Investigating protein-protein interactions of the language-related transcription factor FOXP2 in live cells with bioluminescence resonance energy transfer

Transcription factors play central roles in coordinating developmental processes, as evidenced by the increasing number of transcription factor-related developmental disorders being uncovered by next-generation sequencing and genome-wide studies of copy number variation. The action of a transcription factor in regulating gene expression depends on interactions with other transcription factors, co-activators/co-repressors and chromatin modifying and remodeling complexes. Transcription factors are commonly regulated by post-translational modifications. However the study of protein-protein interactions and post-translational modifications of transcription factors by common techniques such as co-immunoprecipitation and mass spectrometry is hampered by the difficulty in preserving interactions and modifications through cell lysis. To circumvent this issue, we developed a Bioluminescence Resonance Energy Transfer (BRET) assay, which allows protein-protein interactions to be observed in live cells. In this assay, a protein of interest is expressed as a fusion with luciferase from Renilla reniformis, and its putative interaction partner as a fusion with Yellow Fluorescent Protein (YFP). Upon addition of a cell-permeable substrate, the distance-dependent non-radiative transfer of energy from luciferase to YFP is quantified by measurement of light emission at two wavelengths to assess the interaction between the two fusion proteins. To validate the utility of this assay for investigating transcription factor interactions, we confirmed homodimerization of the FOXP2 transcription factor, haploinsufficiency of which causes a rare and severe speech and language disorder, as well as interaction of FOXP2 with other members of the FOXP family. We also confirmed the interaction between FOXP2 and multiple candidate interactors identified through yeast two-hybrid assays, including the autism-related transcription factor TBR1, the co-repressors CtBP1 and CtBP2, and post-translational modification enzymes of the PIAS family. The role of PIAS enzymes in sumoylation – the covalent modification of proteins with Small Ubiquitin-like Modifier (SUMO) proteins – led us to further explore this process, which is notably difficult to investigate because of the dynamic and labile nature of the modification, which is also typically present on only a minor fraction of molecules of a given protein. Combining the BRET assay with gel-shift techniques we demonstrated that FOXP2 is sumoylated. Finally, we used the BRET assay to examine the effects of etiological FOXP2 variants in speech and language disorder on protein-protein interactions and post-translational modification. In summary, the BRET assay is a sensitive, reliable and potentially high-throughput technique for exploring protein biology in the context of live cells. We have demonstrated applications of the assay in validating putative protein-protein interactions, assessing post-translational modifications, and investigating functional effects of protein variants identified in patient cohorts. These investigations have provided novel insights into the function of the FOXP2 transcription factor in neurodevelopment and into the etiology of FOXP2-related speech and language disorder