Megakaryocytic differentiation of hematopoietic stem cell by NF-E2 transcription factors.

NF-E2是一种异源二聚体，由一个谱系特异性亚基p45和一个普遍存在的亚基p18组成，属于小的Maf家族。为了了解NF-E2转录因子在造血干细胞巨核细胞分化中的作用，我们利用逆转录酶介导的聚合酶链反应（RT-PCR）研究了纯化的早期造血祖细胞（HPCs）在液体培养系统中诱导单系巨核细胞分化的转录因子表达。在巨核细胞分化过程中，诱导p45 NF-E2及其相关转录因子Nrf1和Nrf2的表达。此外，我们发现p18 （mafK）和mafG在慢性髓性白血病（CML）巨核细胞分化过程中也被诱导。特别是诱导了更丰富的mafG表达。这些结果表明Nrf1和Nrf2以及p45作为异源二聚体在巨核细胞分化中发挥重要作用。接下来，我们研究了Nrf1和Nrf2来自具有小maf的异源二聚体的可能性。我们构建了mafK、mafG、p45、Nrf1和Nrf2的原核表达质粒。重组蛋白用Ni-NTA区纯化，并进行电泳凝胶迁移量测定（EGMSA）。EGMSA显示小Maf蛋白与Nrf1和Nrf2形成异源二聚体，并识别NF-E2基序。

NF-E2 is a heterodimer composed of a lineage-specific subunit p45 and a ubiquitous subunit p18 which belongs to small Maf family. In order to understand the role of NF-E2 transcription factors in megakaryocytic differention of hematopoietic stem cells, we have studied the expression of the transcription factors in purified early hematopoietic progenitor cells (HPCs) induced to unilineage megakaryocytic differentiation in a liquid culture system by reverse transcriptase mediated polymerase chain reaction (RT-PCR). Expression of p45 NF-E2 and its related transcription factors, Nrf1 and Nrf2, was induced during megakaryocytic differentiation. Furthermore, we found that p18 (mafK) and mafG were also induced during megakaryocytic differention using chronic myelogenous leukemia (CML) blasts. Especially expression of mafG was induced more abundantly. These results indicated that Nrf1 and Nrf2 as well as p45 play an important role in megakaryocytic differentiation as heterodimers with small Mafs.We next studied the possibility that Nrf1 and Nrf2 from heterodimers with small Mafs. We construct prokaryotic expression plasmids for mafK,mafG,p45, Nrf1 and Nrf2. Recombinant proteins were purified with Ni-NTA regin and Electrophoretic gel mobility shift assays (EGMSA) was performed. EGMSA showed that small Maf proteins form heterodimers with Nrf1 and Nrf2 and recognize the NF-E2 motif.