An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion.

Kizhakke Mattada Sathyan,Fabiana M Duarte,Warren D Anderson,Leighton Core,Michael J Guertin,Brian D Mckenna

doi:10.1101/gad.328237.119

Kizhakke Mattada Sathyan, Fabiana M Duarte + Show 4 more

Open Access

https://doi.org/10.1101/gad.328237.119

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Abstract

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding downstream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in nonplant systems. However, tagging proteins at their endogenous loci results in chronic auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the auxin response transcription factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems but is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin, and we found that expression of the ARF PB1 (Phox and Bem1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transcriptional profiling indicates that ZNF143 activates transcription in cis and regulates promoter-proximal paused RNA polymerase density. Rapidly inducible degradation systems that preserve the target protein's native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.

Highlights

The function of proteins can be studied in cells using RNAi depletion, loss-of-function mutants, temperaturesensitive mutations, small molecule inhibitors, CRISPR interference, or nucleic acid aptamers
GENES & DEVELOPMENT 33:1441–1455 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/19; www.genesdev.org transduction system: (1) transport inhibitor response 1 (TIR1), a subunit of a ubiquitin ligase complex that binds to the target substrate; (2) the auxin response transcription factors (ARF), which directly regulate gene expression; and (3) auxin/indole-3-acetic acid (Aux/IAA) proteins, which are destabilized in the presence of auxin-mediated ubiquitination
ARF-PB1 interacts with the AIDtagged protein to prevent degradation in the absence of auxin; the tagged protein’s abundance is more representative of native levels

Summary

Introduction

The function of proteins can be studied in cells using RNAi depletion, loss-of-function mutants, temperaturesensitive mutations, small molecule inhibitors, CRISPR interference, or nucleic acid aptamers. In the presence of auxin, TIR1 interacts with domain II of Aux/IAA to facilitate ubiquitination and degradation of Aux/IAA (Gray et al 2001; Dharmasiri et al 2003), which liberates ARFs to regulate transcription This AID system was engineered to function outside plant cells (Nishimura et al 2009). Excluding AHR-responsive genes from downstream differential expression analyses is critical when investigating the activity of AID-tagged transcriptional regulators These improvements enhance the robustness, sensitivity, and specificity of the AID system. We applied ARF-rescued AID-mediated rapid degron depletion to the transcription factor ZNF143 to identify the primary ZNF143 response genes and define a functional role of ZNF143 in transcriptional regulation

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