[生物技術] 利用生長素系統來調控蛋白質的降解。20191106 Genes & Development

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

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

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 systemin 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.

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改良型生長素降解系統，保存了蛋白質先天的表現量，並且可以利用生長素來快速且專一的移除目標蛋白質

摘要

快速的影響蛋白質的功能，可以讓科學家在避開下游、因為時間累積，而產生增幅反應的情況下，研究目標基因的主要分子機制。生長素誘導降解的系統（AID）已被開發，在非植物系統中來作為可以誘導蛋白質的快速降解。然而，被標記的蛋白質在內生的表現位置，會被蛋白質降解系統緩慢的分解。為了改善這個缺點，在改良的系統中，我們表現了生長素反應轉錄因子（ARF）。在前一代的系統中，雖然ARF有其重要性，但並沒有被包含在其中。在植物體內，在沒有生長素的情況下，ARF會直接與生長素降解系統互動，並且，我們發現當表現ARF的PB1區域，可以抑制帶有生長素降解系統標記蛋白質的持續分解。此外，生長素所引起的蛋白質降解，在新的系統中也較前一代來的迅速。為了在能夠定量的情況下，測試新的ARF-AID系統的靈敏度，我們測量了當我們快速調降ZNF143轉錄因子後，全基因體轉錄的變化。整體轉錄的變化顯示，ZNF143會在目標基因上啟動轉錄並且調控靠近啟動子區域的RNA聚合酶的密度。能夠維持蛋白質的原始表現量與型態，並且快速誘導蛋白質降解的系統，可以專一的並且在特定的時間點內建定目標蛋白質的失調，會為生物系統的研究帶來新的革命性變革

原文出處： http://genesdev.cshlp.org/content/early/2019/08/22/gad.328237.119