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, 71 (4), 540-553.e4

Pericentromere-Specific Cohesin Complex Prevents Meiotic Pericentric DNA Double-Strand Breaks and Lethal Crossovers

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Pericentromere-Specific Cohesin Complex Prevents Meiotic Pericentric DNA Double-Strand Breaks and Lethal Crossovers

Mridula Nambiar et al. Mol Cell.

Abstract

In most eukaryotes, meiotic crossovers are essential for error-free chromosome segregation but are specifically repressed near centromeres to prevent missegregation. Recognized for >85 years, the molecular mechanism of this repression has remained unknown. Meiotic chromosomes contain two distinct cohesin complexes: pericentric complex (for segregation) and chromosomal arm complex (for crossing over). We show that the pericentric-specific complex also actively represses pericentric meiotic double-strand break (DSB) formation and, consequently, crossovers. We uncover the mechanism by which fission yeast heterochromatin protein Swi6 (mammalian HP1-homolog) prevents recruitment of activators of meiotic DSB formation. Localizing missing activators to wild-type pericentromeres bypasses repression and generates abundant crossovers but reduces gamete viability. The molecular mechanism elucidated here likely extends to other species, including humans, where pericentric crossovers can result in disorders, such as Down syndrome. These mechanistic insights provide new clues to understand the roles played by multiple cohesin complexes, especially in human infertility and birth defects.

Keywords: DNA double-strand break; Rec11; STAG3; chromosome missegregation; cohesin; crossover; heterochromatin; infertility; meiosis; pericentric repression.

Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Meiosis-specific linear-element protein Rec10 tethered to pericentric regions overcomes repression of meiotic recombination
(A) Schematic representation of the composition of proteins present during meiosis at the pericentric regions in S. pombe. (B) Model for initiation of meiotic recombination by DSB formation across chromosomal arms in S. pombe. See text for explanation. (C) Cartoon of telomeric cluster during the horsetail stage of meiosis. (D) Fluorescent microscopic images of meiotic horsetail cells (marked with white arrowheads) expressing Rec10-GFP, Rec10-GFP-CD or Rec10MUT-GFP-CD (green). Rec10MUT is a recombination-deficient missense mutant (R184F D185T). cen3 (red) is at the nuclear pole opposite the telomeric cluster, which also binds CD due to heterochromatin. cen3 is labeled with tdTomato at the left innermost repeat (imr). BF, bright field. Yellow, colocalized signals when merged. Images are representative of at least 20-30 cells examined. (E) Assay for recombination across the pericentric region on chromosome 3 (cen3). cnt, central kinetochore-binding region; imr, innermost repeat; otr, outer repeat. Recombinants across cen3 were measured as Ura+ His and Ura His+, and chromosomal arm recombinants (cen3-ade6) as His+ dark red (ade6-M26) and His light red (ade6-52). o, absence of the ura4+ or his3+ insertion. (F, G) Recombinant frequency (RF) across cen3 (F) and the cen3-ade6 arm interval (G). Data are mean ± SEM (n=4 to 8 experiments, assaying 654 to 3733 spore colonies). ****, p <0.0001 (two-tailed Fisher’s exact test). In this and all subsequent figures bar colors correspond to those of the recombination proteins in Figure 1B, with wild type (wt) as black. See also Figure S1, Tables S1, S2 and S3.
Figure 2
Figure 2. Linear element protein Rec10 tethered to pericentric regions induces DSB formation
(A) Pulse-field gel electrophoresis showing cen3 DSBs at the indicated times after induction of meiosis in a rad50S mutant. The ~125 kb cen3 fragment from BglI digestion was detected on a Southern blot with a probe at the right end of the cen3 fragment (upper images). On the same blots DSBs were measured at the chromosomal arm hotspot ade6-3049 using a probe at the left end of the ~58 kb ade6 fragment. *, repetitive rDNA BglI fragment (10.9 kb) from cross-hybridization. Signals were analyzed by PhosphorImager. (B) Quantification of the blots in Figure 2A, using ImageQuant software. Bar colors correspond to those of the recombination proteins in Figure 1B.
Figure 3
Figure 3. Pericentric recombination mediated by pericentric-tethered Rec10 resembles chromosomal arm recombination
(A) Recombinant frequency (RF) across cen3 in strains expressing Rec10-CD in the absence of Rec8, Rec11, Rec25, Rec27, Spo11, CK1 or Swi6 as indicated. The RFs of rec10-CD rec11Δ and rec10-CD CK1Δ are not statistically different (p value >0.05; two-tailed Fisher’s exact test). (B) RF across cen1 (with flanking genetic markers as in Figure 1E) in wild-type, dcr1Δ and rec10-cd strains. Data are mean ± SEM (n=3 to 6 experiments, assaying 583 to 1284 spore colonies; for dcr1Δ and rec10-cd rec8Δ, n=2 experiments, assaying 749 and 707 spore colonies, respectively, and error bars indicate range). *, p = 0.0287 (two-tailed Fisher’s exact test); **, p = 0.002; ***, p <0.001; ****, p <0.0001; “ns”, p > 0.05 (not significant). Bar color (red) corresponds to that of Rec10 in Figure 1B, with wt and dcr1Δ in black. See also Tables S1 and S4.
Figure 4
Figure 4. Other linear element proteins tethered to pericentric regions also activate pericentric recombination in a Rec10-dependent manner
(A, B) RF across cen3 (A) and the cen3-ade6 interval (B). Data are mean ± SEM (n=3 to 6 experiments, assaying 546 to 878 spore colonies). ***, p <0.001 (two-tailed Fisher’s exact test); ****, p <0.0001. rec27-cd is not significantly different from wt (p=1.000) for RF at cen3 (Table S1). Bar color (blue) corresponds to that of Rec25 and Rec27 in Figure 1B. (C) Fluorescent microscopic images of meiotic horsetail cells (marked with white arrowheads) expressing Rec25-GFP, Mug20-GFP, or Rec27-GFP, each with or without CD. BF, bright field; LinE, linear element protein (Rec25, Rec27, or Mug20). Images are representative of at least 30-100 cells examined. See also Table S2.
Figure 5
Figure 5. Meiosis-specific cohesin subunit Rec11 tethered to pericentric regions promotes recombination during meiosis but only in the absence of Swi6 or when fused directly to Rec10
(A) Fluorescent microscopic images of meiotic horsetail cells (marked with white arrowheads) expressing Rec11-GFP or Rec11-GFP-CD in the presence and absence of Swi6. Images are representative of at least 25-100 cells examined. (B) RF across cen3 in the indicated mutants. Bar color (yellow) corresponds to that of Rec11 in Figure 1B. (C) Fluorescent microscopic images of meiotic horsetail cells (marked with white arrowheads) expressing Rec11-Rec10-GFP-CD in the presence and absence of Swi6. BF, bright field. Images are representative of at least 30-50 cells examined. (D) RF across cen3 in the indicated mutants. Yellow and red parts of the bars reflect the fusion of Rec11 (yellow) and Rec10 (red) in Rec11-Rec10-CD as in Figure 1B. Data are mean ± SEM (n = 3 to 9 experiments, assaying 577 to 1947 spore colonies). *, p = 0.0186 (two-tailed Fisher’s exact test); ****, p <0.0001; “ns”, p >0.05 (not significant). See also Figure S2 and Table S5.
Figure 6
Figure 6. Both mitotic cohesin subunit Psc3 and Swi6 (HP1) repress meiotic recombination by blocking Rec11 at the pericentromeres
(A) RF across cen3 in the indicated mutants. psc3Δ swi6Δ is synthetically lethal and thus could not be tested. Data are mean ± SEM (n = 3 to 8 experiments, assaying 364 to 1529 spore colonies). (B) Frequency of dissected asci (tetrads) with 4, 3, 2 or 1 viable spore(s) in wild type, rec10-cd homozygous (+/+), rec10-cd heterozygous (+/−) and rec11-cd (homozygous) crosses. (C) Segregation errors in h90 mes1Δ cells at the end of MI in wt, rec10-cd and rec10-cdW104A strains (n >200 cells for each genotype). cen3 is labeled with tdTomato at the left innermost repeat (imr). Cells undergoing normal segregation show cen3 foci in both nuclei, while those with MI non-disjunction show two cen3 foci in the same nucleus. Premature separation of sister chromatids (PSSC) leads to more than one focus in the same nucleus. *, p = 0.0126 (two-tailed Fisher’s exact test); **, p = 0.0055; ***, p <0.001; ****, p <0.0001; “ns”, p >0.05 (not significant). Bar colors correspond to those of the recombination proteins in Figures 1A and 1B, with wt in black; striped bars indicate heterozygosity. See also Figure S2, Tables S3, S5, S6, S7 and S8.
Figure 7
Figure 7. Model for repression of meiotic recombination in pericentric regions
Histone H3K9me (orange flags) in pericentric regions binds the chromodomain (CD)-containing protein (Swi6) responsible for enriching cohesin subunits (Rec8-Psc3) specifically in pericentric regions. Both Psc3 and Swi6 exclude Rec11 from the pericentric regions and thereby prevent it from binding Rec8 and thus to recruit Rec10. Absence of Rec10 precludes activation of Spo11 complex to form DSBs that initiate recombination during meiosis.

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