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

7.项目总结/摘要 在所有的真核生物中，最大的核体是核仁，它是一个相分离的、非膜结合的核体。 专门合成核糖体RNA并将其组装成核糖体的细胞器。另夕h 核仁的外部是与多个特异性DNA位点相互作用的枢纽，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，核仁相关的DNA在着丝粒重复序列中高度富集。着丝点 染色体附着在有丝分裂纺锤体上的位点，对于正确的染色体是至关重要的。 种族隔离几种核仁蛋白与着丝粒-核仁相互作用有关， 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，并且在癌细胞中大大增加。 然而，调节这些相互作用的机制仍然未知。 癌细胞不仅表现出增加的着丝粒-核仁相互作用，它们还经常含有一种 核仁周区室（PNC），一种复杂的细胞学特征，在非肿瘤细胞中不存在。PNC是 位于核仁表面，含有多种RNA种类和RNA结合蛋白。我们 在此证明，这些机构也包含特定的DNA位点，其中一些编码非编码RNA 保留在PNC中。一种名为metarrestin的候选癌症治疗剂是基于其对癌症的治疗能力而分离出来的。 解离PNC; metarrestin目前处于临床试验中，基于其减少人类转移的能力， 肿瘤异种移植实验。重要的是，对于这个提议，我们已经观察到metarrestin也干扰了 着丝粒-核仁相互作用 我们还提出了数据，着丝粒-核仁相互作用的扰动后，暴露在巨噬细胞 细菌脂多糖（LPS），先天免疫系统的典型刺激物。我们也证明了 这种反应在抑制特定信号传导途径时被阻断。这些变化伴随着 改变H3 K27 me 3的核分布，这是一种兼性染色体组蛋白修饰特征， 异染色质 总而言之，这个提议的中心主题是， 相互作用对于理解染色体错误分离、转移和先天免疫是重要的。 我们计划进行一系列的协同实验，以了解更多的潜在机制。比如我们 将测试新着丝粒的着丝粒活动是否会产生核仁关联，或者相反， 是着丝粒卫星重复序列的一个特性，与活性无关。我们将采取候选人和公正的 寻找核仁相互作用所需的着丝粒蛋白的方法。我们将描述 metarrestin影响DNA基因座与PNC和核仁的关联，我们将定义涉及PNC和核仁的顺式作用基因座。 PNC协会。我们将描述信号传导介导的干扰所需的信号通路， 巨噬细胞中的核仁-着丝粒相互作用这些研究的结果将允许随后的测试 的普遍性。例如，巨噬细胞中的信号成分是否也在肿瘤细胞中起作用， 接受了治疗性的异羟肾上腺素吗通过这种方式，这一合作提案将把来自 不同的实验系统，以回答有关核组织之间的基本联系的问题 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.