CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS

描述（由申请人提供）：感光细胞功能的发育和维持需要精确调节感光细胞特异性基因的表达。本研究的长期目标是确定调节感光细胞基因转录的分子机制以及转录失调在感光细胞疾病中的作用。转录调控涉及网络转录因子及其靶基因之间的相互作用。我们正在研究一种感光细胞特异性转录因子，锥-杆同源盒（Crx），它对许多感光细胞基因的转录至关重要，并与感光细胞退行性疾病有关。我们已经确定了10 Crx相互作用蛋白（CIP）的表达普遍存在于许多细胞或具体（优先）在光感受器。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。在此更新申请中，我们建议使用体内和体外方法来测试这一假设。在目标#1中，我们将专注于两个特定的CIP，核受体Nr 2 e3和锌指转录因子Sp 4，并在小鼠视网膜中使用免疫共沉淀和共表达研究来表征它们与Crx在体内的相互作用。我们还将使用细胞转染来确定这些相互作用是否对视杆细胞或视锥细胞特异性基因（如视蛋白和目标#2中鉴定的基因产物）的转录具有功能意义。在目标#2中，我们将使用染色质免疫沉淀试验（ChIP）来鉴定各种受Crx和CIP调节的体内靶标，这些靶标对光感受器的功能和存活很重要。候选感光基因靶标将使用PCR检测，引物对应于特定基因的调控区，而新靶标将通过筛选基因组阵列（ChIP-阵列）或通过克隆（ChIP-克隆）鉴定。目标#3是基于我们最近的发现，即几种光感受器基因的调控区上的组蛋白是超乙酰化的（激活转录的染色质修饰），Crx与共济失调蛋白-7或CBP/p300相互作用，这是与催化组蛋白乙酰化的共激活复合物相关的普遍存在的CIP。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，并且通过与普遍存在的CIP相互作用的Crx来促进。我们将确定组蛋白乙酰化的程度是否影响感光基因的表达，通过修改这个过程中的调节因素。例如，组蛋白去乙酰化酶抑制剂应增加组蛋白乙酰化，从而增加视网膜母细胞瘤细胞中的光感受器基因转录，而Crx或共济失调蛋白-7突变应减少Crx-/-或SCA 7小鼠视网膜中的组蛋白乙酰化和光感受器基因转录。我们还将确定Crx是否招募参与体内组蛋白乙酰化的共激活因子复合物。在这三个目标中，我们将使用感光细胞疾病的小鼠模型来研究Crx及其相关因子中的致病突变如何改变这些调节蛋白在体内的正常功能。这些研究将导致一个新的水平的理解的分子机制，在体内调节感光细胞特异性基因表达，并将提供新的方法来设计感光细胞疾病的治疗。

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.