFP::COSA-1 (green). E. Fraction of meiotic nuclei with less than 6, 6, or greater than 6 GFP::COSA-1 foci in control animals and pch-2 mutants 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 and 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. Boxes indicate late pachytene, the area where GFP::COSA-1 foci are analyzed, and diakinesis, where bivalents 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). D. Oocytes from control animals and pch-2 mutants stained for DAPI (magenta). Scale bar is 4um. E. Number of DAPI stained bodies in meiotic nuclei at different timepoints of auxin treatment in control animals and pch-2 mutants. 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) and pch-2 mutants (blue) from the transition zone to late pachytene, 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) and pch-2;him-3R93Y mutants (pink) 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) 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 and pch-2 mutants. The line represents a rolling average of 2 rows. G. Swarm plot showing number of GFP::MSH-5 foci in control (yellow) and pch-2 mutant (blue) 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 and negative 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. 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-1T452A mutants 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-1T452A and pch-2;syp-1T452Amutant worms stained for DAPI (magenta). Scale bar is 4um. D. Swarm plot showing number of DAPI stained bodies in control animals (blue), pch-2 (yellow), syp-1T452A (maroon), and pch-2;syp-1T452A (light blue) 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-3H96Yand 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) and pch-2; htp-3H96Y (green) mutants from the transition zone to late pachytene, normalized to 100. The line represents a rolling average of four rows.

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.