CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS

描述（由申请人提供）：视杆细胞和视锥细胞光感受器的发育和维持需要光感受器基因的协调调节表达。这种调节发生在多个水平上，需要转录调节蛋白网络的作用。这种调控网络以锥-杆同源盒蛋白CRX为中心，并结合了光受体特异性转录因子（TF）和一般染色质调节剂。我们研究的最终目标是确定这种调控蛋白网络如何与共表达靶基因的染色质相互作用，以实现协调调控的空间和时间表达，从而更好地了解感光细胞发育和疾病。在上一个资助期间，我们发现CRX通过招募能够催化组蛋白乙酰化和组织调控区之间基因内相互作用的共激活剂来调节靶基因染色质构型。在新的资助期内，我们将把我们的转录调控视野扩展到三个维度：我们假设视杆细胞和视锥细胞基因作为簇共同调控，需要在细胞核中由特定的TF和一般表观遗传调节剂介导的适当的高阶染色体组织。为了验证这一假设，Aim 1将通过评估缺失视杆细胞、视锥细胞及其前体中的三种催化酶MLL 1、MLL 2和MLL 3的影响，确定组蛋白H3赖氨酸4甲基化的作用，这是乙酰化下游发生的阳性标记;目的2将揭示共调控的感光基因及其共调控的染色体内和染色体间的特异性相互作用，使用最先进的表观遗传学技术定位于视杆细胞核与视锥细胞核。为了确定这些染色体组织是否有助于亚型规范和维护过程中的转录协同调节，染色体相互作用，TF结合，组蛋白标记和转录的配置文件将在不同的发育年龄进行研究。我们还将通过分析缺乏特定转录因子或表观遗传调节因子的突变视网膜来确定转录调控网络是否介导特定的染色体相互作用。这项研究将提供第一个有针对性的评估感光基因的转录共调控，并确定潜在的分子和细胞机制。所获得的新知识将对开发基于干细胞的细胞替代疗法以及为其他治疗干预提供新靶点产生影响。

DESCRIPTION (provided by applicant): Development and maintenance of rod and cone photoreceptors require coordinately regulated expression of photoreceptor genes. This regulation occurs at multiple levels and requires the action of a network of transcription regulatory proteins. This regulatory network is centered on the cone-rod homeobox protein CRX and incorporates both photoreceptor-specific transcription factors (TFs) and general chromatin modulators. The ultimate goal of our research is to determine how this regulatory protein network interacts with the chromatin of co-expressed target genes to achieve coordinately regulated spatial and temporal expression, providing a better understanding of photoreceptor development and disease. During the last grant period, we discovered that CRX regulates target gene chromatin configurations by recruiting co-activators capable of catalyzing acetylation of histones and organizing intra-gene interactions between regulatory regions. In the new grant period, we will extend our vision of transcriptional regulation to three dimensions: We hypothesize that rod and cone genes are co-regulated as clusters, requiring appropriate higher-order chromosome organization in the nucleus that is mediated by specific TFs and general epigenetic modulators. To test this hypothesis, Aim 1 will determine the role of histone H3 lysine 4 methylation, a positive mark that occurs downstream of acetylation, by assessing the effects of deleting three catalytic enzymes, MLL1, MLL2 and MLL3 in rods, cones and their precursors; Aim 2 will reveal specific intra- and inter-chromosome interactions of co-regulated photoreceptor genes and their co-localization in rod vs. cone nuclei using state-of-the-art epigenetic technologies. To determine if these chromosome organizations contribute to transcription co-regulation during subtype specification and maintenance, profiles of chromosome interactions, TF binding, histone marks and transcription will be studied at various developmental ages. We will also determine if the transcription regulatory network mediates the specific chromosome interactions by analyzing mutant retinas lacking specific TFs or epigenetic modulators. This study will provide the first targeted assessment of transcriptional co-regulation of photoreceptor genes and determine the underlying molecular and cellular mechanisms. The new knowledge gained will have implications in developing stem cell-based cell replacement therapy as well as providing new targets for other therapeutic interventions.