Nuclear Architecture, NcRNAs and Epigenetics in Transcriptional Regulation by ER

描述(申请人提供)：雌二醇通过雌激素受体α(ER？)起作用，对女性生殖功能的发育和动态平衡是必不可少的，对许多转录程序施加关键控制力，包括这些调节增殖和新陈代谢。ER在基因激活中介导辅活化子/辅抑制子交换的分子机制已经被很好地阐明。内质网、染色质修饰物和特定基因座的动态定位对于建立精确的转录调控和实现正确的基因表达模式都是必不可少的。然而，目前还不清楚这些因子是如何在不同的亚核结构中进行空间调控的，以及这种调控可能如何有助于基因调控。)我们最近的发现证明了信号诱导的转录单位从转录抑制区到允许环境的重新定位。我推测，非编码RNA(NcRNAs)TUG1和NEAT2通过选择性地与ER？靶基因启动子上存在的甲基化和未甲基化的Polycomb 2蛋白(PC2)相互作用，控制ER？靶基因从转录抑制多聚体(PCGS)重新定位到染色质间颗粒的基因激活环境。为了实现这个长期目标，我建议从三个具体的目标来研究这一假说：1)定义非组蛋白甲基化/去甲基化事件和ncRNAs作为一种新的分子策略，负责全基因组的ER？转录程序；2)确定分别位于PCGS和染色质间颗粒的两个ncRNAs，TUG1和NEAT2，在控制这两个亚核结构之间ER靶基因的重新定位方面的潜在作用，这取决于PC2甲基化对雌激素的反应；3)研究ncRNA在调节组蛋白密码的“读取器”，如PC2，识别组蛋白尾部修饰的能力中的潜在变构作用。为了实现这些目标，我建议通过使用特定抗体的CHIP-SEQ分析来确定甲基化和未甲基化PC2在全基因组范围内的位置，并结合Gro-Seq、RNA-Seq分析和siRNA击倒策略来确定PC2或其他活跃的“读取器”和ncRNAs在控制ER转录组中的功能作用。我还将研究在E2刺激下，ER调节基因从PcG小体到染色质间颗粒的潜在重新定位以及潜在的机制，通过TUG1或NEAT2 ncRNAs的结合指导PC2识别组蛋白尾部修饰的偏好。综上所述，这项研究评估了一种新的概念，即ER？靶基因位点在功能上不同的核结构结构之间的重新定位，以及几个在ER？靶基因调控中发挥关键作用的新的调节器的作用。这些发现将为药物开发提供一些有趣的治疗意义的创新目标。根据广泛的初步数据，我有信心在获奖期间实现这些目标，并成为一名独立调查员。 与公众健康相关：众所周知，雌激素在促进乳腺发育、调节转录和新陈代谢以及许多病理条件方面发挥着重要作用。虽然对雌激素受体的作用有很多了解，但其他未被探索的分子策略似乎有助于雌激素功能的关键方面。本申请中拟议的研究从一个创新的角度剖析了雌激素受体靶基因表达的控制，该创新观点结合了核结构的三维背景、表观遗传修饰和新的非编码RNA，这将揭示使用小RNA靶向ncRNA转录本治疗雌激素相关疾病的新方法。

DESCRIPTION (provided by applicant): Estradiol, which acts through the Estrogen Receptor alpha (ER¿), is essential for the development and homeostasis of female reproductive function, exerting critical control of many transcriptional programs, including these regulatory proliferatio, and metabolism. The molecular mechanism used by ER¿ to mediate coactivator/corepressor exchanges in gene activation has been reasonably well elucidated. ER¿, chromatin modifiers and dynamic positioning of particular loci are all essential for establishing precise transcriptionl regulation and achieving the correct patterns of gene expression. However, it is not clear how these factors are spatially regulated in distinct subnuclear structures and how this regulation may contribute to gene regulation. ) Our recent findings demonstrated a signal-induced relocation of the transcription units from a transcriptional repressive compartment to a permissive environment. I hypothesize that non-coding RNAs (ncRNAs) TUG1 and NEAT2 control ER¿ target genes to relocate from the transcriptional repressive Polycomb bodies (PcGs) to the gene activation milieu of the interchromatin granules, by selectively interacting with methylated and unmethylated Polycomb 2 protein (Pc2) present on ER¿ target gene promoters. To address this long-term goal, I propose to investigate the hypothesis from three specific aims: 1) to define non- histone methylation/demethylation events and ncRNAs as a novel molecular strategy responsible for genome- wide ER¿ transcriptional programs; 2) to determine the potential role of two ncRNAs, TUG1 and NEAT2, located in PcGs and interchromatin granules, respectively, in controlling relocation of ER¿ target genes between these two subnuclear structures, depending on the status of Pc2 methylation in response to estrogen; 3) to investigate the potential allosteric role of ncRNA in modulating the ability of "readers" of the histone code, such as Pc2, to recognize histone tail modifications. To achieve these aims, I propose to define the genome-wide location of methylated vs. unmethylated Pc2 by ChIP-Seq analysis using specific antibodies and the functional roles of Pc2 or other active "readers" and ncRNAs in controlling the ER¿ transcriptome by a combination of GRO-Seq, RNA-Seq analysis and siRNA knockdown strategies. I will also investigate the potential relocalization of ER¿-regulated genes from PcG bodies to interchromatin granules upon E2 stimulation and the underlying mechanisms, by which the binding of TUG1 or NEAT2 ncRNAs directs the preference of Pc2 recognition of histone tail modification. Taken together, the proposed study evaluates a new concept that ER¿ target gene loci relocate between functionally distinct nuclear architectural structures and the roles of several novel modulators that play a critical role in ER¿ target gene regulation. These findings will provide innovative targets for drugs development for a number of intriguing therapeutic implications. Based on the extensive preliminary data, I am confident of accomplishing these aims in the period of the award and emerging as an Independent Investigator. PUBLIC HEALTH RELEVANCE: It has been well established that estrogen plays important roles in promoting mammary gland development, regulating transcription and metabolism, and many pathological conditions. While much is understood about actions of Estrogen Receptor, additional unexplored molecular strategies appear to contribute to the critical aspect of estrogen function. The proposed studies in this application dissect the control of estrogen receptor target gene expression from an innovative view that incorporates three-dimensional context of nuclear architecture, epigenetic modifications and novel non-coding RNAs, which will uncover new approaches to treat estrogen-related disease using small RNAs to target ncRNA transcripts.