The FOXP2-TBR1 interaction and its role in the regulation of DNA binding

Blane, Ashleigh Anne
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Forkhead Box P2 (FOXP2) and T–Brain Related Protein 1 (TBR1) are transcription factors associated with neurological development and disorders such as autism spectrum disorder. FOXP2 and TBR1 each contain highly conserved DNA binding domains, the forkhead domain (FHD) and the T–Box domain (T–Box), respectively. TBR1 and FOXP2 appear to interact via the T–Box domain of TBR1 and either the FHD or N–terminal region of FOXP2. Mutations in both proteins have been implicated in autism and some of these result in the disruption of the interaction between the two proteins. The aim of this study was to determine whether the TBR1 T–Box domain and the FOXP2 FHD interact and the subsequent effect of the interaction on DNA binding. Both proteins were successfully expressed in E. coli cells and purified using liquid chromatography. The tertiary and quarterly structure of each protein was evaluated using tryptophan fluorescence and size exclusion chromatography. The proteins were confirmed to be functional by assessing their DNA binding functions using electrophoretic mobility shift assays. As the DNA binding properties of the TBR1 T–Box have never been characterised, DNA binding to T–box was further characterised using isothermal titration calorimetry (ITC) and single molecule Förster resonance energy transfer (smFRET) using DNA containing a single site (SSL) and two palindromic sites (PAL). Finally, fluorescence anisotropy (FA) was used to assess the effect of the T–Box–FHD interaction on each protein’s DNA binding function. Both T–Box and FHD were found to be monomeric in solution and had a folded structure with DNA binding capabilities. T–Box bound both SSL and Pal DNA by interacting with both the major and minor grooves, like other T–Box domains have exhibited in crystal structures. T–Box bound the SSL DNA with a 10X greater affinity than Pal DNA, likely because of interactions that occur in regions flanking the binding site. Furthermore, this work found evidence to suggest that although T–Box can occupy both sites on Pal DNA, T–Box binds these sites independently rather than dimerising in solution and then binding as a dimer. Finally, FA studies showed that the FHD–T–Box interaction disrupts the interaction of the FHD with DNA whereas T–Box can interact with DNA and FHD simultaneously. This work provides insight into a complex regulatory pathway that can cause neurological disorders such as ASD when mis–regulation occurs
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2021