Utilizing the Chicken Embryo to Decode the Transcriptional Regulation of FOXE3

Miami University Oxford, Oxford OH

Investigators

Abstract

The discovery of precisely how transcription factors (TFs) function to regulate transcriptional programs during development is of fundamental importance for understanding cellular identity and organogenesis. The stereotypical architecture, anatomical simplicity and wealth of previous developmental information makes the lens a particularly attractive tissue in which to tease out these discoveries. The advent of genome-wide sequencing strategies to identify the epigenetic state of chromatin within specific cell types has made it possible to identify cis-regulatory elements (CREs) that control gene expression within these cells. Using these strategies, CREs that putatively regulate the lens TF gene, Foxe3 have been discovered in the mouse genome. The expression of FOXE3 is largely restricted to the lens and homozygous loss of function mutations result in aphakia and/or microphthalmia in all vertebrate species where these mutations have been identified, including humans. As such, FOXE3 represents an attractive target to elucidate how TFs utilize cis-regulatory elements to achieve tissue-specific gene expression. Furthermore, while studies in mice have identified Foxe3 CREs that contain specific TF motifs and bind to specific TFs, to date the relative importance of any of these identified motifs in regulating Foxe3 expression in vivo is lacking. The current proposal seeks to exploit the developmental advantages of the chick lens to decode the cis-regulatory grammar that dictates FOXE3 expression in both the chick and mouse lens. Building on recent information that uncovered genome wide chromatin accessibility and the epigenetic active enhancer modification (H3K27ac) four putative FOXE3 CREs have been identified in the chick genome. This proposal will annotate the mouse and chick FOXE3 CREs for the presence of known TF binding motifs. The proposal seeks to employ recombinant retroviral vectors to functionally test the requirements of these CREs to drive reporter gene expression in the chick lens. The proposal will utilize retroviral reporter constructs to determine if specific TF motifs, discovered within essential FOXE3 CREs, are essential to drive lens-specific expression. In addition, a lens-specific CRISPR based genome editing strategy will be developed in the chick lens to screen candidate TFs for their requirement to drive the normal expression pattern of the endogenous FOXE3 gene in developing chick lenses. The central hypothesis tested in this proposal is that regions of non-coding open chromatin near the FOXE3 gene in both mice and chickens comprise promoter and/or enhancer elements that interact with TFs sufficient to specifically drive gene expression to the developing chick lens. The success of this proposal will not only directly test the role of specific CREs and TFs for their role in regulating FOXE3 expression in vivo but it also promises to enhance the utility of the chick lens for screening gene and enhancer function by developing CRISPR-based genome editing strategies.