AML1/RUNX1 Works as a Negative Regulator of c-Mpl in Hematopoietic Stem Cells

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 AML1 (RUNX1) is a family member of heterodimeric transcription factors named core binding factors. AML1 was originally identified at a breakpoint on human chromosome 21 in the t(8;21) translocation and known as the most common targets of chromosomal translocations in human leukemia (1, 2). In addition to chromosomal translocations, recent reports have shown the importance of point mutations of AML1 in hematological malignancies, such as acute myelogenous leukemia (AML) 2 and myelodysplastic syndromes (MDS) (3). The Runt domain of AML1 is utilized for DNA binding and heterodimerization with a partner PEBP2␤/CBF␤. Although PEBP2␤ by itself does not bind to the DNA, the association with PEBP2␤ is necessary for AML1 to elicit its biologic activity (4 -6). AML1 can regulate the transcription of the target gene both positively and negatively through the binding to the consensus DNA sequence, TGT/cGGT, possibly dependent on the cellular context and/or its target gene. For example, it positively regulates the expression cytokines and their receptors in myeloid and lymphoid lineage cells (7-12), whereas it negatively regulates CD4 transcription in immature thymocytes (13). Several experiments using conventional and conditional gene targeting in mice demonstrated that AML1 is essential for the early step in definitive hematopoiesis (14). North et al. (15) revealed that AML1 is required for the generation of hematopoietic stem cells (HSCs) from the vitelline and umbilical arteries and from the aorta-gonad-mesonephros (AGM) region. In addition, AML1 is necessary for the transitions from the stage of doublenegative (DN)2 to DN3 and DN3 to DN4 in the T-lymphocyte development (9, 17). Furthermore, AML1 plays an important role in the maturation of megakaryocytes and platelet production. AML1 deletion in adult mice led to the impaired polyploidization of megakaryocytes and low platelet production (17, 18), whereas the number of megakaryocyte progenitors was not altered in these mice, suggesting that AML1 is indispensable for the terminal maturation of megakaryocytes. Also, the hereditary loss-of-function mutation of AML1 or

Histone Lysine-specific Demethylase 1 (LSD1) Protein Is Involved in Sal-like Protein 4 (SALL4)-mediated Transcriptional Repression in Hematopoietic Stem Cells

“…However, in the same assay, the EBF1 mRNA levels were not changed accordingly (data not shown). This may suggest that SALL4 affects downstream gene expression differently according to specific cell types, which is similar to some other genes in previous report (30). Interestingly, we also monitored that SALL4 represses the expression level of CEBPA, which may be due to unidentified functional site and/or involvement of other machinery.…”

C-terminal mutation of RUNX1 attenuates the DNA-damage repair response in hematopoietic stem cells

“…This mutant was originally identified in a patient with MDS, and we and others previously showed it functions as a dominant-negative mutant over WT RUNX1 (RUNX1b) by inhibiting its DNA-binding activity. 20,21 RUNX1dC resembles in size an isoform of RUNX1, RUNX1a, (Figure 1a), which also has an inhibitory effect against RUNX1b. 22 Two days after gene transduction, we evaluated accumulations of DSBs using an antibody recognizing the phosphorylated histone variant H2AX at serine 139 (gH2AX).…”

“…We had to transfect high amount of DNA into test cells; therefore we utilized the adherent cell line, 293T, to perform the luciferase assay. 20 Single transduction experiment of RUNX1dC revealed RUNX1dC showed reduced GADD45A-reporter activity compared with the transduction of RUNX1b (10-fold activation in RUNX1b and 5-fold activation in RUNX1dC). In the case of the D171N mutant, the D171N mutant did not show the significant GADD45A-reporter activity (3.3-fold) compared with back-bone RUNX1 mutation attenuates DNA-damage response Y Satoh et al reporter activity (2.9-fold).…”

Transcriptional Regulation of Platelet Formation: Harnessing the Complexity for Efficient Platelet Production In Vitro

“…They therefore proposed a model whereby RUNX1 controls early MK differentiation genes in collaboration with GATA1, whilst for genes which are more typically expressed in later stages, RUNX1 regulation is effected in collaboration with FOS/ JUN family members and ETS TFs [106]. Another example of how RUNX1 can regulate the expression of the same gene, but with different partners, is exemplified by the analysis of the promoter for myeloproliferative leukaemia (MPL, virus oncogene, the TPO receptor) where RUNX1 interacts with the SIN3A corepressor complex in haematopoietic stem and progenitor cells, whilst it forms a complex with the transcription activator EP300 (E1A-binding protein p300) on the same promoter in MKs [107]. The co-occupancy of RUNX1 and EP300 on the promoter region of multiple key MK genes was later confirmed in a genome-wide ChIP-Seq study using primary murine MKs [108].…”

“…2.5.3.3) or the selective binding of TAL1 to active GATA1 TF complexes (see Sect. 2.5.2) described above [107,197]. In addition, TFs regulate not only expression of other partners with which they cooperate and physically associate but also their own expression levels.…”