AML1/RUNX1 works as a negative regulator of c-Mpl in hematopoietic stem cells.
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.
Satoh
Yusuke
Y
Department of Hematology and Oncology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Matsumura
Itaru
I
Tanaka
Hirokazu
H
Ezoe
Sachiko
S
Fukushima
Kentaro
K
Tokunaga
Masahiro
M
Yasumi
Masato
M
Shibayama
Hirohiko
H
Mizuki
Masao
M
Era
Takumi
T
Okuda
Tsukasa
T
Kanakura
Yuzuru
Y
D000818:Animals; D050676:Core Binding Factor Alpha 2 Subunit / Q000378:metabolism / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000166:cytology; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D053628:Receptors, Thrombopoietin / Q000235:genetics* / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology
D050676:Core Binding Factor Alpha 2 Subunit; C481270:Mpl protein, mouse; C493728:RUNX1 protein, human; D053628:Receptors, Thrombopoietin; C493726:Runx1 protein, mouse; C082218:MPL protein, human