Crossover Position Drives Chromosome Remodeling for Accurate Meiotic Chromosome Segregation

Abstract

Interhomolog crossovers (COs) are a prerequisite for achieving accurate chromosome segregation during meiosis [1, 2]. COs are not randomly positioned, occurring at distinct genomic intervals during meiosis in all species examined [3-10]. The role of CO position as a major determinant of accurate chromosome segregation has not been previously directly analyzed in a metazoan. Here, we use spo-11 mutants, which lack endogenous DNA double-strand breaks (DSBs), to induce a single DSB by Mos1 transposon excision at defined chromosomal locations in the C. elegans germline and show that the position of the resulting CO directly affects the formation of distinct chromosome subdomains during meiotic chromosome remodeling. CO formation in the typically CO-deprived center region of autosomes leads to premature loss of sister chromatid cohesion and chromosome missegregation, whereas COs at an off-centered position, as in wild type, can result in normal remodeling and accurate segregation. Ionizing radiation (IR)-induced DSBs lead to the same outcomes, and modeling of IR dose-response reveals that the CO-unfavorable center region encompasses up to 6% of the total chromosome length. DSBs proximal to telomeres rarely form COs, likely because of formation of unstable recombination intermediates that cannot be sustained as chiasmata until late prophase. Our work supports a model in which regulation of CO position early in meiotic prophase is required for proper designation of chromosome subdomains and normal chromosome remodeling in late meiotic prophase I, resulting in accurate chromosome segregation and providing a mechanism to prevent aneuploid gamete formation.

Keywords: C. elegans; DNA double-strand break; DNA repair; Mos1; chromosome remodeling; chromosome segregation; crossover; germline; meiosis.

Figure 1.. The position of a single DSB/CO affects late prophase I chromosome remodeling.

(A) Schematic drawing of the single inducible DSB assay. Blue lines represent the chromosomes and the red boxes correspond to the Mos1 transposon inserted at different chromosomal locations. After heat shock-induced DSB formation by transposon excision and subsequent repair via homologous recombination (HR), diakinesis nuclei were analyzed for the presence of either a single asymmetric or symmetric bivalent (all other chromosomes were univalents given that this analysis was performed in a spo-11 mutant background that lacks endogenous DSBs). (B) Schematic drawing of the locations of the Mos1 transposon insertions on chromosomes III, II and V. 1 indicates the location of ttTi1730, 2 the location of ttTi23808, 3 the location of ttTi2384, 4 the location of ttTi13055 and 5 the location of ttTi17604 on chromosome III. 6 indicates the location of ttTi5605 on chromosome II and 7 the location of ttTi35006 on chromosome V. Numbers 1 and 4 indicates DSBs occurring in an off-center position, 2, 6 and 7 indicates DSBs occurring in a center position, 5 indicates a DSB occurring in a subtelomeric position and 3 indicates a DSB occurring at the border of the center region. The physical (Mb) and genetic (cM) maps are shown one above of the other for every chromosome. Genetic maps are based on Rockman and Kruglyak [42]. The yellow rectangles represent the recombination frequencies on the chromosome arms compared to frequencies on the center region in blue. (C) Quantification of bivalent formation for chromosome III by immunoFISH analysis of late diakinesis oocytes following Mos1 transposon excision (+ or − heat shock; HS) from the indicated locations on chromosome III. All lines carry the spo-11 mutation and either presence or absence of a Mos1 transposon insertion (Mos1⁺ or Mos1⁻, respectively) on chromosome III and presence or absence of the transposase (Tn⁺ or Tn⁻ respectively). Representative immunoFISH images with LAB-1 (magenta), HTP-3 (yellow) and a FISH probe for chromosome III (green). (D) Quantification of asymmetric or symmetric bivalent formation in late diakinesis oocytes based on coimmunostaining for the HORMA domain-containing protein HTP-3 (yellow) and LAB-1 (magenta). Immunofluorescence images from wild type, spo-11;Tn (transposase⁺) mutants and spo-11 mutants in which a single Mos1-induced DSB was generated in one of the following locations on chromosome III: off-center (at either the right or left arms), the center, and the subtelomeric region. Both chromosomal axes are highlighted by HTP-3 while LAB-1 is restricted to the long arm of the bivalents in wild type. Total numbers of DAPI-stained bodies (bivalents/univalents) observed in the oocytes at diakinesis are shown in the first column. Dashed boxes indicate the bivalents/univalents shown at higher magnification. Illustrations on the right depict the chromosome configuration observed at this stage and the localization of HTP-3 (yellow) and LAB-1 (magenta). Long (L) and short (S) arms of the asymmetric bivalent are indicated on wild type. n= number of nuclei scored. Bar, 2 μm. See also Figures S1, S2 and Data S1 and S2.

Figure 2.. A single centered crossover results in premature sister separation.

(A) Immunolocalization of SMC-1 (red) and DAPI (blue) in diakinesis stage oocytes in wild type, spo-11 mutants with a single Mos1 insertion at an off-center position (right arm) on chromosome III, and spo-11 mutants with a single Mos1 insertion at the center of chromosome III after heat shock, all of which underwent emb-30 depletion by RNAi to induce arrest of oocytes before the meiotic division to facilitate this analysis. 12 out of 200 oocytes in spo-11 mutants with a single Mos1 insertion at the center of chromosome III showed premature separation of sister chromatids after heat shock as evidenced by the presence of 14 DAPI-stained bodies (10 univalents + 4 separated sister chromatids) and absence of SMC-1 staining in 4 DAPI-stained bodies (presumably the 4 prematurely separated sister chromatids). (B) Immunofluorescence-FISH images of metaphase to anaphase I nuclei. On the first row are representative images of wild-type nuclei at the metaphase to anaphase I transition with a chromosome III FISH probe (magenta) and tubulin (yellow). The two FISH signals (foci) show separating homologs but joined sister chromatids (n=15/15 nuclei examined). The second and third rows are representative images of metaphase to anaphase I and anaphase I nuclei, respectively, from worms harboring a single Mos1 insertion at an off-center position (right arm) on chromosome III. Both nuclei with separating homologs but joined sister chromatids (n=6/12), as seen in wild type, and nuclei with premature sister separation (n=6/12), as depicted by more than two FISH signals, were detected. The last row depicts anaphase I for worms with a single Mos1 insertion at the center of chromosome III. Sister chromatid separation is evidenced by the presence of more than two FISH signals (n=8/11). Numbers in the first column represent number observed/total number scored. Bar, 2 μm.

Figure 3.. Chromosome remodeling defects are already observed at late pachytene stage and can be rescued by additional exogenous DSBs.

(A) Immunolocalization of LAB-1 in late pachytene on chromosome III following heat shock-induced DSB formation. HTP-3 (yellow), LAB-1 (magenta) and a FISH probe for chromosome III (blue) are shown. Illustrations for each observed localization pattern are shown in the lower panel. Histogram shows the quantification of various LAB-1 localization patterns in late pachytene nuclei subjected to heat shock-induced DSBs at different locations on chromosome III. (B) Crossover precursor marker COSA-1 localizes at high frequency to Mos1-induced CO sites. Late pachytene nuclei of whole mounts hybridized with a FISH probe recognizing chromosome III (magenta) showing the localization of chromosome axis marker HTP-3 (yellow) and GFP::COSA-1 (blue). Illustrations depict the different localizations observed for COSA-1. Histogram shows the quantification of COSA-1 foci in late pachytene nuclei from lines with DSBs induced at the indicated positions on chromosome III (identified by FISH). Similar to other studies, GFP::COSA-1 foci were also detected on chromosomes in spo-11 mutants, potentially reflecting spontaneous DNA lesions [43, 44]. n, number of late pachytene nuclei scored; HS, heat shock. (C) Exogenous DSB formation by γ-IR rescues CO formation and LAB-1 localization defects in late pachytene nuclei subjected to a single Mos1-induced DSB at the center of chromosome III. HTP-3 (yellow), COSA-1 (blue) and LAB-1 (magenta). Histograms show quantifications of COSA-1 foci and LAB-1 localization pattern on chromosome III (identified by FISH). Bars, 2 μm. See also Figure S3 and Data S3, S4 and S5.

Figure 4.. Remodeling defects are independent of the source of DSB formation.

(A) Representative DAPI-stained bodies (blue) showing the localization of HTP-3 (yellow) and LAB-1 (magenta) observed in diakinesis nuclei of spo-11 mutants subjected to γ-IR doses producing 1, 2 or 3 DSBs per chromosome pair. Illustrations are shown below the immunofluorescence images. Bar, 2 μm. Histogram depicting the categories of bivalent/univalent configurations observed at diakinesis after inducing 1 DSB per homolog pair by γ-IR (2.5 Gy), n indicates the number of diakinesis nuclei examined. (B) Quantification of LAB-1 localization in diakinesis nuclei following exposure to indicated γ-IR doses. (C) Histogram showing quantification of the observed number of bivalents and/or univalents at different γ-IR doses. (D) Mathematical modeling used to determine the length of the center region leading to chromosome remodeling defects upon CO formation. (E) Model for how CO position influences late prophase chromosome remodeling and subsequent chromosome segregation. DSBs occur throughout the length of the chromosomes (up to 10 per homolog pair in C. elegans [37]), but only 1 introduced at an off-centered position is preferentially used for CO formation leading to correct chromosome remodeling resulting in asymmetric bivalents and accurate chromosome segregation. In contrast, COs occurring at the center of the chromosomes result in impaired chromosome remodeling resulting in symmetric bivalents. This chromosome configuration lacks LAB-1 localization and this in turn fails to restrict AIR-2/pH3 to a single chromosome axis resulting in premature loss of sister chromatid cohesion and errors in chromosome segregation. See also Figure S4 and Data S5.