Project Summary The response to systemic infection and tissue injury requires the rapid adaptation of hematopoietic stem cells (HSCs) in the bone marrow, which proliferate and divert their differentiation towards the myeloid lineage. Significant interest has emerged in understanding the signals that trigger this emergency hematopoietic program. However, the mechanisms that terminate this response of the HSCs and restore tissue homeostasis remain unknown. The clinical success of proteasome inhibitors, bortezomib, and E3 ubiquitin ligase glues for the treatment of hematologic diseases has made the Ubiquitin pathway a bona fide target for cancer therapeutics. Thus, defining how novel E3 ligases function in the bone marrow and investigating their specific roles in normal and emergency hematopoiesis can lead to novel therapeutic interventions. We have demonstrated that the E3 ubiquitin ligase Spop restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeds in an unresolved manner and the sustained response in the HSCs results in a lethal phenotype reminiscent of hyper-inflammatory syndrome. Our proteomic/biochemical studies demonstrated that Spop restricts inflammation by targeting the signal transducer Myd88 for proteasome-dependent degradation. Myd88 accumulation in conjunction with an inflammatory stimulus leads to Myddosome formation, the hyper-phosphorylation of the Irak4 kinase and activation of a number of transcription factor pathways (NF-kB, Jun, Pu.1, Cebpb). This proposal defines: (a) the transcriptional and chromatin landscape changes imposed during initiation and termination of emergency hematopoiesis in the bone marrow HSC and progenitor cells, (b) the role of the myddosome assembly, signaling and termination in emergency hematopoiesis and gene regulation and (c) the structural details of myddosome assembly and termination. The findings of this grant proposal will uncover HSC-intrinsic mechanisms essential for reestablishing homeostasis following emergency hematopoiesis.