AML1/RUNX1 Works as a Negative Regulator of c-Mpl in Hematopoietic Stem Cells
AML1/RUNX1 Works as a Negative Regulator of c-Mpl in Hematopoietic Stem Cells
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DOI：
10.1074/jbc.m804768200
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发表时间：
2008-10-31
影响因子：
4.8
通讯作者：
Kanakura, Yuzuru
中科院分区：
文献类型：
--
作者：
Satoh, Yusuke;Matsumura, Itaru;Kanakura, Yuzuru
在这项研究中，我们分析了AML 1/RUNX 1在c-mpl启动子调控中的作用。在使用293 T和HeLa细胞的荧光素酶测定中，野生型AML 1通过近端AML结合位点激活c-mpl启动子。与此雅阁一致，电泳迁移率改变分析和染色质免疫沉淀分析证实AML 1与该位点结合。接下来，我们分析了AML 1的功能，使用缺乏C末端的AML 1突变体（AML 1dC），这是最初发现的骨髓增生异常综合征患者。AML 1dC显性负抑制野生型AML 1的转录活性。然而，出乎意料的是，AML 1dC转导的鼠c-Kit（+）Sca 1（+）谱系（-）细胞比模拟转导的细胞更丰富地表达c-mpl mRNA和c-Mpl蛋白，这导致促血小板生成素介导的增殖增强。此外，当AML 1dC在从胚胎干（ES）细胞发育成造血细胞的过程中被诱导表达时，AML 1dC增强了造血干/祖细胞上的c-Mpl表达。此外，我们发现，早期造血细胞来源于AML 1（-/-）ES细胞表达c-Mpl比那些从野生型ES细胞发展更强烈。相反，AML 1dC几乎不影响巨核细胞的c-Mpl表达和成熟。对于AML 1在调节c-mpl启动子中的不同作用的机制，我们发现AML 1在造血干/祖细胞中与c-mpl启动子上的转录抑制子mSin 3A形成复合物，尽管它在巨核细胞中与同一启动子上的转录激活子p300形成复合物。总之，这些数据表明，AML 1可以通过根据细胞类型改变结合伴侣来正向和负向调节c-mpl启动子。
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.