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.