CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS

描述(由申请人提供)：光感受器细胞功能的发展和维持需要光感受器特定基因的精确调控表达。我们研究的长期目标是确定调控光感受器基因转录的分子机制，以及转录失调在光感受器疾病中的作用。转录调控涉及网络转录因子及其靶基因之间的相互作用。我们正在研究一种光感受器特异的转录因子，视锥-杆同源盒(CRX)，它对许多光感受器基因的转录是必不可少的，并与光感受器退行性疾病有关。我们已经确定了10个CRX相互作用蛋白(CIPS)，它们要么在许多细胞中普遍表达，要么在光感受器中特异性地(优先)表达。我们假设CRX和这些CIP的相互作用调节特定类型的光感受器中靶基因的转录。在这一更新应用中，我们建议使用体内和体外方法来检验这一假说。在目标1中，我们将专注于两个特定的CIPS，核受体Nr2e3和锌指转录因子SP4，并通过免疫共沉淀和小鼠视网膜共表达研究，在体内表征它们与CRX的相互作用。我们还将使用细胞转染来确定这些相互作用是否对视杆或视锥特异基因的转录具有功能意义，例如Ospins和Aim#2中确定的基因产物。在Aim#2中，我们将使用染色质免疫沉淀分析(ChIP)来鉴定由CRX和CIPS调控的各种体内靶点，这些靶点对光感受器的功能和生存至关重要。候选的光感受器基因靶点将使用与特定基因的调控区域相对应的引物进行检测，而新的靶点将通过筛选基因组阵列(芯片阵列)或克隆(芯片克隆)来识别。目的#3是基于我们最近的发现，几个光感受器基因的调节区上的组蛋白是超乙酰化的(一种激活转录的染色质修饰)，CRX与ataxin-7或CBP/p300相互作用，这是普遍存在的CIP，与催化组蛋白乙酰化的共激活复合体相关。因此，我们假设，组蛋白的超乙酰化对于激活光感受器基因的转录是重要的，并被CRX与无处不在的CIPS相互作用所促进。我们将通过改变调节这一过程的因素来确定组蛋白乙酰化程度是否影响光感受器基因的表达。例如，组蛋白去乙酰酶抑制剂应该增加组蛋白乙酰化，从而增加视网膜母细胞瘤细胞中光受体基因的转录，而CRX或ataxin-7突变应该减少CRX-/-或SCA7小鼠视网膜中组蛋白乙酰化和光受体基因的转录。我们还将确定CRX是否在体内招募了参与组蛋白乙酰化的共激活复合体。在这三个目标中，我们将使用光感受器疾病的小鼠模型来研究CRX及其相关因子的致病突变如何改变这些调节蛋白在体内的正常功能。这些研究将导致对体内调控光感受器特异基因表达的分子机制的新水平的理解，并将为设计光感受器疾病的治疗方案提供新的途径。

DESCRIPTION (provided by applicant): The development and maintenance of photoreceptor cell function require precisely regulated expression of photoreceptor-specific genes. The long-term goal of our research is to determine the molecular mechanisms regulating the transcription of photoreceptor genes and the role of transcription dysregulation in photoreceptor diseases. Regulation of transcription involves interactions among network transcription factors and their target genes. We are studying a photoreceptor-specific transcription factor, cone-rod homeobox (Crx) that is essential for the transcription of many photoreceptor genes and is associated with photoreceptor degenerative diseases. We have identified ten Crx interacting proteins (CIPs) that are expressed either ubiquitously in many cells or specifically (preferentially) in photoreceptors. We hypothesize that the interactions of Crx and these CIPs regulate transcription of target genes in specific types of photoreceptors. In this renewal application, we propose to test this hypothesis using both in vivo and in vitro approaches. In Aim #1, we will focus on two specific CIPs, the nuclear receptor Nr2e3 and the zinc-finger transcription factor Sp4 and characterize their interactions with Crx in vivo using co-immunoprecipitation and coexpression studies in the mouse retina. We will also use cell transfections to determine if these interactions have functional significance on the transcription of rod- or cone-specific genes, such as opsins and the gene products identified in Aim #2. In Aim #2, we will use chromatin immunoprecipitation assays (ChIP) to identify a variety of in vivo targets that are regulated by Crx and CIPs and that are important for the function and survival of photoreceptors. Candidate photoreceptor gene targets will be detected using PCR with primers corresponding to the regulatory regions of specific genes, while novel targets will be identified by screening genomic arrays (ChIP-Array) or by cloning (ChIP-Cloning). Aim #3 is based on our recent findings that histones on the regulatory regions of several photoreceptor genes are hyper-acetylated (a chromatin modification that activates transcription) and that Crx interacts with ataxin-7 or CBP/p300, ubiquitous CIPs associated with co-activator complexes that catalyze histone acetylation. Thus, we hypothesize that hyper-acetylation of histones is important for activating transcription of the photoreceptor genes and is promoted by Crx interacting with ubiquitous CIPs. We will determine if the degree of histone acetylation affects photoreceptor gene expression by modifying the factors regulating this process. For example, histone deacetylase inhibitors should increase histone acetylation and therefore photoreceptor gene transcription in retinoblastoma cells, and Crx or ataxin-7 mutations should decrease histone acetylation and transcription of photoreceptor genes in the retina of Crx-/- or SCA7 mice. We will also determine if Crx recruits the co-activator complexes involved in histone acetylation in vivo. In each of the three aims, we will use mouse models of photoreceptor diseases to investigate how disease-causing mutations in Crx and its associated factors, alter the normal functions of these regulatory proteins in vivo. These studies will lead to a new level of understanding of the molecular mechanisms that regulate photoreceptor-specific gene expression in vivo and will provide new approaches for designing treatments for photoreceptor diseases.