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- Gadd45g和Pvt1的替代同种型Pvt1alt。在初步研究中，我们已经确定， 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细胞增殖。 邻近基因，即肿瘤抑制基因Gadd45g和癌基因c-Myc，并且Pvt1alt是 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网络中提供了一个未被认识的调节层，并可能介导p53依赖性的 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 在细胞水平上进行一系列全面的实验，这将测试基因抑制是否 原代和转化细胞系中的lncRNA-Gadd45g和Pvt1alt影响p53依赖性转录和 细胞对压力的反应与此同时，我们将研究是否遗传抑制的两个lncRNA中， 如p53所示，在肺癌小鼠模型中肿瘤的发展促进肿瘤的发展。在一起， 这组实验将建立顺式调节lncRNA在以下情况下的生理相关性： p53网络在体外和体内。在目的2和3中，我们将阐明lncRNA的作用机制， Gadd45g和Pvt1alt.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， 为了确定顺式调节的功能元件， lncRNA转录物从其转录行为和从潜在的DNA元件中积累， 轨迹这一系列研究的发现将通过澄清关于中心的争议来推进该领域。 参与局部基因表达控制，并可能阐明顺式调节关系如何在 疾病状态，包括癌症。最后，为了努力确定普遍原则和特定地点原则， 在目的3中，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，通过检查转录的动力学，表观遗传状态和染色质， 建筑在各自的位置。总之，这里提出的研究利用了严格的遗传学， 方法，优雅的功能测定，和最先进的分子工具，以解决顺式- p53肿瘤抑制通路的调节lncRNA。这项工作的广泛意义在于其 有可能扩大我们对转录，表观遗传和染色质重组事件的理解 在对压力的生理反应中发生，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- Gadd45g and an alternative isoform of Pvt1, Pvt1alt. In preliminary studies, we have 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 Gadd45g and the oncogene c-Myc, and that Pvt1alt 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-Gadd45g and Pvt1alt 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- Gadd45g and Pvt1alt. 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.