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

R 环在表观遗传稳定性和衰老中的动态和影响

基本信息

批准号：
10315986
负责人：
Henry Miller
金额：
$ 3.25万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10315986
关键词：
3-Dimensional Address Age Aging Animals Binding Bioinformatics Biology of Aging Camptothecin Cell Aging Cells ChIP-seq Chromatin Chronology Clustered Regularly Interspaced Short Palindromic Repeats DNA Data Development Disease Drug Targeting Elements Embryo Enhancers Ensure Enzymes Epigenetic Process Fibroblasts Future Gene Expression Genes Genetic Transcription Genome Genomic Instability Genomics Hybrids Impairment In Vitro Knowledge Longevity Manuscripts Mus Muscle Nerve Degeneration Noise Pathologic Phenotype Physiological Preparation Proteins Publishing RNA RNA Degradation RNA analysis Research Role Structure System Testing Training Untranslated RNA age related aged anti aging career cofactor endonuclease epigenetic marker epigenetic therapy epigenome epigenomics helicase in vivo innovation mouse model new therapeutic target novel overexpression preservation prevent promoter restoration sight restoration therapeutic target tissue regeneration transcriptome sequencing

项目摘要

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.

项目概要简介：越来越多的证据支持表观遗传失调是一个关键驱动因素的观点。事实上，最近的研究表明表观遗传标记可以准确预测实际年龄，并且可以预测体内年龄。表观遗传重编程可以延长衰老动物的寿命并实现组织再生。大卫·辛克莱小组最近的证据表明，表观遗传“噪音”的诱导通过使用非诱变双链断裂（ICE 小鼠模型）导致加速衰老表型在生理和细胞水平（ICE MEF），例如暗示的细胞身份丧失、表观遗传衰老。在 ICE 小鼠中诱导的结果是关键增强子、驱动基因的表观基因组结构的失调最近的证据表明增强子是通过与靶基因启动子的 3D 相互作用来表达的。转录成非编码 RNA 种类，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 与增强子 DNA 形成一种称为“R 环”的结构（一种在最近的一项研究中，我们的实验室证明了 STAG2（一种蛋白质）。有助于维持增强剂稳定性和细胞身份，还在体外结合 R 环并与其共定位增强子，表明 STAG2 结合 eRNA R 环我们还发现（未发表）STAG2 保护 R-环。此外，我发现 DHX9 是一种蛋白质，它可以防止 RNA:DNA 解旋酶 RNaseH1 的降解。调节 R 环的形成，是 ICE 小鼠肌肉中最过度表达的基因，我发现了 eRNA 的证据综上所述，ICE MEF 中增强子的 R 环失调，这些发现表明 STAG2 具有保护作用。 eRNA R 环随着年龄的增长保持年轻增强子的稳定性因此，我与这种失调作斗争。生理 R 环通过损害年轻增强子稳定性来驱动表观遗传衰老。目标 1：阐明 eRNA R 环在表观遗传衰老过程中增强子稳定性的机制。与表观遗传衰老中增强子失调相关的 eRNA R 环我将使用 CRISPR 系统。操纵这些 R 环并评估对增强子稳定性和细胞衰老表型的影响。目标 2：确定 STAG2/R 环相互作用对保存年轻表观基因组的影响。通过操纵 STAG2 和 RNaseH1 来观察 ICE 细胞中的细胞衰老和表观遗传噪声，我将评估 STAG2 与 ICE MEF 中表观遗传衰老的差异结合。结论和意义：带着这些目标，我将阐明生理性 eRNA R 环（以及 STAG2/R 环相互作用）在增强子稳定性随衰老的机制中具有重要意义。他们是第一个解决 R 环在增强子稳定性或衰老中的生理作用的人，并且他们也有可能揭示恢复年轻表观基因组的新药物靶点。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。