The role of the TET-dependent DNA demethylation pathway in photoreceptor development and pathology

项目摘要 视网膜色素变性(RP)和相关遗传性光感受器营养不良的特点是进行性 感光器丧失，导致视力下降，甚至失明。虽然这些疾病是由 各种基因的突变，Rho、USH2A、PRPH2、RP1、CNGB1、EYS、PDE6A、PDE6G、 PDE6C、PDE6H、GNA1和NR2E3占相当数量的病例。我们最近表演了一场 人和小鼠胚胎视网膜(主要含有视网膜)的全基因组DNA甲基化分析 祖细胞[RPC])、出生后的小鼠RPC和成熟的光感受器。我们发现， 上述基因的启动子在从胎儿体内分离的DNA中高度甲基化(高度甲基化) 视网膜和RPC。在RPC分化过程中，这些启动子的甲基化水平显著降低 转化为光感受器，并伴随相应基因表达的增加。它一般都是 认为启动子区域的DNA甲基化可以抑制基因的表达，而DNA去甲基化应该 发生，以允许基因表达。RPC过程中上述基因启动子去甲基化失败 分化成光感受器可能会降低甚至消除它们的活性，从而导致光感受器 在基因组DNA中没有任何突变的营养不良。因此，不仅DNA突变，视网膜也是如此- DNA的特殊表观遗传学变化可能参与了RP及相关疾病的发病机制。 说明了解光感受器过程中DNA去甲基化途径的重要性 发展。Tet蛋白家族在DNA去甲基化和DNA去甲基化过程中起重要作用。 调节各种物种的眼睛发育和神经发生。我们的数据和其他实验室的结果 表明Tet依赖的DNA去甲基化途径控制着光感受器的发育。这个 这个项目的目标是详细了解Tet驱动的DNA去甲基化是如何 通路规定RPC分化为光感受器，并研究其 活动会导致光感受器死亡和视网膜退化。使用严格的实验设计，我们将 根据我们的特定目标探索这一途径：1)确定Tet依赖的DNA 去甲基化途径是祖细胞和光感受器前体之间的“垂直”表观遗传“开关” 视网膜发育中的命运；2)决定Tet依赖的DNA去甲基化途径是否起作用 作为视杆和视锥感光细胞表型之间的“水平”表观遗传“开关”；3)决定 Tet酶需要具有DNA结合域的转录因子作为Tet结合伙伴来指定 光感受器发育过程中去甲基化和激活的靶基因。为了达到这些目标， 我们将使用动物模型以及广泛的生化、分子和表观遗传学方法，努力 以获得稳健和公正的结果。在项目完成后，我们预计结果将揭示 表观遗传机制在光感受器正常和病理发育中的作用。

Project Summary Retinitis pigmentosa (RP) and related inherited photoreceptor dystrophies are characterized by progressive photoreceptor loss, resulting in poor vision or even blindness. While these disorders are caused by the mutations of various genes, mutations in RHO, USH2A, PRPH2, RP1, CNGB1, EYS, PDE6A, PDE6G, PDE6C, PDE6H, GNAT1, and NR2E3 account for a substantial number of cases. We recently performed a genome-wide DNA methylation analysis of human and murine fetal retinas (which mostly contain retinal progenitor cells [RPCs]), postnatal murine RPCs, and mature photoreceptors. We discovered that the promoters of all of the genes above were highly methylated (hypermethylated) in DNA isolated from fetal retinas and RPCs. The methylation of these promoters was significantly reduced during RPC differentiation into photoreceptors and accompanied by an increased expression of the corresponding genes. It is generally accepted that DNA methylation in promoter regions silences gene expression, while DNA demethylation should occur to allow gene expression. Unsuccessful demethylation of the promoters of the genes above during RPC differentiation into photoreceptors may reduce or even eliminate their activity, leading to photoreceptor dystrophies without any mutations in the genomic DNA. Thus, not only mutations in DNA but also retina- specific epigenetic changes in the DNA may contribute to the pathogenesis of RP and related diseases, indicating the importance of understanding the DNA demethylation pathway during photoreceptor development. The ten–eleven translocation (TET) protein family has a vital role in DNA demethylation and regulates eye development and neurogenesis in various species. Our data and the results of other laboratories indicate that the TET-dependent DNA demethylation pathway controls photoreceptor development. The objectives of this project are to gain a detailed understanding of how the TET-driven DNA demethylation pathway specifies the differentiation of RPCs into photoreceptors, and to investigate how irregularities in its activity lead to photoreceptor death and retinal degeneration. Using a rigorous experimental design, we will explore this pathway in accordance to our specific aims: 1) determine whether the TET-dependent DNA demethylation pathway acts as a “vertical” epigenetic “switch” between progenitor and photoreceptor precursor fates in the developing retina; 2) determine whether the TET-dependent DNA demethylation pathway functions as a “horizontal” epigenetic “switch” between rod and cone photoreceptor phenotypes; 3) determine whether TET enzymes require transcription factors with DNA binding domains acting as TET binding partners to specify target genes for demethylation and activation during photoreceptor development. To reach these objectives, we will employ animal models and a wide range of biochemical, molecular, and epigenetic approaches, striving to obtain robust and unbiased results. Upon the completion of the project, we expect the results to reveal the role of epigenetic mechanisms in photoreceptor normal and pathological development.