Roles and mechanisms of cis-regulatory IncRNAs in the p53 tumor suppressor pathway

项目摘要/摘要 顺式调控的长非编码RNA(LncRNAs)具有独特的调控基因表达的能力 相邻的基因。在剂量补偿的背景下进行历史研究，有越来越多的证据表明 顺式调节的lncRNAs也可能在正常生理和疾病发展中发挥广泛的作用。 然而，它们的功能和行动机制的特点仍然很差。这项研究建议 本文旨在通过对两个lncRNAs的研究来了解顺式调控在癌症生物学中的作用。 Gadd45和PVT1的另一种亚型Pvt1b。在初步研究中，我们确定了这些lncRNA 是由中枢肿瘤抑制因子p53直接调节的，它们在顺式作用下调节它们的 邻近基因，即肿瘤抑制基因Gadd45和癌基因Myc，以及Pvt1b参与其中 在抑制克隆生长方面。基于这些令人兴奋的数据，我们假设顺式调控的lncRNA 在P53网络中提供一层不受重视的调节层，并可能介导P53依赖的应激 反应和肿瘤抑制功能。为了解决这一假设，在目标1中，我们建议执行 在细胞水平上的一系列综合实验，这些实验将测试lncRNA的遗传抑制- Gadd45和Pvt1b在原代和转化细胞系中对P53依赖的转录和细胞的影响 对压力的反应。同时，我们将检查这两个lncRNAs在发育过程中是否存在遗传抑制 小鼠肺癌模型中的肿瘤促进了肿瘤的发展，如P53所示。加在一起，这套 实验将在p53的背景下建立顺式调控的lncRNAs的生理学相关性 体外和体内的网络。在目标2和目标3中，我们将阐明lncRNA-Gadd45的作用机制。 和Pvt1b。目标2应用了一种创新的基因方法来进行转录特异性降解，这是我们开发的， 为了通过解离lncRNA的积累来确定顺式调控的功能元件 转录本来自其转录行为和基因座中潜在的DNA元件。由此得出的结论 一系列研究将通过澄清关于局部基因表达的核心角色的争议来推动该领域的发展 控制，并可能阐明顺式调节关系如何在疾病状态下被破坏，包括 癌症。最后，为了努力确定独联体监管的普遍原则以及特定地点的特点，在 目的3，我们将确定顺式作用的lncRNAs是否影响局部转录和表观遗传事件 分别检测转录动力学、表观遗传状态和染色质结构 精神错乱。总而言之，这里提出的研究利用了严格的遗传方法、优雅的功能 分析，和最先进的分子工具，以解决顺式调控的lncRNA对p53的贡献 肿瘤抑制途径。这项工作的广泛意义在于它有可能扩大我们的理解 在生理过程中发生的转录、表观遗传和染色质重组事件 对压力做出反应，并在癌症中经常感到不安。

Project Summary/Abstract Cis-regulatory long noncoding RNAs (lncRNAs) have the unique capability to regulate the expression of neighboring genes. Historically studied in the context of dosage compensation, there is mounting evidence that cis-regulatory lncRNAs may also play widespread roles in normal physiology and disease development. However, their functions and mechanisms of action have remained poorly characterized. The study proposed herein aims to understand the role of cis-regulation in cancer biology by focusing on two lncRNAs, lncRNA- Gadd45 and an alternative isoform of Pvt1, Pvt1b. In preliminary studies, we established that these lncRNAs are directly regulated by the central tumor suppressor factor p53, that they act in cis to regulate their neighboring genes, namely the tumor suppressor Gadd45 and the oncogene Myc, and that Pvt1b is involved in suppressing clonal growth. Based on these exciting data, we have hypothesized that cis-regulatory lncRNAs provide an unappreciated layer of regulation in the p53 network and may mediate p53-dependent stress responses and tumor suppressive functions. To address this hypothesis, in Aim 1, we propose to perform a comprehensive series of experiments at the cellular level, which will test whether genetic inhibition of lncRNA- Gadd45 and Pvt1b in primary and transformed cell lines affects p53-dependent transcriptional and cellular responses to stress. In parallel, we will examine whether genetic inhibition of the two lncRNAs in developing tumors in a mouse model of lung cancer promotes tumor development, as shown for p53. Together, this set of experiments will establish the physiological relevance of cis-regulatory lncRNAs in the context of the p53 network in vitro and in vivo. In Aims 2 and 3, we will elucidate the mechanism of action of lncRNA-Gadd45 and Pvt1b. Aim 2 applies an innovative genetic approach for transcript-specific degradation, developed by us, in order to determine the functional element of cis-regulation by dissociating the accumulation of the lncRNA transcript from the act of its transcription and from the underlying DNA elements in the locus. Findings from this set of studies will advance the field by clarifying a controversy about the central player in local gene expression control and may elucidate how cis-regulatory relationships become disrupted in disease states, including cancer. Finally, in an effort to define universal principles as well as locus-specific features of cis regulation, in Aim 3, we will determine whether cis-acting lncRNAs impact local transcriptional and epigenetic events by examining the kinetics of transcription, the epigenetic state, and the chromatin architecture in their respective loci. In summary, the studies proposed here take advantage of rigorous genetic approaches, elegant functional assays, and state-of-the art molecular tools to address the contribution of cis-regulatory lncRNAs to the p53 tumor suppressor pathway. The broad significance of this work lies in its potential to expand our understanding of the transcriptional, epigenetic, and chromatin re-organization events that take place during the physiological response to stress and become frequently perturbed in cancer.