Regulation of the lens transcriptome and chromatin architecture by FOXE3

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Revelation of novel molecular mechanisms that regulate transcriptional networks controlling cellular differentiation provides essential information relevant both to understanding organogenesis and for reprogramming cells for regenerative therapies. The ocular lens provides a simple, self-contained tissue with characteristic patterns of differentiation-specific gene expression to model how transcription factors regulate chromatin landscapes to direct specific transcriptional networks through cooperative interactions with enhancers, promoters, and other regulatory protein complexes. The forkhead transcription factor, FOXE3, is an abundant transcription factor expressed in the early lens forming ectoderm, and maintained in the lens epithelium, downstream of PAX6 expression. In fact, mutations in FOXE3 mirror many of the ocular phenotypes (Peters anomaly, cataracts, reduced epithelia proliferation and fiber cell differentiation) resulting from deficiencies in PAX6 or AP-2, the two other abundantly expressed transcription factors in lens epithelium. However, little information exists concerning how FOXE3 regulates lens development. An RNA-seq analysis in lenses from a newly created Foxe3 allele identified numerous differentially regulated genes. These included downregulation of many classical lens identity genes (including Bfsp1, Bfsp2, Dnase2b and multiple crystallins) and upregulation of many genes associated with neural and or retina differentiation (including Nr2e1, Otx2, Ascl1, Tbx3, Rax, Vsx2, Lhx2 and Six6). This surprising shift in gene expression in FOXE3 deficient lenses, coupled with the structural similarity of FOXE3 to several pioneer transcription factors that can act as key drivers of cellular differentiation and reprogramming, led to the hypothesis that transcriptional regulation of gene expression in lens mediated by FOXE3 is determined by its dynamic interactions with chromatin resulting in its presence in both open and closed chromatin through cluster of adjacent cis-regulatory sites and trans- acting DNA-binding transcription factors in the promoters and enhancers. Two specific aims will test this hypothesis. 1) To determine how the loss of FOXE3 function affects chromatin landscape and gene control in the lens, combinations of ATAC-seq, scRNA-seq and bulk RNA-seq will be employed on Foxe3 null lenses. 2) To determine the cis-regulatory grammar of FOXE3-bound promoters and enhancers in lens, FOXE3 binding sites in lens chromatin will be discovered using CUT&RUN followed by bioinformatic analysis to identify the consensus FOXE3 binding motif and adjacent transcription factor binding motifs. This information will be integrated to discover direct FOXE3 transcriptional targets, which will be validated by RT-qPCR and luciferase assays. The fundamental information gained by these approaches will fuel broader and systematic analysis of molecular mechanisms of gene control by FOX transcription factors focused on their impact on chromatin structural dynamics for tissue differentiation and cellular reprogramming.