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Molecular and Cellular Biology, August 2009, p. 4103-4115, Vol. 29, No. 15
0270-7306/09/$08.00+0     doi:10.1128/MCB.00090-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development{triangledown} ,§

Hui Huang,1 Ming Yu,1 Thomas E. Akie,1 Tyler B. Moran,1 Andrew J. Woo,1 Nathan Tu,1 Zachary Waldon,2 Yin Yin Lin,2 Hanno Steen,2 and Alan B. Cantor1*

Department of Pediatric Hematology/Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts,1 Department of Pathology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts2

Received 20 January 2009/ Returned for modification 21 February 2009/ Accepted 16 May 2009

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.

* Corresponding author. Mailing address: 300 Longwood Ave., Karp 7, Children's Hospital Boston, Boston, MA 02115. Phone: (617) 919-2026. Fax: (617) 730-0222. E-mail: alan.cantor{at}childrens.harvard.edu

{triangledown} Published ahead of print on 26 May 2009.

§ Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, August 2009, p. 4103-4115, Vol. 29, No. 15
0270-7306/09/$08.00+0     doi:10.1128/MCB.00090-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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