LncRNA mechanism of heart failure

项目摘要 基因组包含大量的非编码DNA和RNA元件，其数量远远超过蛋白质- 编码基因，提出了一个全新的视角转录电路。之间 非编码DNA元件，增强子是通过激活靶基因启动子的DNA片段， 作用于基因启动子，与它们与靶基因的距离或方向无关。许多 最近发现增强子编码非编码RNA，产生了一类新兴的 增强子RNA（eRNA）。心力衰竭的eRNA机制，特别是在其与 eDNA和染色质，在很大程度上是未知的。我们最近发现了一种心脏特异性eRNA 保护心脏免于病理性肥大和衰竭。该计划将重点关注新的 小鼠遗传模型，以确定这种新鉴定的eRNA在控制 增强子功能，三维染色质环，心脏基因表达，和病理 肥厚考虑到RNA可以被化学修饰并作为治疗药物递送， 该项目的成功将为设计新的机制治疗奠定基础。 目的1：确定eRNA在心脏保护中的体内充分性。我们将使用转基因技术， 敲入/敲出小鼠遗传学技术，以确定这种eRNA的心脏保护作用。方法 包括CRISPR激活的诱导型和心肌细胞特异性eRNA修饰，经主动脉 收缩、组织病理学、超声心动图、压力-容积环和分子标记物研究。 目的2：确定eRNA如何与其同源eDNA在三维染色质环中相互作用 和基因调控。我们将确定eRNA与其同源物之间的分子相互作用 eDNA在形成eRNA-eDNA杂交双链体和染色质成环到靶基因以进行转录中的作用 不同病理生理条件下的调节。方法包括小鼠遗传学、染色质 构象捕获、定量PCR、解旋酶测定、电迁移率变动测定和DNA-RNA 双重免疫沉淀。 目的3：确定正常和患病人类心脏中eRNA-eDNA的相互作用。我们将使用人类 心脏组织和iPS衍生的心肌细胞，以确定eRNA-eDNA的进化保守性 相互作用和eDNA三维染色质环到靶基因。方法包括 核糖体分析、体外转录和翻译、qPCR、染色质分级分离和 构象捕获、DNA-RNA双链体免疫沉淀和基于iPS的技术。

Project Summary The genome contains numerous non-coding DNA and RNA elements that far outnumber protein- coding genes, presenting a drastically new perspective of the transcriptional circuits. Among noncoding DNA elements, enhancers are the DNA fragments that activate target gene promoter by acting on gene promoters, independent of their distance or orientation to target genes. Many enhancers are recently found to encode noncoding RNAs, giving rise to an emerging class of enhancer RNAs (eRNAs). The eRNA mechanisms of heart failure, particularly at its interface with eDNA and chromatin, are largely unknown. We recently discovered a cardiac-specific eRNA capable of protecting the heart against pathological hypertrophy and failure. This program will focus on new mouse genetic models to define the molecular function of this newly identified eRNA in controlling enhancer function, 3-dimensional chromatin looping, cardiac gene expression, and pathological hypertrophy. Given that RNAs can be chemically modified and delivered as a drug for therapy, the success of this program will lay down a foundation for designing new mechanism-based therapy. Aim 1: Determining the in vivo sufficiency of an eRNA in cardioprotection. We will use transgenic and knock in/out technology of mouse genetics to define the cardioprotective role of this eRNA. Methods include CRISPR-enabled inducible and cardiomyocyte-specific eRNA modifications, transaortic constriction, histopathology, echocardiography, pressure-volume loop, and molecular marker studies. Aim 2: Defining how an eRNA interacts with its cognate eDNA in 3-dimensional chromatin looping and gene regulation. We will determine molecular interactions between an eRNA and its cognate eDNA in forming eRNA-eDNA hybrid duplex and in chromatin looping to target genes for transcription regulation under different pathophysiological conditions. Methods include mouse genetics, chromatin conformation capture, quantitative PCR, helicase assay, electric mobility shift assays, and DNA-RNA duplex immunoprecipitation. Aim 3: Defining eRNA-eDNA interactions in normal and diseased human hearts. We will use human heart tissues and iPS-derived cardiomyocytes to define the evolutionary conservation of eRNA-eDNA interactions and eDNA three-dimensional chromatin looping to target genes. Methods include ribosome profiling, in vitro transcription and translation, qPCR, chromatin fractionation and conformation capture, DNA-RNA duplex immunoprecipitation, and iPS-based technology.