Abstract
In this study, we analyzed the roles for AML1/RUNX1 in the regulation of the c-mpl promoter. Wild-type AML1 activated the c-mpl promoter through the proximal AML-binding site in luciferase assays using 293T and HeLa cells. In accord with this result, electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that AML1 bound to this site. Next, we analyzed the function of AML1 using a mutant of AML1 lacking the C terminus (AML1dC), which was originally found in a patient with myelodysplastic syndromes. AML1dC dominant-negatively suppressed transcriptional activity of wild-type AML1. However, unexpectedly, AML1dC-transduced murine c-Kit(+)Sca1(+)Lineage(-) cells expressed c-mpl mRNA and c-Mpl protein more abundantly than mock-transduced cells, which led to the enhanced thrombopoietin-mediated proliferation. Moreover, when AML1dC was induced to express during the development of hematopoietic cells from embryonic stem (ES) cells, AML1dC augmented the c-Mpl expression on hematopoietic stem/progenitor cells. Furthermore, we found that early hematopoietic cells that derived from AML1(+/-) ES cells expressed c-Mpl more intensely than those that developed from wild-type ES cells. In contrast, AML1dC hardly affected c-Mpl expression and maturation of megakaryocytes. As for the mechanism of the different roles of AML1 in the regulation of the c-mpl promoter, we found that AML1 forms a complex with a transcription repressor mSin3A on the c-mpl promoter in hematopoietic stem/progenitor cells, although it forms a complex with a transcription activator p300 on the same promoter in megakaryocytic cells. Together, these data indicate that AML1 can regulate the c-mpl promoter both positively and negatively by changing the binding partner according to cell types.
Highlights
The t(8;21) translocation and known as the most common targets of chromosomal translocations in human leukemia [1, 2]
We found that early hematopoietic cells that derived from AML1ϩ/Ϫ embryonic stem (ES) cells expressed c-Mpl more intensively than those that developed from WT
Early hematopoietic cells that derived from AML1ϩ/Ϫ ES cells expressed c-Mpl more intensively than those that developed from WT ES cells
Summary
Reagents and Antibodies—Recombinant human (h) interleukin-6 (hIL-6), murine (m) IL-3, murine stem cell factor (mSCF), human thrombopoietin (hTPO), human erythropoietin, and an anti-mouse c-Mpl monoclonal antibody were provided by Kirin Brewery Co. (Tokyo, Japan). One more probe contained the proximal putative AML1-binding sequence in the human c-mpl promoter (Ϫ135/Ϫ116, numbered from the first ATG). Retrovirus Transfection into Murine Hematopoietic Stem/ Progenitor Cells—Purified KSL cells were cultured in DMEM containing 10% FBS, mSCF (100 ng/ml), and hTPO (100 ng/ml). Two days after retrovirus infection, Mock-, AML1dC-, and AML1b-transduced cells were cultured in DMEM containing 2% FBS without cytokines for 4 h. Similar results were obtained from luciferase assays using HeLa cells (Fig. 1B) These results suggest that AML1 may regulate the expression of c-mpl through the proximal AML1-. Colony Assays—Two days after retrovirus infection, GFPϩ compared with the nuclear extract from AML1b-untransfected cells (1000 cells/35-mm dish) were cultured in the methylcellu- 293T cells (Fig. 2A, lane 1), that from AML1b-transfected cells lose media M3234 (Stem Cell Technologies, Vancouver, British formed two additional complexes with the c-mpl (Ϫ135/Ϫ116).
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