Molecular underpinnings of photoreceptor transcriptional regulation by CRX and NRL

两个关键的转录因子，同源结构域蛋白CRX和碱性亮氨酸拉链蛋白NRL， 在光感受器分化和动态平衡过程中处于基因调控的中心。CRX是 指定有丝分裂后光感受器前体对发育的承诺是必不可少的 而NRL决定视杆细胞的命运。在编排 光感受器发育的转录程序，CRX和NRL在功能上和 身体上通过蛋白质与蛋白质的直接相互作用。光感受器转录缺陷 CRX和NRL编码基因突变导致严重的视网膜疾病 包括视网膜色素变性、视锥视杆细胞营养不良和Leber先天性黑发。尽管我们 对CRX和NRL的生物学和转录网络的深入了解， 对这些转录因子的功能和独特协同作用的机械性洞察 原子水平是不够的。在拟议的研究中，我们试图确定晶体和溶液 CRX和NRL各自的DNA结合络合物的结构，以及 CRX/NRL/DNA三元复合体。尽管这些转录因子中的突变已经被发现，但它们 与关键基因的调控没有机械联系。机械的预测来自 CRX和NRL致病突变如何改变DNA结合的结构 顺式调节元件的特异性将在后续分析中得到验证，包括高- 吞吐量方法，如SPEC-SEQ。这些研究将增进我们对 CRX和NRL的功能，定义了它们协同的分子性质，并允许我们描绘 突变的CRX和NRL蛋白导致视网膜疾病的特定机制。我们 假设CRX和NRL的结构最终与它们的顺式调控复杂 这些元素将使治疗方法能够有针对性地设计，通过调节 在特定启动子上的转录活动。

Two key transcription factors, homeodomain protein CRX and basic leucine zipper protein NRL, are at the center of gene regulation during photoreceptor differentiation and homeostasis. CRX is essential for specifying commitment of postmitotic photoreceptor precursors to the development of photoreceptor cells, whereas NRL determines the rod cell fate. In orchestrating the transcriptional program of photoreceptor development, CRX and NRL cooperate functionally and physically via a direct protein-protein interaction. Defects in photoreceptor transcriptional regulation due to mutations in the genes encoding CRX and NRL cause severe retinal diseases including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite our advanced understanding of the biology and transcriptional networks of CRX and NRL, mechanistic insight into the functions and unique synergy of these transcription factors at the atomic level is lacking. In the proposed studies, we seek to determine the crystal and solution structures of the individual DNA-bound complexes of CRX and NRL, as well as the structure of the ternary CRX/NRL/DNA complex. Although mutations in these TFs have been identified, they have not been mechanistically linked to regulation of key genes. The mechanistic predictions from the structures on how disease-causing mutations in CRX and NRL may alter DNA-binding specificity at cis-regulatory elements will be validated in the follow-up assays, including high- throughput approaches such as Spec-seq. These studies will enhance our knowledge of the functions of CRX and NRL, define the molecular nature of their synergy, and allow us to delineate specific mechanisms whereby mutant CRX and NRL proteins cause retinal diseases. We hypothesize that ultimately the structures of CRX and NRL complexed with their cis-regulatory elements will enable targeted design of therapeutics to treat visual disorders via modulation of transcriptional activities at specific promoters.