PU.1 immortalizes hematopoietic progenitors in a GM-CSF-dependent manner.
OBJECTIVE
The Ets family transcription factor PU.1 is essential for both myeloid and lymphoid development. PU.1 was discovered because of its involvement in murine erythroleukemia. We previously described that infection with a retroviral vector encoding PU.1 immortalizes fetal liver progenitor cells in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. In this study, we sought to characterize PU.1-immortalized progenitor (PIP) cells.


METHODS
PIP cells were characterized using microscopy, reverse-transcriptase polymerase chain reaction analysis, and flow cytometric analysis. In addition, progenitors were immortalized with a retrovirus containing a PU.1 cDNA flanked by loxP sites. The differentiation potential of immortalized progenitors was tested by Cre-mediated excision of the proviral PU.1 cDNA.


RESULTS
PIP cells are blastlike in morphology and express cell surface markers indicative of myeloid development. Immortalization of progenitor cells requires both an acidic activation domain and an intact DNA-binding domain of PU.1. Gene expression analysis of PIP cells demonstrated the expression of genes of both myeloid and erythroid lineages. Proliferation of PIP cells was GM-CSF dependent and restricted. Upon Cre-mediated excision of proviral PU.1 cDNA, increased expression of myeloid and erythroid-specific genes was observed; as well as the appearance of both macrophages and erythrocytes in culture.


CONCLUSIONS
We demonstrate that ectopic expression of PU.1 is sufficient to immortalize a hematopoietic progenitor with myeloid and erythroid differentiation potential in response to GM-CSF. These data highlight the importance of the level of PU.1 expression at critical stages of hematopoiesis.
Houston
Isaac B
IB
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA.
Huang
Kelly J
KJ
Jennings
Serena R
SR
DeKoter
Rodney P
RP
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D008099:Liver / Q000166:cytology; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics; D000099232:Proto-Oncogene Protein Spi-1
D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011518:Proto-Oncogene Proteins; D013629:Tamoxifen; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1