Regulation of retrotransposable element activity in Drosophila.

项目总结(项目2) 本项目的重点是利用果蝇模型系统来研究逆转座子的作用 在衰老过程中发生的细胞功能障碍的进展中的元素(RTE)，并发展成 抑制RTE活动和延长健康寿命的发明。保持压抑性的异质- 染色质随着年龄的增长而下降，导致rtes在物种间的表达和动员增加，如 种类繁多，如果蝇和老鼠。我们假设RTES的表达和动员的增加 导致体细胞基因组损伤的累积，导致细胞和有机体的损失 动态平衡，从而促进衰老。我们将使用强大的分子和基因工具以及短暂的生命 以确定RTES对衰老的影响，特别是我们将在整个 生物体的寿命，这在人类是不可能的，而且是令人望而却步的时间- 在任何哺乳动物模型系统中大规模消耗和昂贵。一种机械论的确认 RTE活动与衰老之间的关系将为制定干预措施奠定基础 减少衰老过程中的RTE活性和基因组损伤，并应延长健康寿命。 这项提议的目的是检验以下假设：(I)染色质调节的抑制的丧失 RTE活性随年龄增长与DNA损伤增加和基因组不稳定有关；(Ii) 随着年龄的增长，sirtuin导致RTE活性升高，这可以通过增加sirtuin活性来逆转；以及(Iii) 抑制体细胞中RTE活性的干预措施可以延长健康寿命。 为了更好地了解RTE活性、基因组损伤和寿命之间的关系，以及 开发能够延长健康寿命的新干预措施，在目标1中，我们将开发高通量DNA 排序方法(与项目1合作，核心B)，部署可用的和新的RTE记者 (与核心B合作)测量老年果蝇的RTE动员，并开展一项 基因筛查以确定参与抑制RTES的新基因和途径。我们将研究 (I)年龄、(Ii)饮食限制(DR)和(Iii)稳定异染色质的遗传干预的影响，如 随着SU(Var)3-9、HP1a、DICER-2、Piwi表达的增加或Adar表达的减少，对RTE活性和动员的影响。 在目标2中，为了确定Sir2和SIRT6在衰老过程中抑制RTE活性的作用，我们将结合 在目标1中开发和使用的工具，通过增加或 减少Sir2和SIRT6活动的表达(与项目3和核心B合作)。在《目标3》中，我们将 (1)确定目标1和目标2中制定的遗传干预措施对苍蝇寿命的影响 (与项目1和3以及核心B合作)，以及(Ii)测试核苷逆转录酶的效果 特异性反转录转座子的抑制物(NRTI)和RNAi敲除对RTE动员和寿命的影响 (与项目1和3以及核心B合作)。

PROJECT SUMMARY (PROJECT 2) The focus of this project is to exploit the Drosophila model system to study the role of retrotransposable elements (RTEs) in the progression of cellular dysfunction that occurs during aging, and to develop inter- ventions that suppress RTE activity and extend healthy life span. The maintenance of repressive hetero- chromatin declines with age, resulting in increased expression and mobilization of RTEs across species as diverse as fruitflies and mice. We hypothesize that the increase in the expression and mobilization of RTEs leads to an accumulation of damage to the genome of somatic cells, causing loss of cellular and organismal homeostasis, and thus promoting aging. We will use the powerful molecular and genetic tools and the short life span of Drosophila to determine the effects of RTEs on aging, and in particular, we will do this over the entire life span of the organism, something that would not be possible in humans, and would be prohibitively time- consuming and expensive in any mammalian model system on a large scale. Confirmation of a mechanistic relationship between RTE activity and aging will provide the groundwork for developing interventions that diminish RTE activity and genomic damage during aging, and should extend healthy life span. The aims of this proposal are to test the hypotheses that: (i) loss of chromatin-regulated suppression of RTE activity with age is associated with increased DNA damage and genome instability; (ii) decreases in sirtuins with age lead to elevated RTE activity that can be reversed by increasing sirtuin activity; and (iii) interventions that suppress RTE activity in somatic cells can extend healthy life span. To better understand the relationship between RTE activity, genomic damage and longevity, as well as to develop new interventions that can extend healthy life span, in Aim 1 we will develop high throughput DNA sequencing methods (collaboration with Project 1, Core B), deploy available and new RTE reporters (collaboration with Core B) to measure mobilization of RTEs in aging Drosophila, and carry out a forward genetic screens to identify new genes and pathways involved in the suppression of RTEs. We will examine the effects of (i) age, (ii) dietary restriction (DR), and (iii) genetic interventions that stabilize heterochromatin, such as increasing Su(var)3-9, HP1a, Dicer-2, Piwi or decreasing Adar expression, on RTE activity and mobilization. In Aim 2, to determine the role of Sir2 and Sirt6 in the repression of RTE activity during aging, we will combine the tools developed and utilized in Aim 1 to monitor RTE activity with genetic manipulations that increase or decrease the expression of Sir2 and of Sirt6 activity (collaboration with Project 3 and Core B). In Aim 3, we will (i) determine the effects of the genetic interventions developed in Aims 1 and 2 on the life span of flies (collaboration with Projects 1 and 3 and Core B), and (ii) test the effects of nucleoside reverse transcriptase inhibitors (nRTIs) and RNAi knockdowns of specific retrotransposons on RTE mobilization and lifespan (collaboration with Projects 1 and 3 and Core B).