PU.1 - c-Jun Interactions Are Crucial for PU.1 Function in Myeloid Development.

Abstract 3651

Poster Board III-587

The Ets transcription factor PU.1 is a master regulator absolutely required for the differentiation of monocytes, macrophages, and B cells in the fetal liver and in the adult bone marrow. PU.1 drives hematopoietic differentiation partly through direct protein-protein interactions with other transcription factors, such as the AP-1 transcription factor c-Jun. We have shown that c-Jun can be recruited to promoters which do not include AP-1 binding sites, such as the MCSFR promoter, and act as a PU.1-dependent co-activator. To address the functional importance of this interaction, we identified and studied PU.1 point mutants that lost the capability to physically interact with c-Jun while retaining normal DNA binding affinity. These mutants failed to efficiently transactivate a PU.1 target reporter, and, more importantly, were unable to induce monocyte/macrophage differentiation of the PU.1-deficient immature myeloid 503 cell line.

Subsequently, we have generated knock-in mouse models harboring these single point mutations by means of homologous recombination. The mutant mice phenotypically resemble PU.1-deficient mice, have an early block in hematopoiesis, and die perinatally. We show that the mutant PU.1 mRNA and protein is expressed in long-term and short-term hematopoietic stem cells, but that the maturation into lymphoid primed multipotent progenitor (LMPP) and later progenitor populations is severely blocked, leading to an almost complete loss of mature B, T and myeloid cells. Collectively, our data strongly suggest that the PU.1-c-Jun interaction is crucial for normal PU.1 function in vivo during murine fetal hematopoiesis.