Genomic strategies to identify novel gene regulatory mechanisms by ligand-activated transcriptional enhancers

项目总结 真核细胞通过多种途径激活的转录调控程序与细胞环境相互作用 内分泌配体。雌激素受体α(ER-)是核激素受体中的一类，对基因的调控具有重要作用。 是理解配体诱导转录程序的一个重要范式。作为对配体的响应 雌激素受体(-estadiol，E_2)主要与转录增强子结合，而转录增强子是的调控序列 控制着来自遥远的基因组座位的基因表达。雌激素的刺激也导致了快速重组 染色质的三维结构和增强子的协同作用。生物化学和基因组学研究 在过去的十年中，已经确定了许多ER的共同调节因子，也在很大程度上解决了ER的组成- 结合增强子复合体。然而，过去几年的多学科研究挑战了 配体诱导转录增强子激活的传统模型及增强子的作用机制 推动者沟通。活细胞成像研究表明，对急性荷尔蒙信号的反应， ER在细胞核内形成不同的隔间，集中转录机制在强健的E2 激活了转录增强子。从成分上讲，这些“核受体凝集物”是 核糖核蛋白复合体不仅由蛋白质组成，还由非编码增强子RNA(ERNA)组成 对身体特性和活动做出贡献。然而，其他RNA物种在配体中的贡献- 诱导增强子凝集物组装和染色质结构尚不清楚。建议进行的研究 将专注于寻找新的ER的RNA协调节子，以促进核糖核蛋白缩合物的组装和 调控的增强子协同作用。AIM I的中心目标将是识别和表征新的lncRNA 有助于利用生化、基因组学和基因组进行增强子复合体组装和基因调控 工程学。AIM II的重点将是了解配体诱导的空间蛋白的流行和功能作用 增强协作性。初步研究发现，很少有候选蛋白质可能发挥作用。 在这种长距离增强协作性中的机械作用。这些蛋白质的贡献，以及 从AIM I实施的筛选策略中鉴定的长距离增强子协同作用中的lncRNAs 还将接受检查。这些研究承诺揭开增强子组装的新的RNA调节器，并将 为重新认识核糖核蛋白相互作用组学在多种调控中的作用铺平了道路 内分泌信号程序。重要的是，这项提案的完成将产生数据和资源 有必要设计一项更大规模的研究，重点关注这些因素对体内相关性和疾病状况的影响 通过改变荷尔蒙信号程序来产生新的分子玩家。

PROJECT SUMMARY Eukaryotic cells interact with cellular environment through transcription regulatory programs activated by various endocrine ligands. Gene regulation by estrogen receptor alpha (ER), a class of nuclear hormone receptor, has been an important paradigm in understanding ligand-induced transcriptional programs. In response to the ligand -estradiol (E2), ER binds predominantly to the transcriptional enhancers, which are regulatory DNA sequence that control the gene expression from distant genomic loci. E2 stimulation also led to rapid reorganization of three-dimensional chromatin architecture and enhancer cooperativity. Biochemical and genomic studies in the past decade have identified many coregulators of ER, and also have largely resolved the composition of ER- bound enhancer complex. However, multidisciplinary studies over the past few years have challenged the conventional models in ligand-induced activation of transcriptional enhancers and the mechanism of enhancer- promoter communication. Live-cell imaging studies have revealed that in response to acute hormonal signaling, ER form distinct compartments inside the cell nucleus that concentrate transcription machinery at robust E2 activated transcriptional enhancers. Compositionally, these “nuclear receptor condensates” are ribonucleoprotein complexes composed of not only proteins, but also non-coding enhancer RNA (eRNA) that contribute to the physical properties and activity. However, the contribution of other RNA species in ligand- induced enhancer condensate assembly and chromatin architecture is not yet understood. The proposed study will focus on identifying new RNA coregulators of ER that facilitate ribonucleoprotein condensate assembly and regulated enhancer cooperativity. The central objective of AIM I will be to identify and characterize new lncRNAs that contribute to enhancer complex assembly and gene regulation using biochemical, genomics and genome engineering. The focus of AIM II will be to understand the prevalence and functional role of ligand-induced spatial enhancer cooperativity. Preliminary studies have identified few protein candidates that might be playing a mechanistic role in this long-distance enhancer cooperativity. The contribution of these proteins, and the lncRNAs identified from the screening strategy implemented in AIM I, in long distance enhancer cooperativity will also be examined. These studies promise to unravel new RNA regulators of enhancer assembly and will pave the way towards a new understanding of ribonucleoprotein interactome in the regulation of various endocrine signaling programs. Importantly, completion of this proposal will generate the data and resources necessary to design a larger study focusing on the in vivo relevance and disease condition contributed by these new molecular players by altering hormonal signaling programs.