The dynamics and impact of R-loops in epigenetic stability and aging

项目总结 导言：越来越多的证据支持表观遗传失调是 衰老。事实上，最近的研究表明，表观遗传标记准确地预测了实际年龄，而且在体内 表观遗传重新编程可以延长老年动物的寿命，并使组织再生。最引人注目的 来自大卫·辛克莱研究小组的最新证据表明，表观遗传“噪音”的诱导 通过使用非致突变双链断裂(ICE小鼠模型)导致加速衰老表型 在生理和细胞水平(ICE MEF)，例如细胞特性的丧失。有趣的是，表观遗传衰老 ICE小鼠的诱导是由于驱动基因的关键增强子和表观基因组结构的失调 通过与靶基因启动子的3D相互作用进行表达。最近的证据表明，增强剂是 转录成一种非编码的RNA物种，增强子RNA(Erna)，并且Erna支持增强子稳定性。 此外，越来越多的证据表明，Erna形成了一种带有增强子DNA的结构，称为“R环”(an RNA：带有移位的单链DNA的DNA杂交体)。在最近的一项研究中，我们的实验室证明了STAG2，一种蛋白质 它有助于维持增强子的稳定性和细胞特性，也在体外结合R-环并与它们共定位 在增强子上，表明STAG2与Erna R-loop结合。我们还发现(未发表)STAG2保护R- 由RNA：DNA解旋酶RNaseH1降解而成的环。此外，我发现DHX9，一种可以 调节R环的形成，是ICE小鼠肌肉中最高过表达的基因，我发现了Erna的证据 在ICE MEF中，增强子上的R-环调节失调。综上所述，这些发现表明，STAG2可以保护 Erna R环，随着年龄的增长保持年轻增强剂的稳定性。因此，我假设 生理性R环通过损害年轻增强剂的稳定性来推动表观遗传衰老。 目的1：阐明Erna R环在表观遗传衰老增强启动子稳定性中的作用机制。我会揭开 与表观遗传衰老中的增强子失调相关的Erna R环。我将使用CRISPR系统 操纵这些R-环并评估其对增强子稳定性和细胞衰老表型的影响。 目的2：确定STAG2/R环相互作用对保存年轻的表观基因组的影响。我会评估 通过操纵STAG2和RNaseH1在ICE细胞中的细胞衰老和表观遗传噪声，我将评估 ICE MEF中STAG2与表观遗传衰老的差异结合。 结论和意义：带着这些目的，我将阐明生理性Erna R环(和 STAG2/R-环相互作用)在随年龄增加的增强子稳定性机制中。这些目标具有重要意义，因为 他们是第一个解决R-环在增强稳定性或衰老方面的生理作用的人，他们 也有可能为修复年轻的表观基因组揭示新的药物靶点。此外， 完成拟议的培训计划将为我在衰老生物学领域的独立研究生涯做好准备。

PROJECT SUMMARY Introduction: A growing body of evidence supports the notion that epigenetic dysregulation is a key driver of aging. Indeed, recent studies show that epigenetic markers accurately predict chronological age, and that in vivo epigenetic reprogramming can prolong lifespan and enable tissue regeneration in aged animals. Most striking was the recent evidence from the David Sinclair group which demonstrated that induction of epigenetic “noise” through use of non-mutagenic double-strand breaks (ICE mouse model) leads to accelerated aging phenotypes at the physiological and cellular level (ICE MEFs), such as loss of cell identity. Interestingly, the epigenetic aging induced in ICE mice results from the dysregulation of key enhancers, epigenomic structures which drive gene expression via 3D interactions with target gene promoters. Recent evidence indicates that enhancers are transcribed into a non-coding RNA species, enhancer RNA (eRNA), and that eRNA supports enhancer stability. Moreover, mounting evidence reveals that eRNA forms a structure with enhancer DNA called an “R-loop” (an RNA:DNA hybrid with a displaced ssDNA strand). In a recent study, our lab demonstrated that STAG2, a protein which helps maintain enhancer stability and cell identity, also binds R-loops in vitro and co-localizes with them at enhancers, suggesting that STAG2 binds eRNA R-loops. We also find (unpublished) that STAG2 protects R- loops from degradation by the RNA:DNA helicase RNaseH1. Furthermore, I found that DHX9, a protein which regulates R-loop formation, is the top over-expressed gene in ICE mouse muscle and I found evidence of eRNA R-loops at enhancers dysregulated in ICE MEFs. Taken together, these findings suggest that STAG2 protects eRNA R-loops to maintain youthful enhancer stability with age. Therefore, I hypothesize that dysregulation of physiological R-loops drives epigenetic aging by impairing youthful enhancer stability. Aim 1: Elucidate the mechanism of eRNA R-loops in enhancer stability with epigenetic aging. I will uncover eRNA R-loops that are associated with enhancer dysregulation in epigenetic aging. I will use a CRISPR system to manipulate these R-loops and assess the impact on enhancer stability and cellular aging phenotypes. Aim 2: Determine the impact of the STAG2/R-loop interaction in preserving the youthful epigenome. I will assess cellular aging and epigenetic noise in ICE cells with manipulation of STAG2 and RNaseH1, and I will assess the differential binding of STAG2 with epigenetic aging in ICE MEFs. Conclusion and significance: With these aims, I will elucidate the role of physiological eRNA R-loops (and STAG2/R-loop interactions) in the mechanism of enhancer stability with aging. These aims are significant as they are the first to address the physiological role of R-loops in either enhancer stability or in aging, and they also have the potential to reveal novel drug targets for restoration of the youthful epigenome. Furthermore, the completion of the proposed training plan will prepare me for an independent research career in aging biology.