Electronic Theses and Dissertations (PhDs)

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Browsing Electronic Theses and Dissertations (PhDs) by Keyword "DNA-binding"

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    Investigating FOXP2 dynamics, stability, and DNA-binding capabilities
    (University of the Witwatersrand, Johannesburg, 2024) Perumal, Cardon; Fanucchi, Sylvia
    All forkhead box (FOX) transcription factors share a conserved DNA-binding domain called the forkhead domain. They regulate gene expression in different organisms and have widespread biological roles, ranging from embryogenesis to immune regulation. The FOXP subfamily, unlike other FOX transcription factors, have the capacity for dimerisation, an evolutionary fate yet to be fully understood. In particular, the FOXP2 forkhead domain (FHD) has been shown in vitro to form domain-swapped dimers. Additionally, the FOXP2 leucine zipper domain forms heterotypic associations with FOXP1, 2, and 4. These somewhat isolated structural characterisations of FOXP2 have informed the domain-specific functionality of the DNA-binding forkhead domain but the leucine zipper domain has been characterized to a lesser extent, although it is thought to be implicated in FOXP2 DNA-binding as well. Elucidating the structural and functional impact of the leucine zipper on FOXP2 DNA-binding remains challenging, as it is unclear how these motifs work together to achieve binding and whether complexation is a requirement for DNA-binding. Owing to this, the cooperative structural contributions of both the leucine zipper and forkhead domain and the effect of DNA on the structure and stability of these domains have not been considered. Consequently, the aim of this study was to gain a comprehensive understanding of the FOXP2 DNA-binding mechanism by comparing the structure, stability, and dynamics of both the leucine zipper domain and the forkhead domain in the presence and absence of DNA. Assessed here, for the first time, is the conformational dynamics of the FOXP2 leucine zipper domain and FHD flanking disordered regions using hydrogen-deuterium exchange mass spectrometry. The results confirm the binding of DNA to the FHD recognition helix as well as the change in dynamics of the interlinking loop region. Additionally, the FOXP2 folding mechanisms and stability for each domain was characterised, revealing a 2-state unfolding mechanism for the forkhead domain and a 3-state mechanism for the longer LeuZip variant. Fluorescence anisotropy studies revealed that the LeuZip variant bound DNA with a higher affinity than the forkhead domain. The findings of this study highlight the structural significance of the leucine zipper domain and unstructured regions and the functional cooperativity of the FOXP2 domains investigated