Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

Abstract

The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.Over the past 2 decades, a number of transcription factors/ cofactors have been identified that play essential roles in megakaryocytic differentiation. These include GATA-1 (46, 57), GATA-2 (4), Friend of GATA-1 (FOG-1) (55), NF-E2 p45 (47), mafG and mafK (39), SCL/Tal1 (30), GABP␣ (41), FLI-1 (17, 49), ZBP-89 (62), and RUNX-1 (14, 18). Yet, how these transcription factors act together to coordinate terminal megakaryocytic maturation remains incompletely understood. Moreover, there is increasing evidence that terminal megakaryocyte maturation is coordinated with localization at vascular sinusoidal niches within the bone marrow (1,21,26). How signaling events related to these spatial cues, as well as more-traditional cytokine-mediated transduction pathways, intersect with these key megakaryocyte transcriptional regulators also remains unclear.The transcription factor RUNX-1 belongs to a family of proteins that share a conserved 128-amino-acid runt homology domain, which mediates DNA binding and interaction with the cofactor CBF-␤ (for a review, see reference 20). RUNX-1 Ϫ/Ϫ mice die between embryonic day 12.5 (E12.5) and E13.5 due to central nervous system hemorrhage and failure of all definitive hematopoiesis (38, 59). The latter cause of death is due to a defect in the emergence of hematopoietic stem cells from the aorta-gonadal-mesonephros region during embryogenesis (31,34,64). Conditional knockout studies of mice demonstrate a specific role for RUNX-1 in megakaryocyte differentiation during ...

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

“…Low-ploidy MK has been observed in KO of other transcription factors, such as GATA1 or FLI-1 (refs 37,38). As these two transcription factors may be associated with RUNX1 in large transcription complexes 39,40 , they may similarly act on MYH10 expression. The observation that two non-muscle myosin II isoforms are present in MK and have distinct roles opens new avenues of research; for example, the MYH9/MYH10 ratio could be used as a marker of MK differentiation and dysfunction.…”

Novel function of PITH domain-containing 1 as an activator of internal ribosomal entry site to enhance RUNX1 expression and promote megakaryocyte differentiation

“…Phosphorylation of STAT1, a known signaling molecule responsible for megakaryocyte differentiation, was also increased upon PMA stimulation and PITHD1 did not affect PMA-induced STAT1 phosphorylation. In addition, we measured the expression of several transcription factors that played important roles in megakaryocyte differentiation [18][19][20]. We discovered that PITHD1 overexpression increased RUNX1 protein but not for GATA1, FLI1, or GABPa (Fig.…”

Inducible Fli-1 gene deletion in adult mice modifies several myeloid lineage commitment decisions and accelerates proliferation arrest and terminal erythrocytic differentiation

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