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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012543-20
Application #
9852448
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Neuhold, Lisa
Project Start
2000-03-01
Project End
2023-01-31
Budget Start
2020-02-01
Budget End
2021-01-31
Support Year
20
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Washington University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Ruzycki, Philip A; Zhang, Xiaodong; Chen, Shiming (2018) CRX directs photoreceptor differentiation by accelerating chromatin remodeling at specific target sites. Epigenetics Chromatin 11:42
Brightman, Diana S; Grant, Rachel L; Ruzycki, Philip A et al. (2018) MLL1 is essential for retinal neurogenesis and horizontal inner neuron integrity. Sci Rep 8:11902
Rajagopal, Rithwick; Zhang, Sheng; Wei, Xiaochao et al. (2018) Retinal de novo lipogenesis coordinates neurotrophic signaling to maintain vision. JCI Insight 3:
Potter, Chloe; Zhu, Wanqiu; Razafsky, David et al. (2017) Multiple Isoforms of Nesprin1 Are Integral Components of Ciliary Rootlets. Curr Biol 27:2014-2022.e6
Ruzycki, Philip A; Linne, Courtney D; Hennig, Anne K et al. (2017) Crx-L253X Mutation Produces Dominant Photoreceptor Defects in TVRM65 Mice. Invest Ophthalmol Vis Sci 58:4644-4653
Brightman, Diana S; Razafsky, David; Potter, Chloe et al. (2016) Nrl-Cre transgenic mouse mediates loxP recombination in developing rod photoreceptors. Genesis 54:129-35
Occelli, Laurence M; Tran, Nicholas M; Narfström, Kristina et al. (2016) CrxRdy Cat: A Large Animal Model for CRX-Associated Leber Congenital Amaurosis. Invest Ophthalmol Vis Sci 57:3780-92
Ruzycki, Philip A; Tran, Nicholas M; Kefalov, Vladimir J et al. (2015) Graded gene expression changes determine phenotype severity in mouse models of CRX-associated retinopathies. Genome Biol 16:171
Tran, Nicholas M; Chen, Shiming (2014) Mechanisms of blindness: animal models provide insight into distinct CRX-associated retinopathies. Dev Dyn 243:1153-66
Masuda, Tomohiro; Zhang, Xiaodong; Berlinicke, Cindy et al. (2014) The transcription factor GTF2IRD1 regulates the topology and function of photoreceptors by modulating photoreceptor gene expression across the retina. J Neurosci 34:15356-68

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