Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
- University of Edinburgh, United Kingdom
- Hospital for Sick Children, Canada
- Michigan State University, United States
Abstract
Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and condensin II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.
Data availability
Proteomics data underlying Figure 6B have been submitted to the PRIDE database under accession PXD032374. All other primary data, including flow cytometry files, fluorescence imaging and uncropped western blot scans are available through the DRYAD digital repository at https://doi.org/10.5061/dryad.g1jwstqt9. Requests for the Rosa26Tir1 transgenic mouse line should be addressed to Bin Gu (guibin1@msu.edu), and requests for the Ncaph- and Ncaph2-AID:Clover lines should be addressed to Andrew Wood (Andrew.j.wood@ed.ac.uk).
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Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degronsDryad Digital Repository, doi:10.5061/dryad.g1jwstqt9.
Article and author information
Author details
Jennifer Margaret Brisbane
Alex von Kriegsheim
Funding
Medical Research Council
- Lewis Macdonald
- Gillian C Taylor
- Jennifer Margaret Brisbane
- Ersi Christodoulou
- Lucy Scott
- Andrew J Wood
Wellcome Trust (102560/Z/13/Z)
- Lewis Macdonald
- Gillian C Taylor
- Jennifer Margaret Brisbane
- Ersi Christodoulou
- Lucy Scott
- Andrew J Wood
Canadian Institutes of Health Research (JR FDN-143334)
- Janet Rossant
- Bin Gu
Medical Research Council (MC_PC_21040)
- Andrew J Wood
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal work was approved by a University of Edinburgh internal ethics committee and was performed in accordance with institutional guidelines under license by the UK Home Office. AID knock-in alleles were generated under project license PPL 60/4424. Rosa26Tir1 knockin mouse lines were generated under the Canadian Council on Animal Care Guidelines for Use of Animals in Research and Laboratory Animal Care under protocols approved by the Centre for Phenogenomics Animal Care Committee (20-0026H). Experiments involving double transgenic animals were conducted under the authority of UK project license PPL P16EFF7EE
Reviewing Editor
- Guillaume Pavlovic, PHENOMIN, Institut Clinique de la Souris (ICS), CELPHEDIA, France
Publication history
- Preprint posted: January 13, 2022 (view preprint)
- Received: February 18, 2022
- Accepted: May 19, 2022
- Accepted Manuscript published: June 23, 2022 (version 1)
- Version of Record published: July 11, 2022 (version 2)
Copyright
© 2022, Macdonald et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
eLife 11:e77987.
https://doi.org/10.7554/eLife.77987
Further reading
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Regulatory networks underlying innate immunity continually face selective pressures to adapt to new and evolving pathogens. Transposable elements (TEs) can affect immune gene expression as a source of inducible regulatory elements, but the significance of these elements in facilitating evolutionary diversification of innate immunity remains largely unexplored. Here, we investigated the mouse epigenomic response to type II interferon (IFN) signaling and discovered that elements from a subfamily of B2 SINE (B2_Mm2) contain STAT1 binding sites and function as IFN-inducible enhancers. CRISPR deletion experiments in mouse cells demonstrated that a B2_Mm2 element has been co-opted as an enhancer driving IFN-inducible expression of Dicer1. The rodent-specific B2 SINE family is highly abundant in the mouse genome and elements have been previously characterized to exhibit promoter, insulator, and non-coding RNA activity. Our work establishes a new role for B2 elements as inducible enhancer elements that influence mouse immunity, and exemplifies how lineage-specific TEs can facilitate evolutionary turnover and divergence of innate immune regulatory networks.
