Cis-regulatory elements (CREs) are critical sites of transcription factor (TF) binding to the genome that orchestrate the expression of genes necessary for normal cellular function. Mutations within CREs can disrupt TF binding and cause inherited human diseases including disorders of vision. The genomic location and function of CREs that are necessary for human vision is largely unknown. This gap in knowledge is a significant obstacle toward understanding the genetic regulation of normal human vision and to identifying disease-causing mutations with CREs. The long-term goal for our research is to understand how genetic variation within CREs shapes the structure and function of the retina and contributes to human vision. The focused objective of this proposal is to determine the mechanisms by which CREs regulate essential gene expression in photoreceptor cells and to determine how genetic mutations within CREs lead to retinal disease. The central hypothesis driving this work is that discrete DNA sequences within CREs are required to regulate essential photoreceptor gene expression and that CRE mutations that disrupt evolutionarily conserved TF binding sites contribute to inherited visual disorders. To test this hypothesis we are pursuing the following specific aims: 1) Determine the activity of human photoreceptor CREs in human retinal organoids using ATAC- Seq, ChIP-Seq and RNA-Seq to compare them to CREs we have previously identified from adult and developing human retinas. This will demonstrate the utility of organoids for studying photoreceptor CREs in their native cellular-genomic context. 2) Test the function of patient-derived variants in human photoreceptor CREs. Using high-throughput AAV-based reporter assays we will determine which CREs sequences are sufficient to drive cell-type-specific expression in the mouse retina and human retinal organoids and determine the consequence of sequence variants on CRE activity. 3) Determine the mechanisms by which multiple CREs regulate the expression of a critical photoreceptor transcription factor, NRL. CRISPR/Cas9-based approaches will target specific CREs at the NRL locus to reveal the contribution of each CRE to the expression of this essential gene and to serve as a case study for the regulation of other essential genes. The contribution of this research will be to elucidate the mechanisms by which CREs regulate genes that are necessary for human photoreceptor function and survival. This work will enable the systematic identification and interpretation of genetic variants within CREs and therefore improve genetic diagnostics for unexplained retinal disease. By opening up the non-coding genome to functional analyses it will be possible for the first time to determine the mechanisms by which individual CREs regulate specific genes that are critical for photoreceptor cell function in a high-throughput and comprehensive manner. This will enable discovery of genetic contributions to human vision and inherited visual diseases that have thus far been inaccessible.
The proposed project is relevant to public health as it will elucidate key genetic mechanisms that underlie the function of the human retina and directly enable diagnosis of inherited visual diseases that currently have unknown genetic origin. The research will achieve this objective by functionally characterizing non-coding regulatory regions of the human genome that are critical for human vision, but that are not currently possible to study using available resources. The novel insights gained by completion of this study will immediately facilitate the discovery and interpretation of new disease-causing mutations and enable the development of therapies to treat visual disorders that affect people of all ages.