The Function of AML1 (RUNX1) C-Deletion Mutant in Hematopoietic Stem/Progenitor Cells.
Authors: Satoh Yusuke , Matsumura Itaru , Ezoe Sachiko , Tanaka Hirokazu , Yokota Takafumi , Ishikawa Jun , Oritani Kenji , Kanakura Yuzuru 
Published: April 2020

AML1 (RUNX1) is a family member of heterodimeric transcription factors named core binding factors (CBFs). AML1 binds to its target DNA through the RUNT domain and is frequently involved in chromosomal translocations associated with human leukemias. Also, AML1 was found to be mutated in a substantial fraction of myelodysplastic syndrome (MDS) patients. Although most of AML1 mutations identified in AML patients were located in the N-terminal region, the C-terminal mutaions yielding mutant AML lacking the C-terminal domain (AML1dC) were frequently observed in MDS patients. Based on the fact that the haploinsufficiency of AML1, which results from heterozygous missense mutation of the AML1 gene, causes familial platelet disorder (FPD) with predisposition to AML, and that the conditional deletion of the AML1 gene using the Cre-LoxP system in adult mice results in the failure of megakaryopoiesis and subsequent platelet production, we speculate that AML1 might be involved in the thrombopoietin (TPO)/c-Mpl (its receptor) system, which is a major regulator of this process. Also, we examined the mechanism through which AML1dC affects the function of wtAML1. At first, we examined the role of AML1 in the regulation of the c-mpl promoter with luciferase assays. In 293T cells, we found that wild-type AML1 (wtAML1) activated the c-mpl promoter by 3.5 fold. This effect was dose-dependently inhibited by a dominant-negative form of AML1, AML1-MTG8. In addition, we found that AML1dC inhibited the function of wtAML1 with efficiencies similar to AML1-MTG8 (or with lesser efficiencies than AML1-MTG8). Next, we tried to determine the element responsive to AML1 in the c-mpl promoter. Using various mutants of the c-mpl promoter, we found that wtAML1 activates the element between −135 and −116, which contains the typical AML1-binding sequence. Furthermore, we confirmed that wtAML1 bound to this element in electrophoretic mobility shift assays using nuclear extract of 293T cells transfected with wtAML1. Also, in this assay, we found that AML1dC bound to the consensus DNA sequence more strongly than wtAML1 and inhibited the DNA-binding of wtAML1 competitively. Next, we examined the roles of AML1 in the c-Mpl expression in hematopoietic cells by expressing AML1dC in the OP-9 system, in which hematopoietic cells develop from embryonic stem (ES) cells during the coculture with OP-9 cells. In contrast to the results obtained from 293T cells, when AML1dC was inducibly expressed with the Tet-off system, c-Mpl was expressed more intensely in Lin−Sca1+ hematopoietic stem/progenitor cells than mock-transfected Lin−Sca1+ cells, indicating that AML1 is a negative regulator of the c-Mpl expression in hematopoietic stem/progenitor cells. In contrast, the surface expression of c-Mpl in mature megakaryocytes was hardly affected by AMLdC in the OP-9 system. We also examined the effects of AML1 on the c-Mpl expression in normal murine bone marrow Lin−Sca-1+ cells by expressing AML1dC with the retrovirus system. As a result, we again found that AML1dC enhanced the c-Mpl expression in murine Lin−Sca-1+ cells. Together, our results indicate that AML1 regulates the c-Mpl expression both positively and negatively according to cell types (cf, 293T cells, hematopoietic stem/progenitor cells, and megakaroycytes). Particularly, although AML1 was previously reported to positively regulate of the c-Mpl expression in megakaryocytes, it was supposed be a negative regulator in hematopoietic stem/progenitor cells.