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Published on: June 3, 2016
A novel RNAi lethality rescue screen to identify regulators of adipogenesis
Olivier van Beekum1, Yuan Gao, Ruud Berger
1Department of Metabolic Diseases, Netherlands Metabolomics Centre, University Medical Centre Utrecht, Utrecht, The Netherlands.
Abstract:
Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an attractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting.
Insights
This study introduces a novel RNAi screening method to identify adipogenesis regulators. The new system successfully identified UCHL3 as a key deubiquitinase in Peroxisome proliferator activator receptor γ (PPARγ)-mediated adipogenesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Adipogenesis, the process of fat cell differentiation, is crucial for metabolic homeostasis.
- Peroxisome proliferator activator receptor γ (PPARγ) is a key transcription factor regulating adipogenesis.
- Current high-throughput screening methods for adipogenic factors face limitations due to heterogeneous cell populations and dynamic gene expression during differentiation.
Purpose of the Study:
- To develop and validate a novel RNA interference (RNAi) screening method for identifying regulators of adipogenesis.
- To overcome the limitations of existing in vitro adipogenesis models for high-throughput screening.
- To identify novel genes involved in Peroxisome proliferator activator receptor γ (PPARγ)-mediated adipogenesis.
Main Methods:
- Development of a novel RNAi screening system based on lethality rescue, utilizing microRNA expression driven by a PPARγ-responsive promoter.
- Application of the screening system to a deubiquitinase knock down library.
- Validation of identified hits in the context of adipogenesis.
Main Results:
- The novel RNAi screening method was successfully established as a proof-of-principle.
- Screening of a deubiquitinase library identified UCHL3 as an essential deubiquitinase for adipogenesis.
- The system enables high-throughput identification of novel regulators of PPARγ-mediated adipogenesis.
Conclusions:
- A novel, high-throughput RNAi screening method based on lethality rescue has been developed for adipogenesis research.
- This method overcomes key limitations of traditional in vitro adipogenesis models.
- The study identified UCHL3 as a critical deubiquitinase in PPARγ-driven adipogenesis, highlighting its potential as a therapeutic target.
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