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

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Drug-Inducible Control of Lethality Genes: A Low Background Destabilizing Domain Architecture Applied to the Gal4-UAS System in Drosophila.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-05-15 , DOI: 10.1021/acssynbio.7b00302
Manjunatha Kogenaru _{1,

2} , Mark Isalan _{1,

2}

Affiliation

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.

中文翻译：

致死基因的药物诱导控制：应用于果蝇 Gal4-UAS 系统的低背景不稳定结构域结构。

不稳定结构域 (DD) 是一种遗传标签，可在多种生物体中通过特定的稳定小分子药物快速、可逆地有条件地控制目的蛋白 (POI) 的丰度水平。 DD标签融合蛋白的量直接影响其分子功能。因此，在没有任何药物的情况下严格调节背景水平非常重要。对于以极低水平发挥作用的蛋白质类别尤其如此，例如参与组织发育的致死基因和某些转录激活蛋白。在这里，我们通过开发精确的定量方法，确定了两种广泛使用的 DD（FKBP 和 DHFR）的非诱导背景和诱导水平。我们表明，两种 DD 在没有药物的情况下都表现出功能背景水平，但程度不同。为了克服这一限制，我们系统地测试了这些 DD (DD-POI-DD) 的双重架构，该架构在非诱导状态下完全抑制蛋白质的功能，同时允许在添加药物时调节功能水平。例如，我们生成了背景水平极低的药物稳定 Gal4 转录激活剂。我们证明，这种功能在果蝇广泛使用的 Gal4-UAS 二分表达系统中发挥作用。通过调节细胞死亡基因，我们证明只有低背景双结构才能严格调节体内致死表型。这些改进的工具将使需要对活细胞和生物体中的蛋白质功能进行极其严格控制的应用成为可能。

更新日期：2018-05-07

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