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).
-
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
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.
Metrics
-
- 7,860
- views
-
- 1,096
- downloads
-
- 22
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
-
- Chromosomes and Gene Expression
The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.
-
- Chromosomes and Gene Expression
Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.
