Infoscience

Thesis

The role of TIF1 gamma during adult murine hematopoiesis

Transforming Growth Factor beta (TGFβ) signaling plays an important role in a variety of cellular processes during embryonic development, adult tissue homeostasis and cancer. TGFβI ligand is a potent inhibitor of early hematopoietic progenitor [1] cells in vitro, however the exact role and mechanisms of this pathway during adult hematopoietic homeostasis remains poorly understood. The Transcriptional Intermediary Factor 1 gamma (Tif1γ) gene encodes a recently uncovered nuclear protein in the TGFβ transduction pathway. Tif1γ is defective in the zebrafish moonshine mutant, which exhibits primitive and definitive hematopoiesis defects [2]. In addition, Tif1γ has been shown to influence differentiation of human erythroid cells in vitro [3]. Recent work in our laboratory has provided genetic evidence that Tif1γ is required for the generation and survival of hematopoietic stem/progenitor cells during murine development (C.Adolphe, K.Shakhbazov et al., manuscript under submission). Given that Tif1γ function is required for embryonic development and null mutants are embryonic lethal [4] (R.Losson unpublished data), we took advantage of a conditional Tif1γ flox allele (R.Losson unpublished data) and combined it with the inducible MxCre line [5] to generate mice in which Tif1γ function can be specifically ablated during adult hematopoiesis. MxCre:Tif1γKO/flox mutants exhibit a decrease in erythroblasts and dramatic increase in granulocytes within the bone marrow. This phenotype is attributable to a massive increase in granulocyte-monocyte progenitors (GMPs) at the expense of common myeloid progenitors (CMPs) and megakaryocyte- erythrocyte progenitors (MEPs), which are severely decreased in Tif1γ mutant bone marrow. In addition, pre-B cells were dramatically decreased in numbers, attributable to an increase in cell death, indicating a pro-survival role for Tif1γ in B lymphocytes. In addition, we performed deletion of Tif1γ in non-competitive and competitive bone marrow transplantation settings, which indicated that only the CMP/MEP phenotype is a non-cell autonomous effect. Two distinct models have been reported suggesting how Tif1γ functions within the TGFβ pathway: one indicating that Tif1γ binds and acts through Smad2/3 and the other that Tif1γ functions as a Smad4 mono-ubiquitin ligase [3, 6, 7]. To genetically address this apparent discrepancy, we generated double Tif1γ/Smad4 hematopoietic specific mutant mice, whose phenotype was surprisingly similar to that observed in Tif1γ single mutants. These data suggest that Tif1γ function is independent of Smad4 in hematopoietic cells. To address the mechanism by which Tif1γ functions, we performed microarray profiling of normal and mutant progenitors, which revealed prostaglandin D2 synthase as a hub gene linking Tif1γ and TGFβ signaling. Finally, we were able to mimic the hematopoietic-Tif1γ null phenotype by providing 16,16-dimethyl prostaglandin D2 to wild-type hematopoietic cells in vitro. In summary, our data provide genetic evidence for a key role of Tif1γ during early myeloid development as well as pre-B cell survival. In addition, we have identified that the majority of Tif1γ function is Smad4 independent. Finally, we identified a novel link between Tif1γ function and prostaglandin metabolism/signaling as a likely mechanism by which Tif1γ regulates hematopoiesis.

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