CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS

描述（由申请人提供）：光感受器特异性基因的精确调控表达对于光感受器的发育和存活至关重要。我们研究的最终目标是确定调节这些基因在哺乳动物视网膜中表达的分子机制，并确定这些机制的破坏如何导致感光细胞疾病。光感受器转录因子网络负责这种调节，并且该网络的关键成员是锥-杆同源框蛋白（Crx）。CRX突变导致人类感光细胞退化。为了了解Crx的作用机制，已经鉴定了几种Crx相互作用蛋白，并且正在研究它们的功能。这些包括三种普遍表达的共激活因子，Gcn 5，Cbp和p300，它们通过组蛋白乙酰转移酶（HAT）活性调节染色质构象。我们的初步数据表明，这些共激活剂的Crx调节途径的重要组成部分。我们推测，协调行动的Crx和它的共激活剂调节染色质的构象，以调节感光基因的表达在视网膜细胞的发育和生存。这一假设将在两个具体目标中得到检验。在目标1中，我们将使用条件性基因敲除小鼠模型确定三种HAT在光感受器发育和存活中的作用。将携带每种共激活因子的floxed等位基因的小鼠与在发育中的（Crx-Cre）或成熟的（Rho-iCre）光感受器中表达Cre的转基因小鼠杂交。在表达Cre的细胞中，floxed基因将被敲除。通过形态学、生物化学和功能测定分析所得突变小鼠的表型。作为一种补充方法，突变的共激活子将异位表达在Y 79视网膜母细胞瘤细胞和新生小鼠视网膜，以确定共激活子的功能是否取决于它们的HAT催化活性或它们与Crx的相互作用。总之，这些分子遗传学方法将揭示Crx调节途径中每个共激活因子的作用和作用机制。除了组蛋白乙酰化，其他几种形式的表观遗传调节，包括组蛋白甲基化和染色体内相互作用，也调节转录。目的2是为了验证这一假设，即差异表观遗传调制调节的杆和锥基因的转录在个别细胞，从而确定感光细胞亚型。对来自小鼠视网膜的分离的、分选的视杆或视锥的染色质免疫沉淀（ChIP）测定将用于将视杆和视锥视蛋白基因上的组蛋白修饰与每种光感受器亚型中的转录激活或抑制相关联。染色体构象捕获（3C）分析将揭示视蛋白基因不同区域之间的染色体内相互作用及其在转录中的重要性。这项研究代表了第一次全面的视网膜表观遗传学研究。这一发现将大大促进我们对视网膜发育、维持和疾病中感光细胞基因表达的理解。 公共卫生相关性：Crx是一种调节许多对光感受器功能重要的基因表达的蛋白质。Crx基因突变导致光感受器变性疾病，导致无法治愈的失明。这项研究调查了Crx如何与其他蛋白质相互作用以保持光感受器健康，以及为什么突变会使光感受器生病，为治疗开发提供了新的见解。

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.