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Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We pr...

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Autores principales: Yunusova, Anastasia, Smirnov, Alexander, Shnaider, Tatiana, Lukyanchikova, Varvara, Afonnikova, Svetlana, Battulin, Nariman
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8599953/
https://www.ncbi.nlm.nih.gov/pubmed/34805274
http://dx.doi.org/10.3389/fmolb.2021.757394
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author Yunusova, Anastasia
Smirnov, Alexander
Shnaider, Tatiana
Lukyanchikova, Varvara
Afonnikova, Svetlana
Battulin, Nariman
author_facet Yunusova, Anastasia
Smirnov, Alexander
Shnaider, Tatiana
Lukyanchikova, Varvara
Afonnikova, Svetlana
Battulin, Nariman
author_sort Yunusova, Anastasia
collection PubMed
description The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system.
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spelling pubmed-85999532021-11-19 Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells Yunusova, Anastasia Smirnov, Alexander Shnaider, Tatiana Lukyanchikova, Varvara Afonnikova, Svetlana Battulin, Nariman Front Mol Biosci Molecular Biosciences The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system. Frontiers Media S.A. 2021-11-04 /pmc/articles/PMC8599953/ /pubmed/34805274 http://dx.doi.org/10.3389/fmolb.2021.757394 Text en Copyright © 2021 Yunusova, Smirnov, Shnaider, Lukyanchikova, Afonnikova and Battulin. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Yunusova, Anastasia
Smirnov, Alexander
Shnaider, Tatiana
Lukyanchikova, Varvara
Afonnikova, Svetlana
Battulin, Nariman
Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_full Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_fullStr Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_full_unstemmed Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_short Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells
title_sort evaluation of the ostir1 and atafb2 aid systems for genome architectural protein degradation in mammalian cells
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8599953/
https://www.ncbi.nlm.nih.gov/pubmed/34805274
http://dx.doi.org/10.3389/fmolb.2021.757394
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