Forkhead box P1 (FoxP1) is a multidomain transcription factor essential for embryonic development. It regulates gene expression by forming complexes with itself and other FoxP family members. Despite its biological importance, the mechanisms driving complex formation and the contribution of individual domains remain poorly understood. Mutations in FoxP1 cause FoxP1 syndrome, a neurodevelopmental disorder with autism spectrum-like features. Defining the physiological spatiotemporal organization of FoxP1 complexes is therefore critical for understanding pathogenic alterations and developing therapeutic strategies. Previous structural studies have characterized FoxP1 interactions in solution. Isolated forkhead (FKH) domains dimerize via domain swapping, while coiled-coil interactions within a leucine zipper (ZIP)-FKH construct suppress domain swapping but promote dimerization. These models advance our view of FoxP1 complexes but overlook contributions from other biomolecules, including DNA. Here, we investigate FoxP1 complexes inside the nuclei of HEK293T cells, providing a more native environment. Using live-cell fluorescence imaging spectroscopy (FIS), we characterize the spatiotemporal configurations of full-length (FL) FoxP1, ZIP-FKH, and FKH fragments via Förster resonance energy transfer (FRET). To resolve nuclear subregions enriched in distinct FRET states, we apply number-and-brightness-based spatial segmentation, uncovering the formation of high-FRET dimers, particularly in the ZIP-FKH construct. We further examine the influence of two pathogenic mutations: R514H, which is known to disrupt DNA binding, and dD358, which avoids dimerization. Strikingly, R514H promotes aggregation and prevents nuclear translocation, while constructs at different FRET states localize to distinct nuclear radial distances, suggesting a link between spatial distribution and dimerization state. Together, our findings provide new insights into the nuclear distribution and configuration of FoxP1 complexes and their pathological perturbations, advancing our understanding of transcription factor regulation in a physiological context.
Lazaro-Alfaro et al. (Sun,) studied this question.