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

• 批准号: 10474329
• 负责人: Henry Miller
• 金额: $ 0.83万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2022-10-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474329
• 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

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.

项目摘要 引言：越来越多的证据支持这样一种观点，即表观遗传失调是遗传缺陷的关键驱动力。 衰老事实上，最近的研究表明，表观遗传标记可以准确地预测实际年龄， 表观遗传重编程可以延长老年动物的寿命并使其组织再生。最引人注目 是最近来自大卫辛克莱小组的证据，该证据表明表观遗传“噪音”的诱导 通过使用非致突变性双链断裂（ICE小鼠模型）导致加速老化表型 在生理和细胞水平（ICE MEF），如细胞身份的丧失。有趣的是， 在ICE小鼠中诱导的基因突变是由关键增强子（驱动基因的表观基因组结构）的失调引起的。 通过与靶基因启动子的3D相互作用表达。最近的证据表明，增强剂 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形成了一种称为“R环”的结构（一种称为“R环”的结构）。 RNA：具有置换的ssDNA链的DNA杂交体）。在最近的一项研究中，我们的实验室证明了STAG 2，一种蛋白质， 它有助于维持增强子的稳定性和细胞特性，也能在体外结合R环并与它们共定位 在增强子，表明STAG 2结合eRNA R环。我们还发现（未发表）STAG 2保护R- 环由RNA：DNA解旋酶RNaseH 1降解。此外，我发现DHX 9是一种蛋白质， 调节R环的形成，是ICE小鼠肌肉中最高的过度表达基因，我发现了eRNA ICE MEF中增强子处的R环失调。总之，这些发现表明STAG 2可以保护 eRNA R环，以保持年轻增强子随年龄增长的稳定性。因此，我推测， 生理R环通过损害年轻增强子稳定性来驱动表观遗传老化。 目的1：阐明eRNA R环在增强子稳定性与表观遗传老化中的作用机制。我会揭开 eRNA R环与表观遗传衰老中增强子失调相关。我会用一个CRISPR系统 操纵这些R环并评估对增强子稳定性和细胞老化表型的影响。 目的2：确定STAG 2/R环相互作用在保持年轻表观基因组中的影响。我将评估 细胞老化和表观遗传噪声在ICE细胞与STAG 2和RNaseH 1的操作，我将评估 STAG 2与ICE MEFs中表观遗传衰老的差异结合。 结论和意义：通过这些目的，我将阐明生理eRNA R环的作用（和 STAG 2/R-环相互作用）在增强子随老化的稳定性机制中的作用。这些目标意义重大， 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，他们 也有可能揭示新的药物靶点，用于恢复年轻的表观基因组。而且 完成这个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。

项目成果

RLSuite: An Integrative R-Loop Bioinformatics Framework.

RLSuite：集成 R-Loop 生物信息学框架。

• DOI: 10.26502/jbsb.5107071
• 发表时间: 2023
• 期刊: Journal of bioinformatics and systems biology : Open access
• 作者: Miller,HE;Montemayor,D;Levy,S;Sharma,K;Frost,B;Bishop,AJR
• 通讯作者: Bishop,AJR

Systematic Analysis of Long Non-Coding RNA Genes in Nonalcoholic Fatty Liver Disease.

• DOI: 10.3390/ncrna8040056
• 发表时间: 2022-07-22
• 期刊: NON-CODING RNA
• 影响因子: 4.3
• 作者: Ilieva, Mirolyuba;Dao, James;Miller, Henry E;Madsen, Jens Hedelund;Bishop, Alexander J R;Kauppinen, Sakari;Uchida, Shizuka
• 通讯作者: Uchida, Shizuka

Current Status of Epitranscriptomic Marks Affecting lncRNA Structures and Functions.

• DOI: 10.3390/ncrna8020023
• 发表时间: 2022-03-28
• 期刊: Non-coding RNA
• 影响因子: 4.3

FibroDB: Expression Analysis of Protein-Coding and Long Non-Coding RNA Genes in Fibrosis.

• DOI: 10.3390/ncrna8010013
• 发表时间: 2022-01-28
• 期刊: Non-coding RNA
• 影响因子: 4.3
• 作者: Ilieva M;Miller HE;Agarwal A;Paulus GK;Madsen JH;Bishop AJR;Kauppinen S;Uchida S
• 通讯作者: Uchida S