Shiming Chen - US grants

Experiments proposed in this application will determine the importance of the Cone-Rod Homeobox (CRX) and its associated regulatory network in photoreceptor gene expression and photoreceptor degenerative diseases. CRX is a novel member of otd/Otx homeodomain transcription factor family that is specifically expressed in photoreceptors. CRX binds to and trans-activates the promoters of many photoreceptor-specific genes. Mutations in CRX are associated with photoreceptor degenerative diseases having a wide age of onset, including cone-rod dystrophy, retinitis pigmentosa and Leber's congenital amaurosis. Our hypothesis is that CRX serves as one-of the key molecules that control the expression of photoreceptor-specific genes and is essential for the normal development and maintenance of the photoreceptors. Two specific aims are proposed to test this hypothesis: Aim I will test how disease-related CRX mutations alter the way that CRX regulates photoreceptor gene expression. Five known CRX homeodomain mutations will be analyzed for their effects on the binding of CRX to DNA and trans-activation of the rhodopsin promoter. Aim 2 will identify and characterize other components of the CRX regulatory pathway, such as proteins that regulate the activity of CRX or co-factors required for CRX function. These proteins are hypothesized to physically interact with CRX and, therefore, will be isolated using protein-protein interaction screens in yeast cells. The resulting positive clones will be characterized by sequence homology to known proteins, expression patterns, and physical and functional interactions with CRX. We will also determine the chromosomal localization of the corresponding human and murine genes. These studies will provide information about the program controlling photoreceptor-specific gene expression and the development and maintenance of photoreceptor function. They are expected to provide information about the molecular basis of photoreceptor degenerative diseases and aid in the selection of targets for therapeutic approaches to these diseases.

DESCRIPTION (provided by applicant): Precisely regulated expression of photoreceptor-specific genes is essential for the development and survival of photoreceptors. The ultimate goal of our research is to identify the molecular mechanisms regulating the expression of these genes in the mammalian retina and to determine how disruption of these mechanisms causes photoreceptor disease. A network of photoreceptor transcription factors is responsible for this regulation, and a key member of this network is the cone-rod homeobox protein (Crx). CRX mutations cause photoreceptor degeneration in humans. To understand the mechanism of action of Crx, several Crx interacting proteins have been identified and their functions are being investigated. These include three ubiquitously expressed co-activators, Gcn5, Cbp and p300, which modulate chromatin conformation via their histone acetyltransferase (HAT) activity. Our preliminary data suggest that these co-activators are important components of the Crx regulatory pathway. We hypothesize that the concerted actions of Crx and its co-activators modulate chromatin conformation to regulate the expression of photoreceptor genes during retinal cell development and survival. This hypothesis will be tested in two specific aims. In Aim 1 we will determine the role of the three HATs in photoreceptor development and survival using conditional knockout mouse models. Mice carrying floxed alleles of each co-activator are being crossed with transgenic mice expressing Cre in developing (Crx-Cre) or mature (Rho-iCre) photoreceptors. In cells that express Cre, the floxed genes will be knocked out. Phenotypes of the resulting mutant mice are analyzed by morphological, biochemical and functional assays. As a complementary approach, mutant co-activators will be ectopically expressed in Y79 retinoblastoma cells and neonatal mouse retinas to determine whether the function of the co-activators depends on their HAT catalytic activity or their interaction with Crx. Together, these molecular genetic approaches will reveal the role and mechanism of action of each co-activator in the Crx regulatory pathway. In addition to histone acetylation, several other forms of epigenetic modulation, including histone methylation and intrachromosomal interactions, also regulate transcription. Aim 2 is designed to test the hypothesis that differential epigenetic modulation regulates the transcription of rod and cone genes in individual cells, thereby determining photoreceptor subtypes. Chromatin immunoprecipitation (ChIP) assays on isolated, sorted rods or cones from mouse retinae will be used to correlate the histone modifications on rod and cone opsin genes with transcriptional activation or repression in each photoreceptor subtype. Chromosome conformation capture (3C) assays will reveal intrachromosomal interactions between different regions of the opsin genes and their importance in transcription. This study represents the first comprehensive epigenetic investigation in the retina. The findings will significantly advance our understanding of photoreceptor gene expression in retina development, maintenance and disease. PUBLIC HEALTH RELEVANCE: Crx is a protein that regulates the expression of many genes important for photoreceptor function. Crx mutations cause photoreceptor degeneration diseases that lead to incurable blindness. This research investigates how Crx interacts with other proteins to keep photoreceptors healthy, and why mutations make photoreceptors sick, providing new insight into therapy development.

? DESCRIPTION (provided by applicant): The cone-rod homeobox transcription factor CRX regulates expression of many photoreceptor genes and is required for photoreceptor development and survival. Human CRX mutations are mostly associated with autosomal dominant (ad) retinopathies: Leber congenital amaurosis (adLCA), cone-rod dystrophy (adCRD) and retinitis pigmentosa (adRP), with variable age of onset and severity. These diseases are currently poorly understood and no treatments are available. Recent in vitro and animal model studies have shed light on the pathologic mechanisms underlying three distinct classes of CRX mutations. Our laboratory has extensively studied Class III mutations, frameshift/nonsense mutations producing C-terminal truncated CRX proteins. These mutant proteins retain DNA binding but lack transcriptional regulatory activity and so interfere with wild- type (WT) protein function. Animal models for Class III include Crx-E168d2 and Crx-Tvrm65 mice and Crx-Rdy cats, all showing similar phenotypes that resemble adLCA or adCRD in humans. These models have revealed an unexpected primary pathogenic defect, overproduction of the mutant mRNA/protein relative to WT. These excessive mutant Crx products amplify the toxic effect of the mutant protein and their level directly correlates with phenotype severity. However, it is unclear how Crx mutations cause this selective overexpression of their own allele. The study we propose here is designed to address this question at the molecular level. Our preliminary results suggest that the normal Crx mRNA is short-lived, but mRNAs carrying Class III mutations are much more stable, indicating that the overproduction of mutant CRX is primarily caused by the increased stability of its transcript. We also discovered that CRX not only binds to DNA, but also acts as a RNA-binding protein (RBP) to bind to both its own transcript and Rhodopsin mRNA. These findings lead us to hypothesize a new role for CRX in regulating mRNA stability, particularly targeting its own transcript. Furthermore, the presence of Class III CRX mutations alters the stability of the mutant allele's transcripts, resulting in mutant mRNA/protein overproduction and subsequent photoreceptor dystrophy. To test these hypotheses, we have designed a set of experiments to map RNA sequences containing the stability codes in WT and mutant Crx mRNA, and to determine how these codes are interpreted by RNA binding proteins, including CRX; to profile other photoreceptor genes subject to CRX-dependent mRNA stability regulation; and to determine the effects of Crx mutations on this regulation in both cultured cells and mouse models. This study will significantly advance our understanding of CRX's multifunctional roles and mechanisms of action, and the effects of disease-causing mutations. It could also have much broader implications for other neurological disorders.

Project Summary    Different human mutations in the photoreceptor-specific gene Crx are linked with multiple retinopathies that vary in their severity and age of onset. How different mutations in this single gene lead to different pathologies is not well understood. Because CRX is a transcription factor, these disease mutations must act by altering the ability of CRX to regulate gene expression. Our goal is to understand how different classes of CRX mutations modify its regulatory function. To achieve this, we will apply a recently developed massively parallel reporter gene technology to systematically and comprehensively measure CRX regulatory function in live retina from multiple mouse knock-in models that carry human CRX disease mutations. We will use the data to train and test a mechanistic, quantitative model that describes how CRX mutations alter protein-DNA and protein-protein interactions to modify gene regulation. In a complementary approach, we will use the knock-in mouse models to determine the effects of CRX disease mutations on its in vivo genome-wide binding, and on the binding of the cooperatively interacting transcription factors OTX2, NRL, and NR2E3. Using these two approaches, we aim to understand how different classes of CRX disease mutations modify gene regulation in photoreceptors, and discover new mechanisms of disease pathogenesis. Our results will provide a strong basis for classifying new CRX mutations, and for designing targeted therapies to treat different genetic forms of retinopathy. 

Project Summary A Molecular Genetics Core will provide customized services for the production of transgenic and knockout mice using the latest available technologies. Supported services include assistance with designing and preparing constructs for gene targeting, viral vectors, establishing genotyping procedures for genetically- modified mice, making recombinant DNA clones, constructing and validating DNA libraries for high-throughput functional screens, in vitro fertilization (IVF) services, microinjection/electroporation of gene targeting constructs and rederivation of mouse strains from cryopreserved stocks or pathogen-contaminated strains, and sperm/embryo cryopreservation. Provision of these support services and resources will greatly enhance the research capabilities of vision investigators at Washington University and will facilitate collaboration among new and established vision scientists.

PROJECT SUMMARY Precisely regulated gene expression is essential for photoreceptor development and maintenance. This process is governed by a genetic program centered on the cone-rod homeobox transcription factor CRX. Mutations in the human CRX gene have been associated with dominant retinopathies with a wide-range of phenotypes and ages of onset. A poor understanding of the mechanism of each individual mutation has made it difficult to develop treatment strategies. To address these challenges, our lab has defined four classes of disease-causing CRX mutations and made mouse models carrying a representative mutation(s) of each class. Up to now, we and others have characterized and reported findings on mouse models for three such classes, proving concordance between the mouse and human conditions due to each mutation. These studies have already provided a deep knowledge of disease pathogenesis. However, the pathogenic mechanism of mutations in the remaining class (Class II) remains to be determined. Class II mutations are linked to the early-onset dominant retinopathies Leber congenital amaurosis (adLCA) and cone rod dystrophy (adCoRD). We have generated mouse lines carrying two individual Class II mutations, Crx-K88N and Crx-E80A, and find that each develops a dominant LCA or CoRD- like phenotype associated with misregulation of photoreceptor gene expression. Because these mutations are located in the CRX homeodomain responsible for DNA binding, we hypothesize that the disease proteins misregulate gene expression by altering CRX?s DNA binding specificity, leading to CRX malfunction at target sites. In Aim 1 of this proposal, we will test our hypothesis in both cell culture and mouse models using cell biology, molecular and functional genomics approaches. Using unbiased high-throughput DNA binding and regulatory function assays, we will determine how these mutations alter CRX?s regulatory activity, leading to misregulation of gene expression and functional deficits in photoreceptors. In Aim 2, we will address the lack of treatment strategies for CRX diseases. We hypothesize that exogenous introduction of the proper amount of normal CRX during a therapeutic window can improve the photoreceptor phenotype in diseased retinae. We have designed a tunable gene augmentation approach that incorporates a tetracycline (doxycycline) switch to turn-on or turn-off therapeutic CRX produced by a transgene integrated within the genome or carried by an adeno associated virus (AAV). We will evaluate phenotypic improvement using established multidisciplinary approaches and expect to see varying degrees of phenotype rescue in different mouse models by CRX augmentation. The outcome of this research will advance our understanding of CRX disease and photoreceptor development, and inform future efforts to treat patients with CRX disease.