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精确调节光感受器特异性基因的表达。我们的长期研究目标是确定光感受器基因转录调控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与其靶基因之间的相互作用。我们正在研究一种光受体特异性转录因子，锥杆同源盒（Crx），它对许多光受体基因的转录至关重要，并与光受体退行性疾病有关。我们已经鉴定了10个Crx相互作用蛋白（cip），它们在许多细胞中普遍表达或在光感受器中特异性（优先）表达。我们假设Crx和这些cip的相互作用调节特定类型光感受器中靶基因的转录。在这个更新申请中，我们建议使用体内和体外方法来验证这一假设。在Aim #1中，我们将重点关注两个特定的cip，即核受体Nr2e3和锌指转录因子Sp4，并通过小鼠视网膜的共免疫沉淀和共表达研究来表征它们与体内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杆状或锥体特异性基因（如视蛋白和Aim #2中鉴定的基因产物）的转录具有功能意义。在Aim #2中，我们将使用染色质免疫沉淀测定（ChIP）来鉴定由Crx和cip调节的各种体内靶点，这些靶点对光感受器的功能和存活很重要。候选的光感受器基因靶点将通过PCR与特定基因调控区域对应的引物进行检测，而新的靶点将通过筛选基因组阵列（ChIP-Array）或克隆（ChIP-Cloning）来鉴定。Aim #3是基于我们最近的发现，几个光受体基因调控区域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，这些普遍存在的cip与催化组蛋白乙酰化的共激活物复合物相关。因此，我们假设组蛋白的超乙酰化对于激活光受体基因的转录很重要，并且通过Crx与普遍存在的cip相互作用促进。我们将通过改变调节这一过程的因子来确定组蛋白乙酰化程度是否会影响光受体基因的表达。例如，组蛋白去乙酰化酶抑制剂会增加视网膜母细胞瘤细胞中的组蛋白乙酰化，从而增加光受体基因转录，Crx或ataxin-7突变会降低Crx-/-或SCA7小鼠视网膜中的组蛋白乙酰化和光受体基因转录。我们还将确定Crx是否在体内招募参与组蛋白乙酰化的共激活物复合物。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在体内改变这些调节蛋白的正常功能。这些研究将使我们对光感受器特异性基因在体内表达调控的分子机制有一个新的认识，并将为设计光感受器疾病的治疗方法提供新的途径。

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.