CRX AND ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS

描述（申请人提供）：杆状和锥状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。这个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（TFs）和一般染色质调节剂。我们研究的最终目的是确定这种调节蛋白网络如何与共表达靶基因的染色质相互作用，以实现协调调节的空间和时间表达，从而更好地了解光感受器的发育和疾病。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的共激活子和组织调控区域之间的基因内相互作用来调节靶基因染色质结构。在新的资助期内，我们将把转录调控的视野扩展到三个维度：我们假设杆状和锥状基因作为集群共同调控，需要在细胞核中由特定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将通过评估在杆状体、锥体及其前体中删除三种催化酶MLL1、MLL2和MLL3的效果，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一个积极标记；目的2将利用最先进的表观遗传学技术揭示染色体内和染色体间共调控光感受器基因的特定相互作用及其在杆状核和锥状核中的共定位。为了确定这些染色体组织是否在亚型规范和维持过程中参与转录共调控，将研究不同发育年龄的染色体相互作用、TF结合、组蛋白标记和转录的谱。我们还将通过分析缺乏特定tf或表观遗传调节剂的突变视网膜来确定转录调节网络是否介导特定的染色体相互作用。本研究将首次对光感受器基因的转录共调控进行有针对性的评估，并确定潜在的分子和细胞机制。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的靶点具有重要意义。

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.