Identification and characterisation of the interaction between FOXP2 and the ligand binding domain of oestrogen receptor α
Date
2023-10
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Witwatersrand, Johannesburg
Abstract
Forkhead box P2 (FOXP2) regulates the expression of various genes and is associated with language and speech, neural development and outgrowth, and cancer. As transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation and cancer progression and outcome. The association between ER1 and FOXP2 was first identified in MCF-7 cells using co-immunoprecipitation. Thereafter, the interaction was characterised biophysically by overexpressing the FOXP2’s DNA-binding forkhead domain (FHD) and N-terminal region (NT), and ER1’s ligand-binding domain (LBD) in E. coli cells. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the thermodynamic parameters and regulation of interaction between FOXP2 FHD and ER1 LBD, respectively. Electrophoretic mobility shift assay was used to investigate the effect of the interaction on FOXP2’s DNA binding ability. Following the successful overexpression and purification of all three proteins, ER1 LBD was found to interact with FOXP2 FHD but not with FOXP2 NT. The affinity of the ER1 LBD for FOXP2 FHD increases with an increase in salt concentration. ITC shows a similar trend and reveals that the interaction is enthalpically favoured at lower salt concentrations but enthalpically opposed at higher salt concentrations. Additionally, the FOXP2-ER1 LBD interaction remains unaffected by the inclusion of oestrogen, but addition of FOXP2 cognate DNA results in inhibition of the formation of the complex. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the DNA simultaneously suggesting a probable role of this interaction in regulating the transcriptional pathway of FOXP2. This study serves as a foundation for further investigation into the interaction between FOXP2 and ER1 in different cell lines and its relevance in FOXP2-mediated outcomes in cancer and neurodevelopmental disorders.
Description
Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Cell Biology (MSc) , to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023.
Keywords
Protein-protein interactions, Protein-DNA interaction, Isothermal titration calorimetry, Transcription factors, UCTD
Citation
Lakhi, Aasiya Yakub. (2023). Identification and characterisation of the interaction between FOXP2 and the ligand binding domain of oestrogen receptor α. [PhD thesis, University of the Witwatersrand, Johannesburg]. https://hdl.handle.net/10539/41766