Regulation of rRNA transcription in mammalian tissues

摘要/项目摘要 核糖体RNA(RRNA)占细胞RNA的90%，核糖体生物发生是其中最重要的一种 细胞内的耗能过程。核心的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 核糖体在不同细胞类型中的产生速率，响应独特的组织特定需求。基因突变 核糖体生物发生蛋白可引起人类特定细胞类型的“核糖病”，表现为 发育异常、特定器官功能障碍或癌症。然而，人们对此知之甚少 差异调控正常细胞核糖体生物发生的转录和表观遗传因素 在完整有机体中的类型。具体地说，还没有人描述核仁的蛋白质组成，也就是 任何原始哺乳动物组织中rRNA转录复合体的组成部分。这表示一个密钥 我们对真核生物生物学理解上的知识鸿沟。 利用定量蛋白质组学和转录因子(TF)在小鼠模型系统中的定位研究，我们 已经确定了核仁的定位和与核糖体DNA(RDNA)丰富的、特异的结合 细胞类型特异性转录因子(PU.1、IRF8、ETV6)，已知对正常发育和生存至关重要，但 其在核糖体生物发生中的作用尚未见报道。在本申请中，我们建议组合使用 识别和剖析特定细胞类型的rRNA调节因子作用的无偏见和有重点的方法 在组织动态平衡方面。我们将通过以下项目实现这一目标： 项目1：发现：我们将在已定义的原代小鼠中使用核仁和rDNA-染色质蛋白质组学 识别具有细胞类型特异性核仁定位和rDNA结合的蛋白质的细胞类型。这样做的目的是 该项目是为了在完整的组织中识别差异rRNA转录的新调节因子。 项目2：机制：我们将使用体外​和体内​降解和染色质捆绑的方法来 剖析rDNA结合的细胞类型特异性转录因子(PU.1、IRF8、ETV6等)在调节 RDNA染色质、rRNA转录和组织动态平衡。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代过程 生物发生进化以满足复杂生物体中不同的组织需求，以及这是如何被破坏的 监管可能会破坏组织的动态平衡，导致疾病。

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.