Photoreceptor development and maintenance require precisely regulated gene expression. This regulation is controlled by the Gene Regulatory Network (GRN) of photoreceptor transcription factors (TFs) and their target non-coding cis-regulatory DNA elements (CREs). Mutations in either TFs or CREs can cause misregulation, leading to photoreceptor diseases. How mutations in coding sequences cause diseases, such as CRX-linked retinopathies, has been extensively studied, and many such mutations have been identified and can be tested for. However, only a few disease-associated mutations in non-coding DNA regulatory elements are known. One example is X-linked blue-cone monochromacy, a red/green color blindness associated with disruption of the Locus Control Region (LCR), a distal CRE of the Red/Green gene array. Little is known about the location or functional importance of other retinal CREs, presenting a bottleneck for identifying and evaluating disease-associated variants in the largely unexplored non-coding portion (99%) of the genome. Thus, there is an urgent need to catalog functional CREs and understand their sequence logic. To this end, we have identified the subset of CRX bound CREs that are Dependent on the activity of CRX to establish an active chromatin state and promote photoreceptor gene expression. We hypothesize that many of these CREs are essential for rod gene expression, cell fate, and survival, and their sequence features determine how they mediate the commands of CRX and/or other TFs. We propose to test this hypothesis in three specific aims using innovative high-throughput functional genomics approaches.
Aim 1 will provide a deep understanding of two previously-identified CREs using loss-of-function studies in mice: By thoroughly characterizing the individual and combined deletions of the two rhodopsin (Rho) enhancers CBR and RER, we will decipher their in vivo roles in regulating endogenous Rho expression, rod cellular function, and health.
Aims 2 and 3 will identify the subset of essential retinal CREs using unbiased high-throughput functional tests in mouse retinas: We will first identify activating CREs that are sufficient to enhance the expression of a reporter gene driven by a minimal rod gene promoter using massively parallel reporter assays (MPRA) (Aim 2); We will then identify active CREs that are necessary for rod gene expression and cell identity using a CRISPR-Cas9 genomic deletion screen (Aim 3). Finally, we will combine the information gained from these two complementary approaches to decipher the sequence logic mediating CRE regulatory function. Ultimately, we will identify the most functionally important photoreceptor CREs, gain a deep understanding of their genetic and epigenetic grammar, and be able to predict the effects of specific disruptions to these CREs on rod gene expression and cell fate. The findings will significantly advance our understanding of normal and pathogenic photoreceptor gene expression. It will also provide new resources for genetic screens and functional testing of disease mutations, and new insights into disease phenotype variability.

Public Health Relevance

This research aims to elucidate the Gene Regulatory Network that dictates where, when and how much photoreceptor genes are expressed, and how disruption of these mechanisms affects photoreceptor health and function. The outcomes will provide new insight into mechanisms underlying phenotypic variability observed within photoreceptor diseases and highlight new targets in the non-protein-coding sequence for genetic studies of disease-causing mutations.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biology of the Visual System Study Section (BVS)
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Neuhold, Lisa
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Washington University
Schools of Medicine
Saint Louis
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Ruzycki, Philip A; Zhang, Xiaodong; Chen, Shiming (2018) CRX directs photoreceptor differentiation by accelerating chromatin remodeling at specific target sites. Epigenetics Chromatin 11:42
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