Loss of cell cycle gatekeeping by CNOT3 impairs hematopoietic stem and progenitor cell division and repopulating activity

Adult mammalian hematopoietic stem cells (HSCs) constitute a heterogeneous population responsible for generating various cell types in the blood throughout adulthood. Gene expression programs underlying regulation of self-renewal and differentiation of HSCs are tightly regulated. However, how post-transcriptional regulation of gene expression influences HSCs and hematopoiesis remains largely unexplored. Here, we report the critical role of CNOT3, a subunit of the CCR4-NOT complex, in regulating hematopoietic stem cells (HSCs) function in adult hematopoiesis. We observed that Cnot3 mRNA is highly expressed in HSCs and CNOT3 ablation in the murine Cnot3 conditional knockout mouse model resulted in anemia, reduced bone marrow cellularity and enhanced extramedullary hematopoiesis in spleen. Deletion of Cnot3 resulted in the early expansion of immunophenotypic HSCs which were then progressively lost over time. Cnot3 knockout hematopoietic stem/progenitor cells (HSPCs) failed to reconstitute hematopoietic systems of recipient animals in transplantation assays. Single-cell RNA sequencing (scRNA-seq) analysis of HSPCs revealed disruptions in lineage development and loss of HSCs. Transcriptomic profiling and cell cycle analysis demonstrated that Cnot3 deletion led to increased cycling activity in HSCs. Our results indicate that CNOT3 is critical for maintenance of homeostasis in HSCs and the hematopoietic system.