Analysis of hematopoietic lineage plasticity by using inducible transcription factors

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In order to investigate the molecular mechanism of differentiation and possible lineage switch or trans-differentiation in various hematopoietic lineages, we generated inducible form of myeloid transcription factors C/EBP α and PU.1 and examined their effect in vitro or in vivo. Full length C/EBP a and PU.1 were fused in frame with ligand binding domain of estrogen receptor (C/EBP α -ER and PU.1-ER) so that they can be activated by 4-hydroxy tamoxifen (4-HT). We subcloned. C/EBP α -ER and PU.1-ER into retrovirus vector, pMX-IRES-GFP and infected virus to BaF3 cells. Interestingly, 4-HT treatment of BaF3/CEBP α -ER, BaF3/PU.1l-ER cells induced growth arrest and apoptosis, indicating that C/EBP α -ER and PU.1-ER are working properly. Next we subcloned Cl EBP α -ER and PU.1-ER in H-2K promoter vector made transgenic mice. We obtained transgene integration in 8 lines for C/ EBP a -ER and 5 lines for PU.1-ER. RT-PCR analysis showed that mRNA is expressed in 5 out of 8 C/EBP α -ER transgenics and only one line expressed C/EBP α -ER protein by western blot. C/EBP α -ER was expressed highly in thymus and spleen, moderately in bone marrow and peripheral blood. Gel-shift assay showed that C/ EBP α -ER protein bound to conserved C/EBP binding sequence in response to 4-HT. With these mice in our hand, we are now planning to investigate how ectopically induced C/ EBP α activity affects differentiation of hematopoietic cells at various developmental stages.