Cullin-5 controls the number of megakaryocyte-committed stem cells to prevent excess megakaryopoiesis and thrombocytosis

Cullin-5 (Cul5) coordinates assembly of cullin-RING-E3 ubiquitin (Ub) ligase (CRL) complexes that include Suppressor of Cytokine Signaling (SOCS)-box-containing proteins. The SOCS-box proteins function to recruit specific substrates to the complex for ubiquitination and degradation. In hematopoiesis, SOCS-box proteins are best known for regulating the actions of cytokines that utilize the JAK-STAT signaling pathway. However, the roles of most SOCS-box proteins have not been studied in physiological contexts and any actions for Cul5/SOCS complexes in signaling by several hematopoietic cytokines, including thrombopoietin (TPO) and interleukin-3 (IL-3), remain unknown. To define additional potential roles for Cul5/SOCS complexes, we generated mice lacking Cul5 in hematopoiesis; the absence of Cul5 is predicted to impair the SOCS-box-dependent actions of all proteins that contain this motif. Here, we show that Cul5-deficient mice develop excess megakaryopoiesis and thrombocytosis revealing a novel mechanism of negative regulation of megakaryocyte-committed stem cells, a distinct population within the hematopoietic stem cell pool that have been shown to rapidly, perhaps directly, generate megakaryocytes, and which are produced in excess in the absence of Cul5. Cul5-deficient megakaryopoiesis is distinctive in being at least in part independent of TPO/Mpl and involves signaling via the beta-common and/or beta-IL-3 receptors, with evidence of deregulated responses to IL-3. This process is independent of the interferon-alpha/beta receptor (IFNARI), previously implicated in inflammation-induced activation of megakaryocyte-committed stem cells, which may suggest distinct regulation of these cells at steady state and under stress. Overall design: LSK cells (Linegage negative, Kit + , Sca1 + bone marrow cells) FACS sorted from wild type and Cul5-/- mice were loaded onto a Chromium Controller (10X Genomics). Single cell 3' v2 transcriptome libraries were obtained following manufacturer's instructions. Samples were pooled and sequenced on NextSeq (Illumina) (75bp kit, paired end, where 98bp were sequenced for genomic read).