Gfi1 Protein Levels Are Upregulated upon Myeloid Differentiation Due to Diminished Proteasomal Degradation.

Abstract Granulocytes and monocytes play an essential role in the innate immune system and the inflammatory system. The generation of these differentiated myeloid cells from hematopoietic stem cells is a tightly regulated process in which transcription factors play an essential role. One of these transcription factors is Growth factor independence 1 (Gfi1). Gfi1 was originally identified as a proviral insertion site in murine leukemia models. Subsequently, it was shown that Gfi1 is essential for the self-renewal and long term reconstituting potential of hematopoietic stem cells and that it regulates T-cell development. In addition, Gfi1 knockout mice are severely neutropenic and exhibit a block in myeloid differentiation resulting in the accumulation of an atypical immature myelo-monocytic cell population. The observed defect in granulocytic differentiation in Gfi1 null mice is in line with Gfi1 point mutations described in patients suffering from severe congenital neutropenia. To further investigate the role of Gfi1 in myelopoiesis, we measured Gfi1 expression during both granulocytic and monocytic differentiation of HL60, NB4 and U937 cells. Upon differentiation of these cells with PMA or retinoic acid Gfi1 mRNA levels declined more than 3-fold. Interestingly, at the same time Gfi1 protein levels increased significantly suggesting that Gfi1 protein expression was regulated in a post-transcriptional manner. Since the ubiquitin proteasome system is one of the main pathways that control protein stability we tested whether Gfi1 was regulated by this system. Proteasome inhibition resulted in an accumulation of cellular Gfi1 protein levels. Moreover, ubiquitination experiments showed that Gfi1 is ubiquitinated and that proteasome inhibition resulted in an accumulation of ubiquitinated Gfi1 species indicating that ubiquitinated Gfi1 is targeted for 26S proteasomal degradation. To study Gfi1 degradation during myelopoiesis we developed a Gfi1 protein degradation assay. This showed that in vitro translated Gfi1 was quickly degraded in lysates from immature myeloid cells in a proteasome-dependent manner since the proteasome inhibitors MG132 and Velcade clearly blocked Gfi1 degradation. Importantly, Gfi1 was not degraded in lysates from differentiated myeloid cells. This suggests that the Gfi1 protein is no longer subject to proteasomal degradation upon terminal myeloid differentiation. To further study this, we treated undifferentiated U937 cells with proteasome inhibitors and observed a clear accumulation of endogenous Gfi1 protein levels, while endogenous protein levels in differentiated U937 did not further increase upon proteasome inhibition. Based on these results we conclude that Gfi1 is upregulated upon myeloid differentiation due to diminished proteasomal degradation despite diminished mRNA levels. Because total proteasome activity was comparable in immature and mature myeloid cells these data strongly suggest that an E3 ubiquitin ligase is responsible for the specific differential degradation. To determine Gfi1 protein stability in primary cells, we performed the degradation experiments with freshly isolated human hematopoietic subsets. This revealed a rapid degradation in immature CD34+ cells and a clear delayed degradation in CD14+ monocytes. Together these data indicate that the ubiquitin proteasome system is a key regulator of Gfi1 protein expression and that Gfi1 may play a role during both terminal granulocytic and monocytic differentiation.