The role of the nucleolus in human genome organization in normal and disease states

7.项目摘要/摘要 在所有真核生物中，最大的核体是核仁，它是一种相分离的、无膜结合的物质。 专门合成核糖体RNA并将其组装成核糖体的细胞器。此外， 核仁的外部是与多个特定DNA基因座相互作用的枢纽，从而有助于 真核细胞核的三维结构。 核仁-基因组相互作用与人类健康的核心过程密切相关。为 例如，核仁相关DNA在着丝粒重复序列中高度丰富。着丝粒，着丝粒 染色体附着在有丝分裂纺锤体上的位置，对正常的染色体是至关重要的。 种族隔离。有几种核仁蛋白与着丝粒-核仁相互作用有关，还有几种 着丝粒蛋白主要存在于间期细胞的核仁中。我们发现核仁- 着丝粒相互作用在细胞分化过程中受到调节，并在癌细胞中大大增加。 然而，调控这些相互作用的机制仍不清楚。 癌细胞不仅表现出着丝粒-核仁相互作用的增加，它们还经常含有一个 核周间隔(PNC)，这是一种复杂的细胞学特征，在非肿瘤细胞中是不存在的。PNC是 位于核仁表面，含有多种RNA物种和RNA结合蛋白。我们 在这里证明这些小体也包含特定的DNA基因座，其中一些编码非编码的RNA 保留在PNC内。一种名为Metarrestin的候选癌症治疗药物被分离出来，这是基于它的能力 分离PNC；Metarrestin目前正在进行临床试验，基于其减少人类转移的能力 肿瘤异种移植实验。对于这项提议，重要的是，我们观察到Metarrestin也会扰乱 着丝粒-核仁相互作用。 我们还提供了巨噬细胞暴露时着丝粒-核仁相互作用受到干扰的数据。 细菌脂多糖(LPS)，天然免疫系统的典型刺激物质。我们还表明， 当特定的信号通路被抑制时，这种反应被阻断。这些变化伴随着 H3K27me3的核分布改变--兼性的组蛋白修饰特征 异染色质。 总之，这项提议的中心主题是，支配着丝粒-核仁的因素 相互作用对于理解染色体错误分离、转移和先天免疫很重要。 我们计划进行一系列协同实验，以更多地了解潜在的机制。例如，我们 将测试新着丝粒的着丝粒活性是否会产生核仁关联，或者是否会 是着丝粒卫星重复的一种特性，与活动无关。我们会选择候选人并且不偏不倚 寻找核仁相互作用所需着丝粒蛋白的方法。我们将描述如何 Metarrestin影响DNA基因座与PNC和核仁的联系，我们将定义涉及的顺式作用基因座 PNC协会。我们将描述信号介导的干扰所需的信号通路。 巨噬细胞中核仁-着丝粒的相互作用。这些研究的结果将为后续的测试做准备 普遍性。例如，当巨噬细胞中的DO信号成分在肿瘤细胞中发挥作用时 用治疗性的Metarrestin治疗吗？通过这种方式，这一协作提案将统一来自 不同的实验系统来回答有关核组织之间基本联系的问题 和人类健康。

7. Project Summary / Abstract In all eukaryotes, the largest nuclear body is the nucleolus, a phase-separated, non-membrane bound organelle specialized for the synthesis of ribosomal RNAs and their assembly into ribosomes. Additionally, the exterior of the nucleolus is a hub for interactions with multiple specific DNA loci, thereby contributing to the three-dimensional architecture of the eukaryotic nucleus. Nucleolus-genome interactions are intimately connected to processes central to human health. For example, nucleolar-associated DNA is highly enriched in centromeric repetitive sequences. Centromeres, the sites of chromosome attachment to mitotic spindles, are fundamentally important for proper chromosome segregation. Several nucleolar proteins have been implicated in centromere-nucleolar interactions, and several centromeric proteins prominently reside in nucleoli in interphase cells. We have found that the nucleolar- centromeric interactions are regulated during cellular differentiation and are greatly increased in cancer cells. However, the mechanisms that regulated these interactions remain unknown. Not only do cancer cells display increased centromere-nucleolar interactions, they also frequently contain a perinucleolar compartments (PNC), a complex cytological feature that is absent in non-tumor cells. PNCs are located on the surface of nucleoli and contain multiple RNA species and RNA-binding proteins. We demonstrate here that these bodies also contain specific DNA loci, some of which encode non-coding RNAs retained within PNCs. A candidate cancer therapeutic termed metarrestin was isolated based on its ability to dissociate PNCs; metarrestin is currently in clinical trials based on its ability to reduce metastasis in human tumor xenograft experiments. Importantly for this proposal, we have observed that metarrestin also perturbs centromere-nucleolar interactions. We also present data that centromere-nucleolus interactions are perturbed in macrophages upon exposure the bacterial lipopolysaccharide (LPS), a canonical stimulus for the innate immune system. We also show that this response is blocked upon inhibition of specific signaling pathways. These changes are accompanied by altered nuclear distribution of the H3K27me3, a histone modification characteristic of facultative heterochromatin. Altogether, the central theme of this proposal is that the factors that govern centromere-nucleolus interactions are important for understanding chromosome missegregation, metastasis, and innate immunity. We plan a series of synergistic experiments to learn more about the underlying mechanisms. For example, we will test whether the centromeric activity of neocentromeres generates nucleolar associations, or if instead that is a property of centromeric satellite repeats regardless of activity. We will take candidate and unbiased approaches to finding centromeric proteins required for nucleolar interactions. We will characterize how metarrestin affects association of DNA loci with PNCs and nucleoli, and we will define cis-acting loci involved in PNC association. We will characterize the signaling pathways required for signaling-mediated disruption of nucleolar-centromeric interactions in macrophages. Results from these studies will allow for subsequent testing of universality. For example, do signaling components in macrophages also operate in tumor cells when treated with the therapeutic metarrestin? In this manner, this collaborative proposal will unite questions from diverse experimental systems to answer questions about the fundamental links between nuclear organization and human health.