Identification of Novel Co-Regulators of the Ecdysone Receptor Using a Yeast Two-Hybrid Assay
Disciplines
Biochemistry | Molecular Biology
Abstract (300 words maximum)
The Ecdysone receptor (ECR) is a nuclear receptor essential for gene regulation during Drosophila development, functioning as a heterodimer with ultraspiracle (USP). While this interaction is well established, the role of additional co-regulators remains unclear. This study aims to identify novel coactivators and corepressors that interact with the ligand-binding domain (LBD) of ECR and influence gene expression. To investigate these interactions, we employed the yeast two-hybrid (Y2H) assay, a widely used method for detecting protein-protein interactions. We fused the ECR LBD to the Gal4 DNA-binding domain (BD) and candidate co-regulators to the Gal4 activation domain (AD). If an interaction occurs, the BD and AD are brought together, activating reporter genes that enable yeast growth on selective media and producing blue colony formation via X-α-Gal hydrolysis. Additionally, resistance to aureobasidin serves as another reporter for interaction specificity. Our results confirmed that the ECR-LBD does not autoactivate the reporter genes, ensuring assay validity. Yeast transformed with LBD alone (Y190 + LBD) showed no growth on SD/-Trp/X-α-Gal/AbA plates, indicating no independent activation. While minimal background activity was observed on SD/-Trp/X-α-Gal plates, colonies remained white. Control experiments demonstrated specificity, with the positive control (Y190 + p53 with TAg) showing strong interaction and blue colonies, while the negative control (Y190 + LAM with TAg) showed no activity. By identifying proteins that modulate ECR activity, this study enhances understanding of steroid hormone signaling. These findings contribute to insights into nuclear receptor regulation in both Drosophila and mammalian systems, shedding light on hormone-driven gene regulation in health and disease.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Rajnish Singh
Identification of Novel Co-Regulators of the Ecdysone Receptor Using a Yeast Two-Hybrid Assay
The Ecdysone receptor (ECR) is a nuclear receptor essential for gene regulation during Drosophila development, functioning as a heterodimer with ultraspiracle (USP). While this interaction is well established, the role of additional co-regulators remains unclear. This study aims to identify novel coactivators and corepressors that interact with the ligand-binding domain (LBD) of ECR and influence gene expression. To investigate these interactions, we employed the yeast two-hybrid (Y2H) assay, a widely used method for detecting protein-protein interactions. We fused the ECR LBD to the Gal4 DNA-binding domain (BD) and candidate co-regulators to the Gal4 activation domain (AD). If an interaction occurs, the BD and AD are brought together, activating reporter genes that enable yeast growth on selective media and producing blue colony formation via X-α-Gal hydrolysis. Additionally, resistance to aureobasidin serves as another reporter for interaction specificity. Our results confirmed that the ECR-LBD does not autoactivate the reporter genes, ensuring assay validity. Yeast transformed with LBD alone (Y190 + LBD) showed no growth on SD/-Trp/X-α-Gal/AbA plates, indicating no independent activation. While minimal background activity was observed on SD/-Trp/X-α-Gal plates, colonies remained white. Control experiments demonstrated specificity, with the positive control (Y190 + p53 with TAg) showing strong interaction and blue colonies, while the negative control (Y190 + LAM with TAg) showed no activity. By identifying proteins that modulate ECR activity, this study enhances understanding of steroid hormone signaling. These findings contribute to insights into nuclear receptor regulation in both Drosophila and mammalian systems, shedding light on hormone-driven gene regulation in health and disease.