Gfi1 Protein Turnover Is Regulated by the Ubiquitin Ligase Triad1.

The transcriptional repressor Growth factor independence-1 (Gfi1) plays an essential role during various stages of hematopoiesis. It is crucial for the self-renewal and long-term reconstituting potential of stem cells, essential for neutrophilic differentiation, and it plays an important role in T-cell and dendritic cell development. Gfi1 has also been implicated in malignant hematopoeisis because the Gfi1 gene is a common proviral integration site in murine leukemia models. We recently found that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Although Gfi1 mRNA levels are low in primary human monocytes, the protein levels are high due to low proteasomal degradation. Conversely, in mature granulocytes Gfi1 mRNA levels are high but protein levels are low due to strong proteasome-mediated turnover. Because Gfi1 plays an important role in normal and malignant hematopoiesis it will be of great interest to identify the ubiquitin ligases that regulate its turnover.

Previously, we showed that the RING finger ubiquitin ligase Triad1 regulates myeloid cell proliferation. Using yeast-two-hybrid assays we found that Triad1 binds the zinc finger region of Gfi1. This interaction was confirmed in co-immunoprecipitation experiments. To study whether the turnover of Gfi1 is regulated by Triad1 we performed ubiquitination assays. To our suprise we found that instead of promoting ubiquitination, Triad1 inhibited Gfi1 protein ubiquitination, also in the presence of proteasome inhibitors. RNAi mediated down regulation of Triad1 protein levels stimulated Gfi1 ubiquitination. Importantly, expression of a Triad1 point mutant (H158A) that fails to bind the ubiquitin conjugating enzyme UbcH7 also inhibited Gfi1 ubiquitination. To study whether the observed diminished ubiquitination by Triad1 affected the turnover of Gfi1 we analyzed Gfi1 protein half-life using the protein synthesis inhibitor cycloheximide. This showed that Triad1 co-expression prolonged the half-life of Gfi1 significantly. We conclude that Triad1 inhibits Gfi1 ubiquitination, resulting in decreased turnover of the protein. As this inhibition also occurs in the presence of proteasome inhibitors and is independent of the ubiquitin ligase activity of Triad1, these data support a model in which Triad1 competes for Gfi1 binding with other ubiquitin ligases that do mark Gfi1 for proteasomal degradation. Currently, we are testing candidate ubiquitin ligases (RING finger and HECT proteins) that were found to associate with Gfi1 in yeast-two-hybrid assays to gain more insight in how the activity of this important transcription factor is regulated.