Novel roles for selected genes in meiotic DNA processing

Abstract

High-throughput studies of the 6,200 genes of Saccharomyces cerevisiae have provided valuable data resources. However, these resources require a return to experimental analysis to test predictions. An in-silico screen, mining existing interaction, expression, localization, and phenotype datasets was developed with the aim of selecting minimally characterized genes involved in meiotic DNA processing. Based on our selection procedure, 81 deletion mutants were constructed and tested for phenotypic abnormalities. Eleven (13.6%) genes were identified to have novel roles in meiotic DNA processes including DNA replication, recombination, and chromosome segregation. In particular, this analysis showed that Def1, a protein that facilitates ubiquitination of RNA polymerase II as a response to DNA damage, is required for efficient synapsis between homologues and normal levels of crossover recombination during meiosis. These characteristics are shared by a group of proteins required for Zip1 loading (ZMM proteins). Additionally, Soh1/Med31, a subunit of the RNA pol II mediator complex, Bre5, a ubiquitin protease cofactor and an uncharacterized protein, Rmr1/Ygl250w, are required for normal levels of gene conversion events during meiosis. We show how existing datasets may be used to define gene sets enriched for specific roles and how these can be evaluated by experimental analysis.

Figure 1. Integration of Datasets to Select Genes with Roles in DNA Processing

See text and Figure S1 and Tables S1, S2, and S3 for details of the selection strategy.

Figure 2. Assessment of YGL250W, SOH1, and BRE5

(A) Schematic representation of Chromosome II from the diploid W303 background which consists of two LYS2 heteroalleles (lys2–5′ndeI⁻ and lys2–3′ndeI⁻). These were used to measure meiotic gene conversion (see Materials and Methods). (B) Spot test of wild type, ygl250wΔ, soh1Δ, and bre5Δ on haploid selection plates and haploid selection plates without lysine to measure meiotic gene conversion. The reduction in meiotic gene conversion of ygl250wΔ, soh1Δ, and bre5Δ was further assessed by random spore analysis (Table 1). (C) Southern blot of DNA isolated from wild type, soh1Δ, and ygl250wΔ SK1 strains containing the ectopic URA3-ARG4 interval on Chromosome III. The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). The full-sized Southern blots are presented in Figure S1. For graphs (D–G), wild type, ygl250wΔ, and soh1Δ are represented by black diamonds, black circles, and white squares, respectively. The corresponding XhoI-digested Southern blots are presented in Figure 3C. (D) Pre-meiotic DNA replication was assessed for synchronized meiotic cultures by fluorescence-activated cell sorting (FACS) and the change from 2c to 4c DNA content was plotted over time. (E) Nuclear divisions (MI and MII) of the synchronized meiotic cultures in (E) were assessed with fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) staining to visualize nuclear division. (F) Molecular analysis for DSB (DSB1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO (CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures.

Figure 3. Further Characterization of VID21, BRE1, LGE1, RMD11, SGF73, and DEF1

Mutants for these genes were made in an SK1 background. The plots on each graph represent wild type (black diamonds), rmd11Δ (white diamonds), bre1Δ (black triangles), lge1Δ (white triangles), sgf73Δ (black circles), def1Δ (white circles), and vid21Δ (black squares). Where error bars are not shown, the time courses are of individual experiments. A total of three experiments were carried out in each case and the data shown are consistent with those obtained in the other experiments. (A) The expression of IME1, a primary transcription factor required for entry into the meiotic cell cycle was assessed. SK1 strains carrying a plasmid that expresses the lacZ reporter gene under the control of the IME1 promoter were grown for synchronous meioses and assessed for lacZ expression via β-galactosidase activity [92]. W303 MAT-a mutant strains for the above genes were assessed for G1 to S phase transition in mitosis after release from α-factor arrest [87]. (B) Pre-meiotic DNA replication was assessed for synchronized meiotic cultures by FACS and the change from 2c to 4c DNA content was plotted over time. See Figure S2 for the raw data of the FACS analysis for meiotic DNA replication. (C) DNA extractions from sporulation time courses were digested with BglII and meiotic DSB formation (DSBIII and IV) at the THR4 hotspot was assessed using Southern blotting and probing techniques [46]. See Figure S3 for the THR4 Southern blots. (D) Nuclear divisions (MI and MII) of the synchronized meiotic cultures in (A) were assessed with fluorescence microscopy using DAPI staining to visualize nuclear division. (E) DNA replication following release from α-factor arrest was assessed via FACS and the change from 1c to 2c DNA content was plotted against time. See Figure S4 for the raw data of the FACS analysis for mitotic DNA replication. (F) The budding index of cells released from α-factor synchrony was assessed by phase contrast microscopy.

Figure 4. Assessment of def1Δ in Meiosis and Mitosis

(A) Immunocytology of nuclear spreads of SK1 wild-type and def1Δ strains after 8 h of sporulation. The meiosis-specific subunit of cohesin, Rec8, was tagged with multiple Haemagglutinin (HA) epitopes. Using antibodies for HA and Zip1 allowed analysis of sister chormatid cohesion and synaptonemal complex formation, respectively. It can be seen in the wild-type example that all 16 chromosomes have long cohesin axes and close to full chromosome synapsis except for the rDNA region on Chromosome XII. Whereas from the first panel for def1Δ it can be seen that axes are aligned but synapsis is minimal. The second and third panels for def1Δ again show aligned axes, but homologues are only partially synapsed. However, as shown in the final panel, synapsis was observed in some meiotic nuclei of the def1Δ strain. Polycomplexes (PCs) of Zip1 were observed in 20% of the nuclei counted for def1Δ at this time point whereas less than 1% PCs were observed for the wild type. (B) Time course of the meiotic nuclei counted using immunocytology for both wild type and def1Δ during meiosis. The def1Δ mutant synapsis phenotype represented in (A) was counted as “aligned” axes in the Rec8 analysis graph. At least 200 nuclei were counted per time point. (C) Ectopic URA3-ARG4 interval on Chromosome III described in Figure 3. XhoI and EcoRI restriction sites are indicated by “X” and “E,” respectively. To detect NCOs, COs, and DSBs, DNA is digested with XhoI and EcoRI then probed with HIS4 sequences (hisU; [44]). For graphs (D–F), wild-type and def1Δ are represented by blue squares and pink diamonds, respectively. The corresponding XhoI and EcoRI double digest Southern blots, the XhoI single digest Southern blots, together with the molecular analyses, are presented in Figure S5. (D) Molecular analysis for DSB (DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (E) Molecular analysis for NCO (NCO1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO (CO1') signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Southern blot of DNA isolated from wild-type and def1Δ SK1 strains containing the ectopic URA3-ARG4 interval on Chromosome III. DNA was digested with XhoI and EcoRI then probed to detect NCOs, COs, and DSBs; mw represents the 1-kb molecular weight marker (Fermentas).