Differentiation-dependent interactions between RUNX-1 and FLI-1 during megakaryocyte development.
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.
Huang
Hui
H
Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115, USA.
Yu
Ming
M
Akie
Thomas E
TE
Moran
Tyler B
TB
Woo
Andrew J
AJ
Tu
Nathan
N
Waldon
Zachary
Z
Lin
Yin Yin
YY
Steen
Hanno
H
Cantor
Alan B
AB
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000166:cytology / Q000196:embryology; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D025941:Protein Interaction Mapping; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D050676:Core Binding Factor Alpha 2 Subunit; C497426:Fli1 protein, mouse; D051789:Proto-Oncogene Protein c-fli-1; C493726:Runx1 protein, mouse; D012694:Serine; D013755:Tetradecanoylphorbol Acetate