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

项目摘要 真核细胞与细胞环境相互作用，通过转录调控程序激活的各种 内分泌配体。雌激素受体α（ER α）是一类核激素受体， 是理解配体诱导的转录程序的重要范例。对配体的反应 雌二醇（E2）主要与转录增强子结合，转录增强子是调节DNA序列 控制着基因表达的基因。E2刺激也导致了细胞的快速重组。 三维染色质结构和增强子协同性。生物化学和基因组研究 在过去的十年中，已经确定了许多ER β的协同调节因子，并且也在很大程度上解决了ER β的组成， 结合增强子复合物。然而，在过去的几年里，多学科的研究已经挑战了 配体诱导的转录增强子激活的传统模型和增强子的机制， 启动子通信。活细胞成像研究表明，在对急性激素信号的反应中， ER β在细胞核内形成不同的隔室，将转录机器集中在强大的E2 激活的转录增强子。在组成上，这些“核受体缩合物”是 核糖核蛋白复合物不仅由蛋白质组成，而且还由非编码增强子RNA（eRNA）组成， 有助于物理性质和活动。然而，其他RNA种类在配体中的贡献- 诱导的增强子缩合物组装和染色质结构尚不清楚。拟定研究 将专注于确定新的RNA辅助调节ER β，促进核糖核蛋白缩合物组装， 调节增强子协同性。AIM I的中心目标是鉴定和表征新的lncRNA 其有助于使用生物化学、基因组学和基因组学增强子复合物组装和基因调控 工程. AIM II的重点将是了解配体诱导的空间表达的患病率和功能作用。 增强子协同性初步的研究已经确定了一些蛋白质候选人，可能发挥作用， 在这种远距离增强器协同性中的机械作用。这些蛋白质的贡献， 从AIM I中实施的筛选策略中鉴定的lncRNA，在长距离增强子协同性中 也将被审查。这些研究有望揭示增强子组装的新RNA调节剂， 为核糖核蛋白相互作用组在各种调节中的新理解铺平了道路。 内分泌信号程序。重要的是，完成这项提案将产生数据和资源， 有必要设计一个更大的研究，重点是体内相关性和疾病状况的贡献，这些 新的分子参与者通过改变激素信号程序。

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.