The Role of Modular Super-Enhancers in the Developing Murine and Human Retina

PROJECT SUMMARY During retinal development, thousands of genes change in their expression as multipotent retinal progenitor cells produce each of the 7 classes of cell types in an evolutionarily conserved birth order. Although it has been well established that large enhancers called super-enhancers (SEs) can play an important role in regulating developmental stage– and cell type–specific gene expression, a major barrier in the field is identifying specific promoter-SE interactions. This is challenging because SEs can act over long distance (up to 1 Mb) and in either orientation. For convenience, investigators often assume that the closest SE is the most relevant, but this is often not the case. Identification and characterization of specific promoter–SE interactions are important because mutations in the non- coding genome can contribute to human disease including retinopathies. To address this major gap in knowledge, we have spent the past 10 years developing a detailed map of chromatin structure in the developing human and murine retinae. This integrated retinal nucleome database (iRNDb) is the largest and most comprehensive map of chromatin structure in the retina with over 2,500 individual files. Recently, we updated the iRNDb (v.2) with the addition of single cell RNA-seq and single cell ATAC- seq to provide developmental stage– and cell type–specific features and HiChIP to provide data on promoter–SE interactions. All our published and unpublished data are shared freely with the biomedical research community on the St. Jude Cloud (https://proteinpaint.stjude.org/iRNDb_v2). Using the iRNDb (v.2), we have identified developmental stage– and cell type–specific promoter–SE interactions for each of the early retinal transcription factor network genes (Vsx2, Lhx2, Otx2, Pax6, Rax, Six3, Six6, Sox2 and Tbx3). The most exciting discovery is that each of the SEs associated with the early retinal transcription factor genes is made up of discrete modules predicted to have developmental stage– and cell type–specific activity. In this proposal, we will test the hypothesis that Six3/SIX3 and Otx2/OTX2 are regulated by modular SEs in human and murine retinae. These genes were chosen because they are essential for retinogenesis and altered expression can cause developmental defects in humans. Our results will fill a fundamental gap in our knowledge about the regulation of early retinal transcription factor network genes and validate our genome-wide map of promoter–SE interactions in the developing retina (iRNDb (v.2)). This research proposal is relevant to human health because it will provide a map of relevant regulatory elements in the non-coding genome that may contribute to developmental defects.