A protocol for transposon insertion sequencing in Schizosaccharomyces pombe to identify factors that maintain heterochromatin.
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ABSTRACT:
SUBMITTER: Li F
PROVIDER: S-EPMC8024771 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community.
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Project description:Kinetochores in multicellular eukaryotes are usually associated with heterochromatin. Whether this heterochromatin simply promotes the cohesion necessary for accurate chromosome segregation at cell division or whether it also has a role in kinetochore assembly is unclear. Schizosaccharomyces pombe is an important experimental system for investigating centromere function, but all of the previous work with this species has exploited a single strain or its derivatives. The laboratory strain and most other S. pombe strains contain three chromosomes, but one recently discovered strain, CBS 2777, contains four. We show that the genome of CBS 2777 is related to that of the laboratory strain by a complex chromosome rearrangement. As a result, two of the kinetochores in CBS 2777 contain the central core sequences present in the laboratory strain centromeres, but lack adjacent heterochromatin. The closest block of heterochromatin to these rearranged kinetochores is ∼100 kb away at new telomeres. Despite lacking large amounts of adjacent heterochromatin, the rearranged kinetochores bind CENP-A(Cnp1) and CENP-C(Cnp3) in similar quantities and with similar specificities as those of the laboratory strain. The simplest interpretation of this result is that constitutive kinetochore assembly and heterochromatin formation occur autonomously.
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Project description:The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis tool in various eukaryotic organisms. Here, we show that PB transposes in the fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We developed a PB-based mutagenesis system for S. pombe by constructing a strain with a selectable transposon excision marker and an integrated transposase gene. PB transposition in this strain has low chromosomal distribution bias as shown by deep sequencing-based insertion site mapping. Using this system, we obtained loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1 from a screen for mutants resistant to the microtubule-destabilizing drug thiabendazole. From another screen for cdc25-22 suppressors, we obtained multiple alleles of wee1 as expected. The success of these two screens demonstrated the usefulness of this PB-mediated mutagenesis tool for fission yeast.
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Project description:Heterochromatin is a conserved feature of eukaryotic genomes and regulates various cellular processes, including gene silencing, chromosome segregation, and maintenance of genome stability. In the fission yeast Schizosaccharomyces pombe, heterochromatin formation involves methylation of lysine 9 in histone H3 (H3K9), which recruits Swi6/HP1 proteins to heterochromatic loci. The Swi6/HP1-H3K9me3 chromatin complex lies at the center of heterochromatic macromolecular assemblies and mediates many functions of heterochromatin by recruiting a diverse set of regulators. However, additional factors may be required for proper heterochromatin organization, but they are not fully known. Here, using several molecular and biochemical approaches, we report that Vgl1, a member of a large family of multiple KH-domain proteins, collectively known as vigilins, is indispensable for the heterochromatin-mediated gene silencing in S. pombe ChIP analysis revealed that Vgl1 binds to pericentromeric heterochromatin in an RNA-dependent manner and that Vgl1 deletion leads to loss of H3K9 methylation and Swi6 recruitment to centromeric and telomeric heterochromatic loci. Furthermore, we show that Vgl1 interacts with the H3K9 methyltransferase, Clr4, and that loss of Vgl1 impairs Clr4 recruitment to heterochromatic regions of the genome. These findings uncover a novel role for Vgl1 as a key regulator in heterochromatin-mediated gene silencing in S. pombe.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2013-07-20 | GSE42850 | GEO
Project description:The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.
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Project description:Fission yeast Swi6 is a human HP1 homolog that plays important roles in multiple cellular processes. In addition to its role in maintaining heterochromatin silencing, Swi6 is required for cohesin enrichment at the pericentromere. Loss of Swi6 leads to abnormal mitosis, including defects in the establishment of bioriented sister kinetochores and microtubule attachment. Swi6 interacts with Dfp1, a regulatory subunit of DBF4-dependent kinase (DDK), and failure to recruit Dfp1 to the pericentromere results in late DNA replication. Using the dfp1-3A mutant allele, which specifically disrupts Swi6-Dfp1 association, we investigated how interaction between Swi6 and Dfp1 affects chromosome dynamics. We find that disrupting the interaction between Swi6 and Dfp1 delays mitotic progression in a spindle assembly checkpoint-dependent manner. Artificially tethering Dfp1 back to the pericentromere is sufficient to restore normal spindle length and rescue segregation defects in swi6-deleted cells. However, Swi6 is necessary for centromeric localization of Rad21-GFP independent of DDK. Our data indicate that DDK contributes to mitotic chromosome segregation in pathways that partly overlap with, but can be separated from both, Swi6 and the other HP1 homolog, Chp2.
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Project description:Distinct chromatin organization features, such as centromeres and heterochromatin domains, are inherited epigenetically. However, the mechanisms that modulate the accuracy of epigenetic inheritance, especially at the individual nucleosome level, are not well-understood. Here, using ChIP and next-generation sequencing (ChIP-Seq), we characterized Ccp1, a homolog of the histone chaperone Vps75 in budding yeast that functions in centromere chromatin duplication and heterochromatin maintenance in fission yeast (Schizosaccharomyces pombe). We show that Ccp1 is enriched at the central core regions of the centromeres. Of note, among all histone chaperones characterized, deletion of the ccp1 gene uniquely reduced the rate of epigenetic switching, manifested as position effect variegation within the centromeric core region (CEN-PEV). In contrast, gene deletion of other histone chaperones either elevated the PEV switching rates or did not affect centromeric PEV. Ccp1 and the kinetochore components Mis6 and Sim4 were mutually dependent for centromere or kinetochore association at the proper levels. Moreover, Ccp1 influenced heterochromatin distribution at multiple loci in the genome, including the subtelomeric and the pericentromeric regions. We also found that Gar2, a protein predominantly enriched in the nucleolus, functions similarly to Ccp1 in modulating the epigenetic stability of centromeric regions, although its mechanism remained unclear. Together, our results identify Ccp1 as an important player in modulating epigenetic stability and maintaining proper organization of multiple chromatin domains throughout the fission yeast genome.
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-07-20 | E-GEOD-42850 | biostudies-arrayexpress
Project description:7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique PolX in fission yeast, incorporates ATP opposite 8oxodG almost exclusively when all nucleotides (ribos and deoxys) are provided at physiological concentrations. Remarkably, this SpPol4-specific reaction could also occur during the NHEJ of DSBs. In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP. These data are the first evidence that ribonucleotides can be used safely for 8oxodG tolerance, suggesting that insertion of the highly abundant ATP substrate could be beneficial to promote efficient and error-free repair of 8oxodG-associated DSBs. Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates. This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.
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