Rice ( <i>Oryza sativa</i>) TIR1 and 5′adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in <i>Schizosaccharomyces pombe</i>

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Abstract

The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing Schizosaccharomyces pombe AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for S. pombe that uses the TIR1 from Oryza sativa (OsTIR1) not fused to Skp1. Furthermore, we demonstrate that degradation efficiency can be improved by pairing an OsTIR1 auxin-binding site mutant, OsTIR1 ^F74A, with an auxin analogue, 5′adamantyl-IAA (AID2). We provide evidence for the enhanced functionality of the OsTIR1 AID and AID2 systems by application to the essential DNA replication factor Mcm4 and to a non-essential recombination protein, Rad52. Unlike AID, no detectable auxin-independent depletion of AID-tagged proteins was observed using AID2.

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Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

Elphège P Nora, Anton Goloborodko, Anne-Laure Valton … (2017)

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, our data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.

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An auxin-based degron system for the rapid depletion of proteins in nonplant cells.

Kohei Nishimura, Tatsuo Fukagawa, Haruhiko Takisawa … (2009)

Plants have evolved a unique system in which the plant hormone auxin directly induces rapid degradation of the AUX/IAA family of transcription repressors by a specific form of the SCF E3 ubiquitin ligase. Other eukaryotes lack the auxin response but share the SCF degradation pathway, allowing us to transplant the auxin-inducible degron (AID) system into nonplant cells and use a small molecule to conditionally control protein stability. The AID system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.

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The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

Liangyu Zhang, Jordan D. Ward, Ze Cheng … (2015)

Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism.

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Author and article information

Contributors

Martin Kupiec: Role: Academic Editor

Maciej Wnuk: Role: Academic Editor

Journal

Journal ID (nlm-ta): Genes (Basel)

Journal ID (iso-abbrev): Genes (Basel)

Journal ID (publisher-id): genes

Title: Genes

Publisher: MDPI

ISSN (Electronic): 2073-4425

Publication date (Electronic): 08 June 2021

Publication date Collection: June 2021

Volume: 12

Issue: 6

Electronic Location Identifier: 882

Affiliations

[1 ]Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK; a.t.watson@ 123456sussex.ac.uk

[2 ]Department of Chemistry, School of Life Sciences, University of Sussex, Brighton BN1 9QJ, UK; S.Hassell-Hart@ 123456sussex.ac.uk (S.H.-H.); J.Spencer@ 123456sussex.ac.uk (J.S.)

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[* ]Correspondence: a.m.carr@ 123456sussex.ac.uk

Author information

Adam T. Watson https://orcid.org/0000-0002-8140-8205

John Spencer https://orcid.org/0000-0001-5231-8836

Antony M. Carr https://orcid.org/0000-0002-2028-2389

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Publisher ID: genes-12-00882

DOI: 10.3390/genes12060882

PMC ID: 8229956

PubMed ID: 34201031

SO-VID: 4acdee22-d483-4927-b9c4-ddb52cfb7297

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Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

Rice ( Oryza sativa) TIR1 and 5′adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in Schizosaccharomyces pombe

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Most cited references 40

Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

An auxin-based degron system for the rapid depletion of proteins in nonplant cells.

The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

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