Regulation of rRNA transcription in mammalian tissues

摘要/项目摘要 核糖体RNA（rRNA）占细胞RNA的90%，核糖体生物合成是细胞内最重要的生物合成之一。 细胞中的能量消耗过程。核心rRNA转录机制在进化上是古老的， 从单细胞真核生物到哺乳动物都高度保守，但高等生物的身体有不同的 核糖体生产率在不同的细胞类型，响应独特的组织特异性需求。突变 核糖体生物发生蛋白引起人类细胞类型特异性的“核糖体病”，表现为： 发育异常、特定器官功能障碍或癌症。然而，人们对 转录和表观遗传因子在正常细胞中差异调节核糖体生物合成 完整的生物体中的类型。具体地说，没有人表征核仁的蛋白质组成，或者 rRNA转录复合物的组成部分，在任何初级哺乳动物组织。这代表着一把钥匙 在我们对真核生物学的理解上存在着知识鸿沟。 在小鼠模型系统中使用定量蛋白质组学和转录因子（TF）图谱研究，我们 已经确定了核仁定位和丰富的，特异性结合核糖体DNA（rDNA）的几个 已知对正常发育和存活至关重要的细胞类型特异性TF（Pu.1、Irf 8、Etv 6），但 其在核糖体生物发生中的作用从未被报道过。在本申请中，我们提出了以下组合： 公正的，以及集中的方法，以确定和剖析的作用，细胞类型特异性rRNA调节 在组织内稳态中。我们将通过以下项目实现这一目标： 项目1：发现：我们将使用核仁和rDNA染色质蛋白质组学在定义的主要小鼠 细胞类型，以鉴定具有细胞类型特异性核仁定位和rDNA结合的蛋白质。这个目标 该项目的目的是鉴定完整组织中rRNA差异转录的新调节因子。 项目2：机制：我们将使用体外和体内降解决定子和染色质拴系方法， 分析rDNA结合细胞类型特异性转录因子（Pu.1，Irf 8，Etv 6等）在调节 rDNA染色质、rRNA转录和组织稳态。这个项目的目标是了解如何 TF-rDNA结合调节正常组织生物学。 这项工作的长期目标是详细了解核糖体的古老过程是如何 生物发生已经进化到满足复杂生物体中不同组织的需要，以及这种破坏是如何发生的。 调节可扰乱组织内稳态并引起疾病。

Abstract / Project Summary Ribosomal RNA (rRNA) comprises 90% of cellular RNA, and ribosome biogenesis is one of the most energy-consuming processes in the cell. The core rRNA transcriptional machinery is evolutionarily ancient and highly conserved from unicellular eukaryotes to mammals, but the bodies of higher organisms have different ribosome production rates in different cell types, responsive to unique tissue-specific demands. Mutations in ribosome biogenesis proteins cause cell-type-specific “ribosomopathies” in humans, manifested by developmental abnormalities, specific organ dysfunctions, or cancers. However, there is little understanding of the transcriptional and epigenetic factors that differentially regulate ribosome biogenesis across normal cells types in intact organisms. Specifically, no one has characterized the protein composition of nucleoli, or the components of rRNA transcription complexes, in any primary mammalian tissue. This represents a key knowledge gap in our understanding of eukaryotic biology. Using quantitative proteomics and transcription factor (TF) mapping studies in a mouse model system, we have identified nucleolar localization and abundant, specific binding to ribosomal DNA (rDNA) of several cell-type-specific TFs (Pu.1, Irf8, Etv6) that are known to be critical for normal development and survival, but whose roles in ribosome biogenesis have never been reported. We propose in this application a combination of unbiased as well as focused approaches to identify and dissect the roles of cell-type-specific rRNA regulators in tissue homeostasis. We will pursue this goal through the following projects: PROJECT 1: DISCOVERY: We will use nucleolar and rDNA-chromatin proteomics in defined primary mouse cell types to identify proteins with cell-type-specific nucleolar localization and rDNA binding. The goal of this project is to identify novel regulators of differential rRNA transcription in intact tissues. PROJECT 2: MECHANISM: We will use ​in vitro and ​in vivo degron and chromatin tethering approaches to dissect the direct roles of rDNA-binding cell-type-specific TFs (Pu.1, Irf8, Etv6, others) in the regulation of rDNA chromatin, rRNA transcription, and tissue homeostasis. The goal of this project is to understand how TF-rDNA binding regulates normal tissue biology. The long-term goal of this work is to gain a detailed understanding of how the ancient process of ribosome biogenesis has evolved to meet diverse tissue needs in complex organisms, and how disruption of this regulation can derange tissue homeostasis and cause disease.