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。 Gadd45G和PVT1的另一种异构体Pvt1alt。在初步研究中，我们已经确定这些 LncRNAs由中心肿瘤抑制因子p53直接调节，它们在顺式作用下调节其 邻近基因，即肿瘤抑制基因gadd45g和癌基因c-Myc，而Pvt1alt是 参与抑制克隆生长的。基于这些令人兴奋的数据，我们假设顺式监管 LncRNAs在P53网络中提供了一层不受重视的调节层，并可能介导P53依赖 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，以测试基因抑制是否 原代和转化细胞系中的LncRNA-gadd45G和Pvt1alt影响P53依赖的转录和 细胞对压力的反应。同时，我们将检查两个lncRNAs的基因抑制是否在 如P53所示，在小鼠肺癌模型中发生肿瘤会促进肿瘤的发展。一起， 这组实验将建立顺式调控lncRNAs在 P53在体外和体内的网络。在目标2和目标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本特异性降解， 由我们开发，以确定顺式调控的功能元件，通过解离 LncRNA转录本在其转录过程中和从潜在的DNA元件中的积累 轨迹。这组研究的发现将通过澄清关于中央银行的争议来推动该领域的发展 在局部基因表达调控中发挥作用，并可能阐明顺式调控关系是如何在 疾病状态，包括癌症。最后，为了努力定义普遍原则以及特定地点 顺式调控的特点，在目标3中，我们将确定顺式作用的lncRNA是否影响局部转录 和表观遗传事件，通过检测转录动力学、表观遗传状态和染色质 建筑在他们各自的地点。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p53抑癌通路的lncRNA。这项工作的广泛意义在于它 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 这发生在对压力的生理反应期间，并经常在癌症中受到干扰。

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.