Genes Important for Schizosaccharomyces pombe Meiosis Identified Through a Functional Genomics Screen

Abstract

Meiosis is a specialized cell division that generates gametes, such as eggs and sperm. Errors in meiosis result in miscarriages and are the leading cause of birth defects; however, the molecular origins of these defects remain unknown. Studies in model organisms are beginning to identify the genes and pathways important for meiosis, but the parts list is still poorly defined. Here we present a comprehensive catalog of genes important for meiosis in the fission yeast, Schizosaccharomyces pombe Our genome-wide functional screen surveyed all nonessential genes for roles in chromosome segregation and spore formation. Novel genes important at distinct stages of the meiotic chromosome segregation and differentiation program were identified. Preliminary characterization implicated three of these genes in centrosome/spindle pole body, centromere, and cohesion function. Our findings represent a near-complete parts list of genes important for meiosis in fission yeast, providing a valuable resource to advance our molecular understanding of meiosis.

Keywords: Schizosaccharomyces pombe; chromosome segregation; meiosis; sporulation.

Figure 1

Overview of the functional genomics screen to identify genes important for meiosis and sporulation in S. pombe. (A) Screen strategy for systematic analysis of mutants. The tester strain carried: the homothallic h⁹⁰ locus, allowing mating type switching, linked to the Nourseothricin (NAT)-resistance cassette NATMX6; rpl42P56Q, which confers recessive cycloheximide-resistance (cyh^R), allowing counterselection against heterozygous diploids; and a GFP label (lacO-ura3⁺ and lacI-GFP-leu2⁺) to follow segregation of chromosome II. The query strain is one of 3285 library strains in which a single nonessential gene has been replaced by KANMX6, conferring resistance to G418. Query and tester strains were mated and allowed to sporulate. Selection for G418-, nourseothricin-, and cycloheximide-resistant leucine and uracil prototrophs allowed the isolation of haploids of mixed mating types carrying the deletion of interest and the GFP-labeled chromosome. These haploids were allowed to mate and sporulate. Meiosis in the absence of a single gene product was assessed visually by scoring the fate of the homozygous GFP label on chromosome II under the microscope. Example patterns of GFP dots in the tetrads are shown. (B) Significantly enriched GO terms associated with the 354 genes whose absence resulted in <20% of cells producing spores in our screen. The percentage of genes fitting this criterion is shown compared to the genome as a whole for each of the GO terms found to be significantly enriched in the no conjugation or low/no sporulation data set. (C) Functional classification of the 354 genes whose absence resulted in <20% sporulation in our screen based on GO terms. The criteria for inclusion in this category was reduced spore formation; therefore the mutant phenotype observed includes mutants that failed to conjugate, enter meiosis, or form asci in the screen. (D) Significantly enriched GO terms associated with the 269 genes whose absence resulted in <90% of tetrads receiving a single GFP dot in each spore. The percentage of genes fitting this criterion is shown compared to the genome as a whole for each of the GO terms found to be significantly enriched in the chromosome segregation defect data set. Note that mutants that produced <20% spores (as shown in C) were not included in this analysis even if they were found to have a chromosome segregation defect. (E) Functional classification based on GO terms of the 269 genes whose absence resulted in a chromosome segregation defect (<90% tetrads with each spore receiving a single GFP dot) in our screen.

Figure 2

Preliminary characterization of genes important for meiosis. To validate hits in the screen, 18 genes were chosen for further analysis. (A and B) The pattern of GFP foci in tetrads carrying homozygous lacO/LacI-GFP on chromosome II was examined after induction of zygotic meiosis. Strains were homothallic (h90), carried the deletion of interest, and were homozygous for lacO/LacI-GFP on chromosome II. (A) Representative images of mutants in the no conjugation/no sporulation class, which includes mutants that fail to conjugate (dbl2Δ), enter meiosis (cdt2Δ, zds1Δ), or form asci (e.g., tpr1Δ, dms1Δ) and a wild type example. Objective used for magnification was ×100. (B) Patterns of segregation of homozygous lacO/LacI-GFP on chromosome II observed after zygotic meiosis of homothallic (h⁹⁰) strains with the indicated genotypes. n=100; N.D., not determined (dms1Δ does not form spores). (C) The pattern of heterozygous lacO/LacI-GFP foci integrated close to cen2 was scored in tetranucleate cells (nuclei were identified by DAPI-staining). Parental h+ and h− haploids, both of which carried the deletion of interest and only one of which carried the GFP label, were mated and induced to undergo zygotic meiosis. n=100; N.D., not determined.

Figure 3

Acb1 is important to prevent premature sister chromatid separation. (A and B) Wild type (AMfy1744) and acb1Δ (AMfy1765) cells carrying mCherry-Atb1 lacO integrated on the arm of both copies of chromosome II and producing LacI-GFP were induced to undergo zygotic (h⁹⁰) meiosis and imaged at 5-min intervals. Representative still images of wild type (A) and acb1Δ (B) cells undergoing meiosis are shown. Arrowhead indicates closely two opposed dots. Bar, 5 μm. (C) Quantification of chromosome segregation errors from movies as in A and B for wild type, acb1Δ, and rec12Δ (AMfy1703) cells undergoing meiosis. NDJ, nondisjunction; SCS, sister chromatid separation; n=50. (D) Analysis of sister chromatid cohesion in mes1Δ cells arrested as binucleate cells before meiosis II. Cells were mes1Δ, carried lacO arrays integrated close to the centromere on both copies of chromosome II, expressed LacI-GFP, and were otherwise wild type (AMfy1786), bub1Δ (AMfy1832), or acb1Δ (AMfy1797). The indicated patterns of GFP foci were scored in 200 binucleate cells of each strain after inducing zygotic (h⁹⁰) meiosis. (E–G) Meiotic recombination is decreased in acb1Δ cells. (E) Schematic of assay showing the positions of markers. (F) Average cross-over recombination frequency during zygotic (h⁺ × h⁻) meiosis for wild type (AMfy1769 × AMfy1778), rec12Δ (AMfy1780 × AMfy1794) and acb1Δ (AMfy1808 × AMfy1810) is shown. Following meiosis, the percentage of colonies that grew on medium lacking either leucine or histidine (but not both) were scored from a total of 234–1443 colonies for each strain and the average of six biological repeats is shown. Error bars representing SD and P values were calculated using t-test (** P < 0.0001, * P < 0.05). (G) Average gene conversion at ade6 is shown for the experiment described in F.

Figure 4

Pof3 is critical for accurate chromosome segregation during meiosis I and meiosis II. (A) Representative images from a movie of pof3Δ zygotic (h⁹⁰) meiosis carrying mCherry-Atb1, lacO integrated on the arm of both copies of chromosome II, and producing LacI-GFP and imaged as described in Figure 3 (strain AMfy1827). (B) Quantification of chromosome segregation errors from movies. Data for wild type and rec12Δ is reproduced from Figure 3C. Cross-over recombination (C) and gene conversion (D) were assayed as described in Figure 3, E–G. The data for wild type and rec12Δ is reproduced from Figure 3, F and G. The data for pof3Δ was generated via a cross between strains AMfy1865 and AMfy1866. Error bars representing SD and P values were calculated using t-test (** P < 0.0001, * P < 0.05).

Figure 5

Dms1 is important for SPB function in S. pombe meiosis. (A) Representative images from a movie of dms1Δ cells carrying mCherry-Atb1, lacO integrated on the arm of both copies of chromosome II and producing LacI-GFP imaged as described in Figure 3 (strain AMfy1553). (B) Quantification of phenotypes observed in dms1Δ cells in movies of meiosis as in A. (C) Analysis of sister chromatid cohesion in mes1Δ dms1Δ cells (AMfy1867) arrested as binucleate cells before meiosis II as described in Figure 3D. Data for mes1Δ are reproduced from Figure 3D. (D) The longest meiosis II spindle length observed was measured in at least 60 cells for wild type and dms1Δ cells from movies as in A. * P = 0.0177. (E) The number and position of SPBs was scored from movies of wild type (AMfy1098) and dms1Δ (AMfy1112) cells carrying Sad1-mCherry and undergoing meiosis II.

Figure 6

Dms1 associates with SPBs during meiosis II. (A) Live cell imaging of Dms1 and SPBs during meiosis. Strain AMfy1845 carrying Dms1-GFP and Sad1-mCherry was induced to undergo zygotic (h⁹⁰) meiosis and images captured at 5-min intervals. Representative images from a movie are shown. Bar, 5 μm. (B) Representative images from live cell imaging showing forespore membrane (FSM; mCherry-Psy1) and Dms1 (Dms1-GFP) localization (AMfy1807). (C) Proteins interacting with Dms1 in meiosis. Diploid strains AMfy1796 (Dms1-GFP) and AMfy781 (no tag control) were harvested 7 hr after induction of azygotic synchronous meiosis and anti-GFP immunoprecipitates were analyzed by mass spectrometry. Proteins found to be specific to the Dms1-GFP sample and where two or more peptides were identified are shown.

Figure 7

Dms1 regulates Spo15 and Plo1 localization during meiosis I. (A–C) Plo1 dissociates from the SPB during prophase I in dms1Δ cells and forms foci consistent with localization to kinetochores in the majority of cells. Representative images from movies of zygotic (h⁹⁰) meiosis are shown for wild-type (A; AMfy1847) and a dms1Δ (B, AMfy1859) cell where presumptive kinetochore localization was not detected. Both strains carry Plo1-GFP and Sad1-mCherry. Bar, 5 μm. Asterisk in A indicates presumed kinetochore foci. (C) Quantification of Plo1 localization during prophase is shown. (D–F) Dms1 is required for Spo15 localization at SPBs during meiosis I. Live cell imaging of Spo15-GFP in wild type (D; AMfy1854) and dms1Δ (E; AMfy1860) cells undergoing zygotic (h⁹⁰) meiosis and also carrying Sad1-mCherry. Asterisk in D indicates presumed kinetochore foci. Arrowheads in E indicate Spo15-GFP at SPBs in meiosis II. (F) Scoring of Spo15-GFP foci in prophase I is shown.