Strains and media
Standard methods were used for the culture, sporulation, genetic crossing and manipulation of S. pombe. Strains were generated through transformations or genetic crosses followed by tetrad dissection. Strains used in this study are listed in Supplementary Table 2. Gene deletion strains were obtained from Bioneer haploid deletion library version 4.0, except for tor1 and gad8, which were deleted using pFA6a-natMX6 construct. Most epitope-tagged strains were generated through PCR module-based methods. GFP or GFP-raf1 was cloned in the pDUAL-Pnmt1 or pUC119-Padh1 vector using In-Fusion cloning. Pombe Minimal Glutamate medium supplemented with adenine, uracil, leucine, histidine and lysine (PMG-5S) was used to overexpress protein under the nmt1 promoter. All other experiments were performed in yeast extract-rich medium supplemented with adenine (YEA) at 30 °C unless otherwise specified. For western blotting analysis, the tor2-ts6 mutant was cultured at 26 °C overnight and then transferred to 30 °C for 16 h. For experiments with antifungal agents, cells were treated with 16 mM caffeine, 0.4 mM fluconazole or 0.3 μM clotrimazole in YEA medium.
Genetic screen
Genetic screens were conducted using a heterothallic strain (mat1M-smt0) harbouring the raf1R576H mutation and a deletion of the local silencer (REIIΔ) adjacent to the mat2P cassette (Fig. 1a). In addition, the strain contained a ura4+ reporter downstream of mat2P (mat2P::ura4+). Exponentially growing cells were treated with the chemical mutagen methylnitronitrosoguanidine at a concentration of 0.5 mg ml−1 for 60 min at room temperature or irradiated with ultraviolet light (UV) at 100 J m−2 using UV crosslinker. Colonies formed on YEA medium were replica plated onto PMG-5S medium and assayed for haploid meiosis by means of iodine staining. Light colonies were restreaked to single colonies and assessed for growth on −Ura and FOA media. Suppressor mutants were subjected to whole-genome sequencing using the Illumina NextSeq500 platform. Genomic sequencing reads were quality trimmed using fastp61 and aligned to the S. pombe ASM294v2.30 reference sequence62 with the BWA aligner63 using default parameters. Duplicate reads in the resulting BAM files were marked using Picard tools (http://broadinstitute.github.io/picard/) ‘MarkDuplicates’. Mutations were called from the duplicate-marked BAM files using samtools ‘mpileup’ and subsequently processed with bcftools64 to generate a single VCF file65 containing mutations identified in the WT and mutant genomes. Mutation impacts were predicted using SnpEff66. Variations that were present in the mutants but absent in the WT controls and were predicted to have ‘HIGH’ or ‘MODERATE’ impact were flagged for further investigation.
Serial dilution assay for heterochromatic silencing
To assess the silencing of the ura4+ reporter in mat1M-smt0 REIIΔ mat2P::ura4+ cells, tenfold serial dilutions of cultures were spotted on uracil-deficient medium and PMG-5S medium supplemented with FOA. The derepression of mat2P was evaluated by exposing the cells to iodine vapour. Cells undergoing haploid meiosis on PMG-5S medium, which results from mat2P derepression, accumulate a starch-like compound and stain dark brown when exposed to iodine vapour. Images of serial dilution plates were presented with Adobe Photoshop v.22.4.2 and Adobe Illustrator v.2024.
RT–qPCR
Total RNA was extracted from 2 × 108 cells using the MasterPure Yeast RNA Purification Kit (Biosearch Technologies) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of DNase-treated RNA using SuperScript III reverse transcriptase with gene-specific primers. The resulting cDNA was subjected to quantitative PCR (qPCR) using iTaq Universal SYBR Green Supermix (Bio-Rad), following the manufacturer’s instructions. The act1+ gene served as the internal control for normalization. Oligonucleotides used for qPCR are listed in Supplementary Table 3.
ChIP–qPCR
For the H3K9me3 ChIP assay, 5 × 108 cells were crosslinked with 3% paraformaldehyde (PFA) for 30 min at room temperature, followed by quenching with 125 mM glycine. For Swi6 or Raf2 or H3K14ub ChIPs, 1 × 109 of cells were incubated for 2 h at 18 °C, fixed in 3% PFA and washed in ice-cold PBS. Cells were treated with 10 mM dimethyl adipimidate at room temperature for 45 min.
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