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

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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.