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). The UPS 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, which contains about 69 E3 ligases, we discovered that Fbxo21 was highly expressed in the HSPC population. Fbxo21 was more highly expressed in the HSPC population 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 both shRNAs against Fbxo21/FBXO21 and a hematopoietic specific conditional Fbxo21 knockout (KO) mouse model. We found that silencing Fbxo21/FBXO21 in HSPCs and the human acute myeloid leukemia (AML) cell line, MOLM-13, leads to a loss in colony formation and differentiation towards the mature myeloid lineage. In our conditional Fbxo21 KO, we also found a decrease in the number of colonies formed after re-plating but found no significant difference in the production of specific hematopoietic cell populations. However upon G-CSF stimulation, we see alterations in the production of hematopoietic cell populations as well as possible changes in downstream signaling. These findings lead to the hypothesis that the ubiquitin E3 ligase FBXO21, and its substrates regulate hematopoietic stem cell maintenance. Deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.