Abstract
Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways, one being the ubiquitin proteasome system (UPS) which controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The substrate recognition component of the UPS is the ubiquitin E3 ligase. Through investigating a specific family of ubiquitin E3 ligases, the Fbox family of proteins, we discovered that Fbxo21 was highly expressed in the HSPC population and was more highly expressed when compared to other Fbox genes. Western blot and qRT-PCR analysis confirmed high expression of FBXO21 in HSPCs, and revealed low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a conditional Fbxo21 knockout (KO) mouse model. We found that silencing FBXO21 in human CD34+ and mouse HSPC cells led to a loss in colony formation and an increase in cell differentiation. In our conditional Fbxo21 KO, we found a decrease in the number of colonies formed after replating and minimal differences in the production of specific hematopoietic cell populations in steady-state hematopoiesis. However upon 5-FU injections, we see a decrease in survival when Fbxo21 is knocked down. When further investigating targets of FBXO21, we found an increase in the activation of proteins involved in cytokine signaling when Fbxo21 is knocked down. FBXO21 has been previously linked to MAPK signaling which leads us to hypothesize that the ubiquitin E3 ligase FBXO21 regulates hematopoietic stem and progenitor cells through cytokine mediated pathways. Deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.