FOXE3 in Creating Lens Chromatin Architecture and Transcriptional Identity - The ocular lens serves as an excellent model for studying cell fate determination, as it consists of two distinct cell types derived from the surface ectoderm: lens epithelial and fiber cells. Lens development requires suppression of pro-neurogenic pathways, distinguishing it from other anterior placodes, such as the olfactory placode, which gives rise to neurons. Mutations in key transcription factors (TFs), including AP-2α, FOXE3, and PAX6, lead to overlapping developmental defects such as aphakia, incomplete lens separation, and fiber cell abnormalities, suggesting shared transcriptional targets and regulatory mechanisms. However, a major gap remains in understanding how FOXE3 functions as a transcriptional regulator and how its activity influences chromatin architecture to suppress pro-neurogenic gene expression during lens formation. Preliminary studies using CUT&RUN identified a de novo FOXE3-binding motif, with strong similarity to FOXD3, and revealed that FOXE3 is required to repress neurogenic gene expression in early lens cells. Additionally, RNA-seq analyses of Foxe3 mutant lenses demonstrated significant differential gene expression linked to pro-neurogenic pathways. This led to the hypothesis that FOXE3 functions as both a transcriptional activator and repressor, coordinating chromatin remodeling to establish and maintain lens cell identity. To test this, state-of-the-art chromatin and transcriptomic technologies, including single- nucleus multiomics (ATAC-seq + RNA-seq), CUT&RUN, and functional assays in chick embryos, to dissect FOXE3’s molecular mechanisms will be conducted. Aim 1 will identify FOXE3 transcriptional targets and determine how chromatin accessibility (DARs) and FOXE3-bound enhancers and promoters are altered in Foxe3 mutant lenses. This will be accomplished through the integration of single-nucleus multiomics and CUT&RUN data to map the FOXE3-dependent regulatory landscape. Aim 2 will determine the functional role of FOXE3 in cell fate decisions by (1) gain and loss of function assays in chick lens cells, (2) testing its ability to suppress neurogenesis in the developing chick retina, and (3) identifying FOXE3-interacting proteins via immunoprecipitation and mass spectrometry. The completion of these aims will provide mechanistic insights into FOXE3’s role in lens development, establish a FOXE3 DNA-binding motif, and identify targets of FOXE3 regulation. These studies aim to reveal how FOXE3 mutations contribute to congenital eye disorders such as aphakia, cataracts, microphthalmia, coloboma, and Peters anomaly. By elucidating the chromatin regulatory mechanisms underlying lens cell identity, this work has the potential to inform therapeutic strategies for human eye diseases linked to transcriptional dysregulation.
