Age-related epigenetic gene silencing in the RPE

描述(申请人提供)：老年性黄斑变性(AMD)是一种由年龄、遗传和环境因素引起的复杂疾病。虽然AMD的遗传学和环境原因正在被大力研究，但衰老在AMD中的确切作用既没有得到很好的理解，也没有得到很好的研究。在其他复杂的与年龄相关的疾病中，如动脉粥样硬化和癌症，表观遗传学提供了关于年龄如何促进疾病发展的基本理解。表观遗传学是对体细胞基因组进行长期共价修饰的研究。这些变化不涉及基因序列的变化，因此不是突变。Cg二核苷酸富集区(CpG岛)基因启动子的甲基化是导致基因沉默的主要表观遗传修饰。这些变化发生在单个细胞中，作为一个随机过程，导致在其他遗传相同的细胞群体中存在显着的异质性。我们假设，在视网膜色素上皮(RPE)中，通过表观遗传机制，作为年龄的一种功能，发挥神经退变、氧化应激或血管生成抑制作用的基因是沉默的。这些变化在RPE细胞群体中是异质性的，导致对邻近的光感受器、Bruchs膜和脉络膜毛细血管的异质性影响。为了验证这一假设，我们将重点研究TIMP3、BRCA1、IGF2和GSTP1。我们已经证明，在小鼠的RPE/脉络膜中，这四个基因在mRNA水平上的表达随着年龄的增长而下调。这四个基因也都有可能与AMD相关的功能。TIMP3是一种血管生成抑制因子，BRCA1和GSTP1是氧化应激抑制因子，IGF2是一种营养因子。最后，这些基因的启动子在小鼠体内都有CpG岛，并在各种人类上皮组织中表现出与年龄相关的甲基化。我们将首先在单个细胞的基础上量化这些基因在小鼠RPE中的年龄相关表达的下降。接下来，我们将研究这些基因启动子在整个小鼠RPE/脉络膜中与年龄相关的甲基化。这些目标的成功完成将使表观遗传学成为研究与年龄相关的人类RPE功能变化的重要新途径，以及将单个RPE细胞的表观遗传学变化与局部病理区域联系起来的技术能力。

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is a complex disease caused by ageing, genetics, and the environment. While the genetics and environmental causes of AMD are being vigorously pursued, the exact role of ageing in AMD is neither well understood nor well studied. In other complex age-related diseases such as atherosclerosis and cancer, epigenetics is providing a basic understanding of how age contributes to disease development. Epigenetics is the study of long term covalent modification of the genome of somatic cells. These changes do not involve alterations in gene sequence and are therefore not mutations. Methylation of gene promoters at CG dinucleotide rich regions (CpG islands) is a major epigenetic modification leading to gene silencing. These changes occur in single cells as a stochastic process resulting in significant heterogeneity in populations of cells which are otherwise genetically identical. We hypothesize that genes functioning as suppressors of neurodegeneration, oxidative stress, or angiogenesis are silenced in the retinal pigment epithelium (RPE) as a function of age by epigenetic mechanisms. These changes are heterogeneous within the population of RPE cells leading to heterogeneous effects on the adjacent photoreceptors, Bruch's membrane, and choriocapillaris. To test this hypothesis, we will focus our initial studies on TIMP3, BRCA1, IGF2, and GSTP1. We have shown that the expression at the mRNA level of all four genes is downregulated with age in the RPE/choroid of the mouse. All four genes also have functions potentially related to AMD. TIMP3 is an angiogenesis inhibitor, BRCA1 and GSTP1 are suppressors of oxidative stress, and IGF2 is a trophic factor. Finally, the promoters of these genes all have CpG islands in the mouse and exhibit age-related methylation in a variety of human epithelial tissues. We will first quantify the age-related decline of expression for these genes on a single cell basis in the mouse RPE. Next we will investigate the age-related methylation of the promoters of these genes in whole mouse RPE/choroid. The successful completion of the these aims will establish epigenetics as an important new avenue for studying age-related changes in human RPE function as well as the technical ability to relate the epigenetic changes in individual RPE cells to localized areas of pathology.