Evaluation of drug-free methods for the detection of gene silencing in <i>Saccharomyces cerevisiae</i>

Shaban, Kholoud; Sauty, Safia Mahabub; Fisher, Ashley; Cheng, Ashley; Yankulov, Krassimir

doi:10.1139/bcb-2022-0243

Cited by 5 publications

(13 citation statements)

References 15 publications

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“…Loss of gene silencing is revealed by the decrease of the percentage of 5-FOA R or the proportion of GFP-negative cells in the mutant relative to wild type isogenic strains. These two reporters had reproduced previously documented effects of various mutants in replication factors or histone chaperones (18,19).…”

Section: Resultssupporting

confidence: 57%

“…Recently, we produced two dual adh4-URA3-HTB1→yEGFP-tel and adh4-URA3-yEGFP←HTB1-tel reporters for the analyses of gene silencing at the VIIL telomere (18,19). These reporters contain the URA3 gene followed by fusion HTB1-yEGFP reporter driven by the Histone H2B promoter (HTB1) in the orientation towards or away from the telomere, respectively (Additional le 1: Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The highly sensitive 5-FOA resistance and CRASH assays do not distinguish which one of the two underlying events leads to the loss of silencing. In this study we revisited the silencing effects of the three pol30 mutants with a routine 5-FOA R assay and a novel drug-free low-sensitivity assay (18,19). Our results reveal positional differences in the effects of the pol30 mutations that lead to transient de-repression or complete epigenetic conversions at the VIIL sub-telomere of S. cerevisiae.…”

Section: Introductionmentioning

confidence: 94%

“…The POL30 and the mutant pol30 strains are identical to the ones published in (12). The derivatives of these strains were produced by PCR-directed deletion of the target genes as described in (19), and were PCR con rmed. The reporter fragments were inserted at the VIIL telomere via electroporation and were con rmed by PCR.…”

Section: Yeast Strains Plasmids and Primersmentioning

confidence: 99%

“…The adh4-URA3-tel construct was identical to the one described in (20). The adh4-URA3-HTB1→yEGFP-tel and adh4-URA3-yEGFP←HTB1-tel reporters were described in (18, 19). All strains are listed in Supplementary Table 1, Additional le 2.…”

Section: Yeast Strains Plasmids and Primersmentioning

confidence: 99%

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Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-fold active/silent state at the sub-telomeres of S. cerevisiae

Sauty

Yankulov

2023

Preprint

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Background Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. In this study, we used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the existence of different states of gene repression at the sub-telomeres of S. cerevisiae. Results We show position dependent transient repression or bimodal expression of reporter genes at the VIIL sub-telomere. We also show that mutations in the replicative clamp POL30 (PCNA) or the destruction of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the destruction of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. We analyze the physical interaction of CAF1 and RRM3 with PCNA and discuss the implications of these findings for our understanding of the stability and transmission of the epigenetic state of the genes. Conclusions There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. We characterise the roles of CAF1, RRM3 and ASF1 in these modes of gene repression. We suggest that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing.

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

Section: Yeast Strains Plasmids and Primersmentioning

confidence: 99%

Section: Yeast Strains Plasmids and Primersmentioning

confidence: 99%

See 3 more Smart Citations

Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-fold active/silent state at the sub-telomeres of S. cerevisiae

Sauty

Yankulov

2023

Preprint

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TOF1 and RRM3 reveal a link between gene silencing and the pausing of replication forks

Shaban,

Dolson,

Fisher

et al. 2023

Curr Genet

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Systematic profiling of subtelomeric silencing factors in budding yeast

Juárez-Reyes

Avelar-Rivas²,

Hernandez-Valdes³

et al. 2023

G3: Genes, Genomes, Genetics

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Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and aging. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

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Evaluation of drug-free methods for the detection of gene silencing in Saccharomyces cerevisiae

Cited by 5 publications

References 15 publications

Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-fold active/silent state at the sub-telomeres of S. cerevisiae

Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-fold active/silent state at the sub-telomeres of S. cerevisiae

TOF1 and RRM3 reveal a link between gene silencing and the pausing of replication forks

Systematic profiling of subtelomeric silencing factors in budding yeast

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