CRX and its Regulatory Network in Retinal Degenerations

描述（由申请人提供）：这是一份竞争性修订申请，以响应通知NOT-OD-09-058“NIH宣布恢复法案资金可用于竞争性修订”。我们资助的R 01项目（5 R 01 EY 012543 -09）研究了同源域转录因子Crx在感光细胞发育和维持中的作用。Crx是光感受器转录因子网络的关键成员，其调节许多光感受器基因的转录，所述光感受器基因对于杆/锥功能是必需的。CRX突变以显性方式与影响视锥细胞和视杆细胞的几种形式的视网膜变性相关，但导致感光细胞疾病的机制尚不清楚。为了开始理解这一机制，我们先前提出了两个具体的目标来检验我们的假设，即Crx通过招募染色质重塑的共激活剂起作用。在目的1中，我们提出在光感受器和异位表达研究中使用loxP/Cre介导的条件性敲除来确定三种Crx相互作用共激活剂的作用。在目标2中，我们提出确定染色质重塑的作用，包括组蛋白修饰和染色体内环的形成，在调节视蛋白基因转录。这两个目标预计将提供深入了解Crx的作用机制，但他们没有直接解决CRX突变如何导致显性疾病。在本修订申请中，我们建议增加目标3，作为前两个目标的延伸，直接解决这个问题。为此，我们创造了两个新的小鼠品系，每个品系都携带一个致病的点突变，R90 W或E168 d2，敲入Crx基因座。与不发展显性感光缺陷的杂合Crx敲除小鼠相反，携带任一敲入等位基因的单拷贝的小鼠显示感光体发育延迟。纯合突变体不发育光感受器外节，并且在P21时失明。随后是快速视网膜变性，比纯合Crx敲除（Crx-/-）小鼠的表型更严重。因此，我们假设R90 W或E168 d2突变通过产生对野生型CRX和/或其他靶蛋白具有显性负效应的故障蛋白而引起疾病。为了验证这一假设，我们将首先仔细表征杂合和纯合突变小鼠的表型，使用形态学，生物化学和电生理学的措施。然后，我们将使用全基因组微阵列和染色质免疫沉淀分析来确定突变CRX的分子靶点。最后，根据我们以前的两个目标和这两个新的子目标获得的知识，我们将分析两个最佳类别的候选蛋白质靶点与突变体Crx的遗传相互作用。这些方法一起将显着推进我们的理解Crx调控网络如何在感光基因的表达，发育，维护和疾病。它还将提供良好表征的新动物模型，其更好地模拟CRX相关疾病的发病机制，并且可用于开发新的治疗性治疗。 公共卫生相关性：人类CRX突变以主导方式导致三种形式的感光细胞疾病，但机制尚不清楚。我们已经产生了两个新的小鼠品系，每个品系都携带了敲入Crx基因座的致病突变。这两个系在杂合和纯合状态下都出现感光细胞缺陷。我们假设这些CRX突变通过显性负性机制引起疾病，并建议通过使用形态学，生物化学和分子遗传学方法彻底表征突变小鼠的表型来验证这一假设。该结果将建立这些小鼠系作为研究CRX相关疾病的发病机制和治疗的新动物模型。

DESCRIPTION (provided by applicant): This is a competitive revision application in response to the Notice NOT-OD-09-058 "NIH Announces the Availability of Recovery Act Funds for Competitive Revision". Our funded R01 project (5R01 EY012543-09) investigates the role of the homeodomain transcription factor Crx in photoreceptor development and maintenance. Crx is a key member of the photoreceptor transcription factor network that regulates the transcription of many photoreceptor genes essential for rod/cone function. CRX mutations are linked in a dominant fashion to several forms of retinal degeneration affecting both cones and rods, but the mechanism that leads to photoreceptor disease is unclear. To begin to understand this mechanism, we previously proposed two specific aims to test our hypothesis that Crx acts by recruiting co-activators for chromatin remodeling. In Aim 1, we proposed to determine the role of three Crx-interacting co- activators, using loxP/Cre-mediated conditional knockout in photoreceptors and ectopic expression studies. In Aim 2, we proposed to determine the role of chromatin remodeling, including histone modifications and intra-chromosomal loop formation, in regulating opsin gene transcription. These two aims are expected to provide insight into Crx's mechanism of action, but they do not directly address how CRX mutations cause dominant disease. In this revision application, we propose to add Aim 3 to address this question directly as an extension of the first two aims. For this purpose, we have created two new mouse lines, each carrying a disease-causing point mutation, R90W or E168d2, knocked into the Crx locus. In contrast to heterozygous Crx knockout mice that do not develop dominant photoreceptor defects, mice carrying a single copy of either knocked-in allele show a delay in photoreceptor development. The homozygous mutants do not develop photoreceptor outer segments and are blind at P21. This is followed by rapid retinal degeneration, more severe than the phenotype of homozygous Crx knockout (Crx-/-) mice. We thus hypothesize that R90W or E168d2 mutation causes disease by producing a malfunctioning protein that has a dominant-negative effect on wild-type CRX and/or other target proteins. To test this hypothesis, we will first carefully characterize the phenotypes of heterozygous and homozygous mutant mice using morphological, biochemical and electrophysiological measures. We will then identify molecular targets of mutant CRX using genome-wide microarray and chromatin immunoprecipitation assays. Finally, based on the knowledge gained from our previous two aims and these two new subaims, we will analyze two best classes of candidate protein targets for their genetic interactions with mutant Crx. These approaches together will significantly advance our understanding of how the Crx regulatory network functions in photoreceptor gene expression, development, maintenance and diseases. It will also provide well-characterized new animal models that better model the pathogenesis of CRX- associated diseases and can be used for developing new therapeutic treatments. PUBLIC HEALTH RELEVANCE: Human CRX mutations cause three forms of photoreceptor disease in a dominant fashion, but the mechanisms are unknown. We have generated two new mouse lines, each carrying a disease-causing mutation knocked into the Crx locus. Both lines develop photoreceptor defects in the heterozygous and homozygous states. We hypothesize that these CRX mutations cause disease by a dominant-negative mechanism, and propose to test this hypothesis by thoroughly characterizing the phenotypes of the mutant mice using morphological, biochemical, and molecular genetics approaches. The outcome will establish these mouse lines as new animal models for investigating pathogenesis and treatments for CRX-linked diseases.