A new versatile system for rapid control of gene expression in the fission yeast <i>Schizosaccharomyces pombe</i>

Zilio, Nicola; Wehrkamp-Richter, Sophie; Boddy, Michael N.

doi:10.1002/yea.2920

Cited by 33 publications

(38 citation statements)

References 30 publications

(38 reference statements)

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“…To this end, we replaced the endogenous SUMO promoter with a weaker constitutive promoter [53]. In stark contrast to sumoΔ cells, those expressing less SUMO ( tetO7-SUMO ) appear wild-type for growth and genotoxin resistance (Fig 2B).…”

Section: Resultsmentioning

confidence: 99%

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion

Nie¹,

Moser²,

Nakamura³

et al. 2017

PLoS Genet

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The posttranslational modifiers SUMO and ubiquitin critically regulate the DNA damage response (DDR). Important crosstalk between these modifiers at DNA lesions is mediated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitinates SUMO chains to generate SUMO-ubiquitin hybrids. These SUMO-ubiquitin hybrids attract DDR proteins able to bind both modifiers, and/or are degraded at the proteasome. Despite these insights, specific roles for SUMO chains and STUbL in the DDR remain poorly defined. Notably, fission yeast defective in SUMO chain formation exhibit near wild-type resistance to genotoxins and moreover, have a greatly reduced dependency on STUbL activity for DNA repair. Based on these and other data, we propose that a critical role of STUbL is to antagonize DDR-inhibitory SUMO chain formation at DNA lesions. In this regard, we identify a SUMO-binding Swi2/Snf2 translocase called Rrp2 (ScUls1) as a mediator of the DDR defects in STUbL mutant cells. Therefore, in support of our proposal, SUMO chains attract activities that can antagonize STUbL and other DNA repair factors. Finally, we find that Taz1TRF1/TRF2-deficiency triggers extensive telomeric poly-SUMOylation. In this setting STUbL, together with its cofactor Cdc48p97, actually promotes genomic instability caused by the aberrant processing of taz1Δ telomeres by DNA repair factors. In summary, depending on the nature of the initiating DNA lesion, STUbL activity can either be beneficial or harmful.

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Section: Resultsmentioning

confidence: 99%

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion

Nie¹,

Moser²,

Nakamura³

et al. 2017

PLoS Genet

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“…Unfortunately, however, the nmt promoter is specific to fission yeast. The Tet‐OFF system is applicable in fission yeast (Zilio et al, ) and we expect that our iAID type set‐up would also work in fission yeast. Notably, because the iAID system simultaneously introduces both tTA and TetR′–Ssn6, cells show dose‐dependent doxycycline sensitivity, and pregrowing cells in the medium containing a low doxycycline concentration can sensitize the cells' response to the restriction condition (Figures , ).…”

Section: Resultsmentioning

confidence: 99%

iAID: an improved auxin-inducible degron system for the construction of a ‘tight’ conditional mutant in the budding yeastSaccharomyces cerevisiae

Tanaka

Miyazawa-Onami

Iida

et al. 2015

Yeast

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Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.

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“…However, tetO/CaMV expression is comparable with only the weakest nmt1 promoter variant and also takes between 9 and 12 h to induce (Forsburg, ; Erler et al ., ). A more recent version of the tetO system has improved its expression kinetics (Zilio et al ., ). Nonetheless, mitochondrial toxicity of tetracycline remains a concern (Moullan et al ., ).…”

Section: Introductionmentioning

confidence: 97%

An estradiol‐inducible promoter enables fast, graduated control of gene expression in fission yeast

Ohira

Hendrickson

McIsaac

et al. 2017

Yeast

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The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β-estradiol-regulated function of the human estrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3EV, turns on quickly, can reach a maximal induction of 20 fold, and exhibits a linear dose response over its entire induction range, with few off target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast.

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A new versatile system for rapid control of gene expression in the fission yeast Schizosaccharomyces pombe

Cited by 33 publications

References 30 publications

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion

iAID: an improved auxin-inducible degron system for the construction of a ‘tight’ conditional mutant in the budding yeastSaccharomyces cerevisiae

An estradiol‐inducible promoter enables fast, graduated control of gene expression in fission yeast

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