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

项目概要/摘要 顺式调节长链非编码RNA（lncRNA）具有调节以下表达的独特能力： 邻近基因在剂量补偿背景下的历史研究中，有越来越多的证据表明， 顺式调节lncRNA也可能在正常生理和疾病发展中发挥广泛作用。 然而，它们的职能和作用机制的特点仍然不清楚。提议进行的研究 本文旨在通过关注两种lncRNA，lncRNA- Gadd 45 β和Pvt 1的替代同种型Pvt 1b。在初步研究中，我们确定这些lncRNA 直接受中央肿瘤抑制因子p53调节，它们顺式作用以调节它们的 邻近基因，即肿瘤抑制基因Gadd 45 β和癌基因Myc，以及Pvt 1b参与 抑制克隆生长。基于这些令人兴奋的数据，我们假设顺式调节lncRNA 在p53网络中提供一个未被重视的调节层，并可能介导p53依赖性应激 反应和肿瘤抑制功能。为了解决这个假设，在目标1中，我们建议执行一个 在细胞水平上进行的一系列综合实验，将测试lncRNA的遗传抑制是否- 原代和转化细胞系中的Gadd 45 β和Pvt 1b影响p53依赖性转录和细胞凋亡 对压力的反应。同时，我们将研究两种lncRNA的遗传抑制是否在发育中起作用。 小鼠肺癌模型中的肿瘤促进肿瘤发展，如p53所示。这一套 实验将确定顺式调节lncRNA在p53基因表达背景下的生理相关性。 在体外和体内的网络。在目的2和3中，我们将阐明lncRNA-Gadd 45抑制剂的作用机制。 Pvt1b目标2应用了我们开发的一种创新的基因方法来进行转录特异性降解， 为了通过解离lncRNA的积累来确定顺式调节的功能元件， 从转录行为和基因座中的潜在DNA元件中转录。时发现的问题 一组研究将通过澄清关于局部基因表达的中心角色的争议来推进这一领域 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 癌最后，为了努力定义cis调节的普遍原则和特定基因座的特征， 目的3，我们将确定顺式作用lncRNA是否影响局部转录和表观遗传事件， 研究转录的动力学，表观遗传状态，以及它们各自的染色质结构， 的位点总之，这里提出的研究利用了严格的遗传学方法，优雅的功能， 分析和最先进的分子工具来解决顺式调节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 转录，表观遗传和染色质重组事件发生在生理过程中， 压力反应，并在癌症中经常受到干扰。

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.