Drug-Inducible Control of Lethality Genes: A Low Background Destabilizing Domain Architecture Applied to the Gal4-UAS System in Drosophila.

Manjunatha Kogenaru,Mark Isalan

doi:10.1021/acssynbio.7b00302

Abstract

Destabilizing domains (DDs) are genetic tags that conditionally control the level of abundance of proteins-of-interest (POI) with specific stabilizing small-molecule drugs, rapidly and reversibly, in a wide variety of organisms. The amount of the DD-tagged fusion protein directly impacts its molecular function. Hence, it is important that the background levels be tightly regulated in the absence of any drug. This is especially true for classes of proteins that function at extremely low levels, such as lethality genes involved in tissue development and certain transcriptional activator proteins. Here, we establish the uninduced background and induction levels for two widely used DDs (FKBP and DHFR) by developing an accurate quantification method. We show that both DDs exhibit functional background levels in the absence of a drug, but each to a different degree. To overcome this limitation, we systematically test a double architecture for these DDs (DD-POI-DD) that completely suppresses the protein’s function in an uninduced state, while allowing tunable functional levels upon adding a drug. As an example, we generate a drug-stabilizable Gal4 transcriptional activator with extremely low background levels. We show that this functions in vivo in the widely used Gal4-UAS bipartite expression system in Drosophila melanogaster. By regulating a cell death gene, we demonstrate that only the low background double architecture enables tight regulation of the lethal phenotype in vivo. These improved tools will enable applications requiring exceptionally tight control of protein function in living cells and organisms.

Highlights

Destabilizing domains (DDs) are genetic tags that conditionally control the level of abundance of proteinsof-interest (POI) with specific stabilizing small-molecule drugs, rapidly and reversibly, in a wide variety of organisms
Various methods have been developed that directly control target protein levels inside living cells.[4−12] These include protein degron systems induced by auxin, light, or destabilizing domains (DDs).[11−14] Here, we mainly focus on the DD-based degron system.[13,14]
We have developed an accurate quantification method to compare different DD constructs more directly, and found that the original dihydrofolate reductase (DHFR)-DD displays less background, whereas the FK506-rapamycin-binding protein (FKBP)-DD can be stabilized fully

Summary

Introduction

Destabilizing domains (DDs) are genetic tags that conditionally control the level of abundance of proteinsof-interest (POI) with specific stabilizing small-molecule drugs, rapidly and reversibly, in a wide variety of organisms. Generic molecular tools that regulate protein stability synthetically at a post-translational level, and in a reversible fashion, are vital for the detailed understanding of conditional functions.[3] To this end, various methods have been developed that directly control target protein levels inside living cells.[4−12] These include protein degron systems induced by auxin, light, or destabilizing domains (DDs).[11−14] Here, we mainly focus on the DD-based degron system.[13,14] The DD strategy involves genetically fusing the protein of interest to a small unstable protein domain. N.B., the maximum s.d. observed for DHFR-eGFP is ±0.38%, most error bars are invisible in (c)

Methods

Results

Conclusion