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A One-step strategy to target essential factors with auxin-inducible degron system in mouse embryonic stem cells.

ABSTRACT: The self-renewal and pluripotency of embryonic stem cells (ESCs) are conferred by networks including transcription factors and histone modifiers. The Auxin-inducible degron (AID) system can rapidly and reversibly degrade its target proteins and is becoming a powerful tool to explore novel function of key pluripotent and histone modifier genes in ESCs. However, the low biallelic tagging efficiency and a basal degradation level of the current AID systems deem it unsuitable to target key pluripotent genes with tightly controlled expression levels. Here, we develop a one-step strategy to successfully target and repress the endogenous pluripotent genes in mouse ESCs and replace their expression with AID fused transgenes. Therefore, this work provides an efficient way for employing the AID system to uncover novel function of essential pluripotent and chromatin modifier genes in ESCs.

SUBMITTER: Li J

PROVIDER: S-EPMC9393215 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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A One-step strategy to target essential factors with auxin-inducible degron system in mouse embryonic stem cells.

Li Jingsheng J Dai Chunhong C Xie Wenyan W Zhang Heyao H Huang Xin X Chronis Constantinos C Ye Ying Y Zhang Wensheng W

Frontiers in cell and developmental biology 20220808

The self-renewal and pluripotency of embryonic stem cells (ESCs) are conferred by networks including transcription factors and histone modifiers. The Auxin-inducible degron (AID) system can rapidly and reversibly degrade its target proteins and is becoming a powerful tool to explore novel function of key pluripotent and histone modifier genes in ESCs. However, the low biallelic tagging efficiency and a basal degradation level of the current AID systems deem it unsuitable to target key pluripoten ...[more]

PMID: 36003152

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Project description:Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.

Dataset Information

A One-step strategy to target essential factors with auxin-inducible degron system in mouse embryonic stem cells.

Publications

A One-step strategy to target essential factors with auxin-inducible degron system in mouse embryonic stem cells.

Similar Datasets

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