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

正常和疾病状态下核仁在人类基因组组织中的作用

• 批准号: 10705594
• 负责人: Daniel Richard Foltz
• 金额: $ 54.48万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-21 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705594
• 关键词: 3-Dimensional; Affect; Architecture; Binding; Biology; Cancer Biology; Cell Nucleolus; Cell Nucleus; Cell Wall; Cells; Centromere; Characteristics; Chromatin; Chromosome Segregation; Chromosomes; Cis-Acting Sequence; Clinical Trials; Complex; Cytology; DNA; Data; Data Set; Disease; Dissociation; Eukaryota; Event; Frequencies; Gene Dosage; Gene Expression; Genetic Transcription; Genome; Genome Stability; Genomics; Goals; Health; Heterochromatin; Histones; Human; Human Chromosomes; Human Genome; Immune; Immune signaling; Infection; Inflammatory; Innate Immune System; Interphase Cell; Investigation; Kinetics; Label; Learning; Link; Lipopolysaccharides; MAP Kinase Gene; Macrophage; Malignant Neoplasms; Maps; Measurement; Mediating; Methods; Mitotic spindle; Modification; Molecular; Mus; Natural Immunity; Neoplasm Metastasis; Normal Cell; Nuclear; Nucleolar Proteins; Organelles; Phase; Prevalence; Process; Property; Proteins; RNA; RNA-Binding Proteins; Repetitive Sequence; Research Personnel; Ribosomal RNA; Ribosomes; Role; Satellite DNA; Series; Shapes; Signal Pathway; Signal Transduction; Site; Stimulus; Stress; Structure; Surface; System; Testing; Therapeutic; Transducers; Untranslated RNA; anticancer treatment; cancer cell; carcinogenesis; carcinogenicity; cell type; cellular targeting; chromatin modification; chromosome missegregation; chromosome movement; deep sequencing; environmental stressor; experimental study; extracellular; functional outcomes; genetic manipulation; genome-wide; genomic locus; histone modification; malignant phenotype; member; monocyte; neoplastic cell; novel; p38 Mitogen Activated Protein Kinase; pathogen; recruit; response; therapeutic lead compound; tool; tumor xenograft

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.

7. 项目总结/摘要 在所有真核生物中，最大的核体是核仁，它是一种相分离的非膜结合体 专门负责合成核糖体 RNA 并将其组装成核糖体的细胞器。此外， 核仁外部是与多个特定 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。为了 例如，核仁相关 DNA 在着丝粒重复序列中高度富集。着丝粒， 染色体附着在有丝分裂纺锤体上的位点，对于正确的染色体至关重要 隔离。一些核仁蛋白与着丝粒-核仁相互作用有关，并且一些 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。 癌细胞不仅表现出着丝粒-核仁相互作用增加，而且还经常含有 核周区室 (PNC)，这是非肿瘤细胞中不存在的复杂细胞学特征。 PNC 是 位于核仁表面，含有多种 RNA 和 RNA 结合蛋白。我们 在此证明这些身体还含有特定的 DNA 基因座，其中一些编码非编码 RNA 保留在 PNC 内。一种名为metarrestin 的候选癌症治疗剂是基于其能够 解离 PNC；基于其减少人体转移的能力，metarrestin 目前正在进行临床试验 肿瘤异种移植实验。对于这个提议来说重要的是，我们观察到metarrestin也会干扰 着丝粒-核仁相互作用。 我们还提供了数据，表明暴露后巨噬细胞中着丝粒-核仁的相互作用受到干扰 细菌脂多糖（LPS）是先天免疫系统的典型刺激物。我们还表明 当抑制特定的信号通路时，这种反应就会被阻断。这些变化伴随着 H3K27me3 的核分布改变，这是兼性的组蛋白修饰特征 异染色质。 总而言之，该提案的中心主题是控制着丝粒-核仁的因素 相互作用对于理解染色体错误分离、转移和先天免疫非常重要。 我们计划进行一系列协同实验，以更多地了解潜在机制。例如，我们 将测试新着丝粒的着丝粒活性是否产生核仁关联，或者是否相反 是着丝粒卫星重复序列的一个属性，与活性无关。我们将公正地挑选候选人 寻找核仁相互作用所需的着丝粒蛋白的方法。我们将描述如何 metarrestin 影响 DNA 基因座与 PNC 和核仁的关联，我们将定义参与的顺式作用基因座 PNC 协会。我们将描述信号介导的破坏所需的信号通路 巨噬细胞中核仁-着丝粒的相互作用。这些研究的结果将有助于后续测试 的普遍性。例如，当巨噬细胞中的信号传导成分也在肿瘤细胞中起作用时， 用治疗性metarrestin 治疗？通过这种方式，该合作提案将汇集来自 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。