A new tool for rapid protein degradation in living animals

Andrew Wood, group leader at the MRC Human Genetics Unit, and his colleagues are developing a new tool to modulate protein levels in vivo.

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Understanding the relationship between genotype and phenotype is essential to understanding the biology of disease. One method for doing this is to alter the function of a gene in a model system - cells in a dish, organoids, or animal models. Generally, this is accomplished by manipulating DNA or RNA in the cell, but these methods have their limitations.

Andrew Wood, group leader at the MRC Human Genetics Unit and lead of the MRC National Mouse Genetics Network (NMGM) Degron Tagging Cluster, has provided a proof of principle for a new way to study protein function in mouse models of development and disease.

New method based on a plant system

Methods that drive the degradation of a target protein in cells and tissues could work much faster than those that prevent the production of new proteins, and they could also limit compensatory mechanisms that arise because of gene deletions. Genetic changes are also difficult to reverse, so a method by which proteins can be removed and subsequently restored to normal levels could be of great benefit to our understanding of the role of proteins at various stages. Therefore, techniques that drive degradation of proteins should be rapid, reversible, and targeted.

The method takes advantage of a chemical-genetic tool known as auxin-inducible degron. It is based on a plant system for protein degradation in which the plant hormone auxin binds a protein, Tir1, which then targets proteins containing a "degron" sequence. These are then tagged for degradation by the mammalian proteasome complex. The system has already revolutionised protein function studies in mammalian in vitro systems, but it has not been possible to degrade tagged endogenous proteins in adult tissues of living animals.

In their research just published in eLife, Andrew's group has developed novel transgenic mouse lines that use this Degron system to rapidly (< 2 hours) and almost completely (> 90%) degrade two protein subunits of condensin complexes, condensin I and condensin II. These proteins are essential for chromosome formation and segregation during mitotic cell division. The researchers found that both protein subunits are required for cell division in progenitor lymphocytes, but not in differentiated peripheral lymphocyte derivatives, where cells can divide with only one subunit present. Although degradation was seen in most cell types tested, problems such as the absence of components of the ubiquitin-proteasome system or poor transport of auxin across barriers within tissues meant that degradation was not possible in a minority of cell types.

Importantly, the team did not detect degradation in the absence of auxin administration, suggesting that the system is not leaky in this case. The amount of target protein, auxin, and Tir1 determined how quickly the proteins could be degraded. This could make the system difficult to use for proteins that are present in very large amounts in the cells.

Being able to rapidly reduce protein levels is really important for studying processes which occur quickly like mitosis, the focus of our lab’s research. As well as helping us understand protein function, the system should also be useful for modelling the mechanism and effects of drugs in disease models, as most drugs target proteins.

 Andrew Wood, Group Leader, MRC Human Genetics Unit and lead for MRC National Mouse Genetics Network’s (NMGM)  Degron Tagging cluster

Development of genetic reporters to evaluate and compare different degron systems

Rapid removal of proteins is key to the study of essential cell cycle proteins such as condensins, because the subsequent changes arising from abnormal cell division can quickly obscure the primary effects of the loss of the degraded protein. Because most drugs target proteins, these tools could also provide a useful way to model the effects of drugs and other therapeutic interventions, in addition to their use for functional studies of proteins in disease models. The Degron Tagging Cluster, which also includes Asier Unciti Broceta of Edinburgh Cancer Research, Joe Marsh of MRC HGU, and Gopal Sapkota and Roland Wolf of the University of Dundee, aims to build on this work and develop a suite of degron models. They will develop genetic reporters to evaluate and compare different degron systems across tissues and disease models. They will use these to test existing ligands and synthesise novel derivatives to identify compounds with favourable pharmacokinetic and pharmacodynamic properties in vivo. 

This study was conducted in collaboration with Dr. Bin Gu (Michigan State University) and Professor Janet Rossant (Sick Kids, Toronto) and supported by UKRI MRC, the Wellcome Trust and the Canadian Institutes of Health Research.

Related Links

Institute of Genetics and Cancer

Andrew Wood Research Group

UKRI National Mouse Genetic Network

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons,  eLife (2022)