Exploring Protein Interactions to Understand Gene Regulation by the Drosophila Ecdysone Receptor
Disciplines
Biochemistry
Abstract (300 words maximum)
The Ecdysone Receptor (EcR) is a nuclear receptor found in invertebrates, such as drosophila, that regulates gene expression during development and reproduction. While it is known that the ligand binding domain (LBD) of the ecdysone receptor binds some accessory proteins besides the ligand to regulate gene expression, protein-protein interactions with the ligand binding domain of the ecdysone receptor is understudied, and identifying the full counsel of these proteins can give major insights into how the system regulates gene expression. This project uses the Yeast Two-Hybrid (Y2H) assay to investigate novel physical interactions between the LBD of EcR and proteins in a drosophila cDNA library. The LBD is fused to the DNA binding domain of Gal4, while a drosophila library of cDNA is fused to the DNA activation domain of Gal4, creating bait and prey proteins respectively. When bait and prey are transformed into yeast, and if they interact, Gal4 transcription factor is successfully reconstituted which then activates reporter genes resulting in blue colonies and resistance to the antibiotic aureobasidin, enabling the identification of novel protein-protein interactions. A preliminary check for autoactivation was completed by transforming our bait into yeast and performing the assay to ensure that reporter genes are not activated by the LBD in the absence of the DNA activation domain. Y2H controls were also performed; the positive control confirmed interaction between known interacting proteins p53 and T-antigen and the negative control confirmed that known non-interacting proteins do not activate reporter genes. By completing the check for potential autoactivation and positive and negative controls of the Y2H assay, it was determined that the assay is functioning as designed, and further experimentation can be completed. The library screening will be completed to determine proteins that positively interact with the LBD of EcR and potentially aid in gene regulation.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Dr. Joanna Wardwell-Ogzo
Exploring Protein Interactions to Understand Gene Regulation by the Drosophila Ecdysone Receptor
The Ecdysone Receptor (EcR) is a nuclear receptor found in invertebrates, such as drosophila, that regulates gene expression during development and reproduction. While it is known that the ligand binding domain (LBD) of the ecdysone receptor binds some accessory proteins besides the ligand to regulate gene expression, protein-protein interactions with the ligand binding domain of the ecdysone receptor is understudied, and identifying the full counsel of these proteins can give major insights into how the system regulates gene expression. This project uses the Yeast Two-Hybrid (Y2H) assay to investigate novel physical interactions between the LBD of EcR and proteins in a drosophila cDNA library. The LBD is fused to the DNA binding domain of Gal4, while a drosophila library of cDNA is fused to the DNA activation domain of Gal4, creating bait and prey proteins respectively. When bait and prey are transformed into yeast, and if they interact, Gal4 transcription factor is successfully reconstituted which then activates reporter genes resulting in blue colonies and resistance to the antibiotic aureobasidin, enabling the identification of novel protein-protein interactions. A preliminary check for autoactivation was completed by transforming our bait into yeast and performing the assay to ensure that reporter genes are not activated by the LBD in the absence of the DNA activation domain. Y2H controls were also performed; the positive control confirmed interaction between known interacting proteins p53 and T-antigen and the negative control confirmed that known non-interacting proteins do not activate reporter genes. By completing the check for potential autoactivation and positive and negative controls of the Y2H assay, it was determined that the assay is functioning as designed, and further experimentation can be completed. The library screening will be completed to determine proteins that positively interact with the LBD of EcR and potentially aid in gene regulation.