SUMMARY FOXP1 syndrome (FOXP1S) is a rare neurodevelopmental disorder causing severe language impairment, motor delays, visual-motor integration deficits, complex psychiatric presentations, repetitive behaviors, sensory impairment, and Autism symptoms. The major pathogenicity mechanism is haploinsufficiency and most identified mutations lie within the DNA binding domain resulting in loss of transcriptional repressor activity. However, the direct binding targets of FOXP1 in neural cells are yet to be elucidated. Our goal is to further our understanding of the disease mechanisms underpinning FOXP1S by determining the transcriptional networks regulated by FOXP1 in human cortical neural progenitor cells (NPCs) and neurons. In other systems, Foxp1 regulates cell cycle dynamics and differentiation by influencing glucose homeostasis, metabolism, angiogenesis, and response to hypoxia. FOXP1 has been shown to contribute to multiple cancers and has been identified as a druggable target as well as a prognostic biomarker. During mouse and human cortical development, Foxp1 is expressed by NPCs and neurons and several studies have sought to determine its role in these different cell populations. In NPCs, Foxp1 promotes symmetric divisions, promoting self-renewal and the generation of early born neurons. In neurons, Foxp1 has been shown to play roles in neuronal differentiation, migration, and morphology. Whilst it has been classified as a transcriptional repressor, evidence supports a role for Foxp1 as a transcriptional activator during brain development. Given its expression in these two distinct cell populations, and our long-term aim to develop an in vitro model of FOXP1S it is important to determine the cell-type specific targets of human FOXP1. Our specific aims are to 1) identify the direct binding targets of FOXP1 in human cortical neural progenitors and neurons and 2) determine the transcriptional changes of direct targets in FOXP1 deficient human cortical NPCs and neurons. Using wildtype and FOXP1 homozygous mutant human induced pluripotent stem cells (iPSCs) we will generate cortical NPCs and neurons using the Dual SMAD inhibition directed differentiation methods. For Aim 1, using CUT&RUN followed by DNA sequencing, we will identify FOXP1 binding motifs in NPCs and neurons, using FOXP1-/- cells to distinguish genuine peaks from background noise. For Aim 2, we will perform bulk RNA Sequencing with wildtype and FOXP1-/- iPSC-derived NPCs and neurons to identify the transcriptional changes of direct target genes (identified in Aim 1) in the absence of FOXP1. This will enable us to determine whether FOXP1 acts as a transcriptional repressor or activator. The experiments outlined in this proposal will enable us to address the fundamental role of FOXP1 in cortical development, and place FOXP1 within transcriptional networks that will inform future studies aimed at developing a 3D in vitro model of the rare disease FOXP1S and identifying therapeutic targets.

PROJECT NARRATIVE. Understanding the downstream activities of FOXP1 is critical to our understanding of normal brain development and the etiology of the rare neurodevelopmental disorder FOXP1 syndrome (FOXP1S). The proposed studies seek to investigate the direct binding targets of FOXP1 to determine its role in neural progenitor cells and neurons. Through these studies we will gain novel insights into the disease mechanisms that underpin FOXP1S, increasing our potential to identify potential therapeutics.