Life Sciences & Biotechnology
Title : | Exploring the role of tandem epigenetic readers in regulating gene expression using an expanded genetic code |
Area of research : | Life Sciences & Biotechnology |
Focus area : | Epigenetics, Molecular Biology |
Principal Investigator : | Dr. Babu Sudhamalla, Indian Institute Of Science Education And Research (IISER) Kolkata, West Bengal |
Timeline Start Year : | 2023 |
Timeline End Year : | 2026 |
Contact info : | s.babu@iiserkol.ac.in |
Details
Executive Summary : | Epigenetic reader modules bind to chemical tags (acetyl, methyl, phosphoryl etc., groups) added to histones to interpret and translate the signal into meaningful functional outcome. Tandem epigenetic readers that contain two adjacent reader domains can simultaneously bind two such chemical modification. But beyond their function in binding different histone modifications, the role of tandem epigenetic readers has not been well explored. This project will explore the role of epigenetic tandem readers in regulating gene expression using the tandem dual bromodomain of TAF1 and the tandem PHD-bromo module of BPTF as a paradigm. First, all the dynamic interacting partners of the tandem reader module will be identified by interaction-based protein profiling (IBPP) approach. The interaction between the epigenetic reader and their ligand are transient, thereby making it impossible to profile their entire interactome by traditional immunoprecipitation methods. Utilizing the amber suppressor mutagenesis, the tandem readers will be engineered to site specifically incorporate a light activable, unnatural amino acids (UAA like AzF, BzF) without altering their overall fold of the protein secondary structure and thermal stability or compromising their ability to bind ligands. Individual domain of the tandem reader will be engineered one at a time. The changes in secondary structure and stability of the tandem reader upon UAA incorporation will be evaluated by Circular Dichroism and Thermal Shift Assay to screen the best mutant for further experiments. The ligand binding ability of the UAA incorporated mutant reader will be validated by Isothermal Titration Calorimetry. After structural validation, the mutant tandem reader will be allowed to crosslink with the cellular proteome. Upon UV irradiation, the light activable group in the UAA will form a reactive intermediate that will form a strong covalent bond with functional groups nearby, thereby capturing even weak and transient interactions. The individual domain-specific interactome of the tandem readers will then be profiled and identified by Mass Spectrometry (LC-MS/MS) based approach. Next, to evaluate the role of individual domains of the tandem reader in regulating gene expression, the mutant tandem readers will be exogenously expressed in human cell lines. Tandem reversible and irreversible cross-linking (TRIC) approach will be employed to first crosslink the protein-DNA interaction by formaldehyde, followed by UV irradiation to irreversibly crosslink the domain specific interacting partner of the tandem reader. Chromatin Immunoprecipitation (ChIP) followed by Polymerase Chain Reaction (PCR) will be performed at the known gene targets of the tandem readers to evaluate the difference in domain-specific interaction at the chromatin. Overall, this project will explore the dynamics of the individual domains of the tandem reader in regulating gene expression. |
Total Budget (INR): | 43,54,799 |
Organizations involved