Abstract
The lineage commitment of multipotent progenitor cells is coordinated by regulatory interactions between transcription factor programs associated with different lineage fates. Accordingly, mutations which interfere with these programs can block differentiation and contribute to leukemogenesis. A thorough understanding of lineage commitment processes should therefore identify targets and strategies for differentiation therapy. We have compared the effects of transient over-expression of the hematopoietic transcription factors PU.1, GATA-1 and SCL on the response of a multipotent hematopoietic progenitor cell line (FDCPmix) to conditions supporting either self-renewal, myeloid differentiation or erythroid differentiation.
Methods: Pure populations of FDCPmix cells productively transfected with transcription factor cDNA/IRES/GFP expression vectors were isolated by FACS sorting and entered directly into liquid media containing either IL-3 (for self-renewal); GM-CSF + G-CSF (for myeloid differentiation) or erythropoietin + hemin (for erythroid differentiation). Semi-solid medium containing IL-3 was used to assess the maintenance of that sub-population of cells which can undergo self-renewal in isolation, and which are assumed to represent the earliest progenitors in an FDCPmix culture. Transcription factor function was confirmed by co-transfection with reporter genes driven by specific target promoters.
Results: The yield of FDCPmix cells from self-renewal medium was markedly enhanced by the transient expression of SCL but reduced by either PU.1 or GATA-1. These differences in yield reflect alterations in survival and/or proliferation over the two day period of transgene expression immediately following transfection. As expected, the transient expression of PU.1 severely reduced the potential of multipotent progenitors to respond to erythroid growth factors, while GATA-1 similarly reduced the response to myeloid growth factors, consistent with an antagonistic relationship between myeloid and erythroid transcription factors during lineage restriction. However, while all three transcription factors demonstrated the appropriate activity on their respective target promoters, PU.1 and GATA-1 also increased general transcriptional activity. Transient expression of GATA-1 actually raised the activity of the cfms (PU.1 target) promoter, while PU.1 activated the serpin 2A (GATA-1 target) promoter. Of the transcription factors tested, only PU.1 consistently reduced the frequency of colony forming cells, suggesting that the earliest multipotent progenitors in an FDCPmix population can be recruited to commitment by PU.1 but not by GATA-1.
Conclusion: These results are consistent with a multi-step process of lineage commitment in which general transcriptional activity in multipotent progenitors is maintained at a low level, base state. An increase in general transcriptional activity would then be required as an early event in commitment, activating the transcription factor networks subsequently responsible for coordinating lineage restriction.
TRIB3 promotes MYC-associated lymphoma development through suppression of UBE3B-mediated MYC degradation
