PU.1 Promotes Monocyte Development in Part By Transcriptionally Activating c-Fos, Egr-1 and Egr-2

PU.1 is an Ets family transcription factor with multiple roles in hematopoiesis. In addition to its critical involvement in regulating the function of hematopoietic stem cells, PU.1 has been shown to regulate myeloid and lymphoid development in a graded manner with high levels of PU.1 favoring monocyte development and low expression levels inducing B cell fate. The molecular mechanism by which PU.1 promotes monocyte development remains incompletely understood. We show here that in murine myeloid 32D and PUER cells, and lineage negative (Lin^-) bone marrow (BM) cells expressing PUER protein in which PU.1 was fused to the estrogen receptor ligand binding domains, treatment with 4-hydroxytamoxifen (4-HT), which activated PU.1, augmented the expression of c-Fos, Egr-1 and Egr-2. In contrast, PU.1 knockdown using shRNAs reduced c-Fos, Egr-1 and Egr-2 expression. c-Fos, Egr-1 and Egr-2 are the immediate early response genes of the Erk1/2 pathway and have been shown to promote monocyte development. A PU.1 mutant defective in c-Jun interaction failed to augment c-Fos, Egr-1 and Egr-2 expression, indicating that PU.1-mediated upregulation of c-Fos, Egr-1 and Egr-2 was dependent on its interaction with c-Jun. Using ChIP and luciferase reporter assays, we further demonstrate that PU.1 transcriptionally activated c-Fos, Egr-1 and Egr-2 through directly binding to their promoters. Activation of PU.1 with 4-HT in 32D, PUER and Lin^- BM cells induced monocyte development. Notably, shRNA-mediated knockdown of c-Fos or Egr-1 resulted in a shift in the development from monocytes to neutrophils in response to PU.1 activation. Knockdown of c-Fos and Egr-1 also affected the expression of other myeloid-specific transcription factors, including C/EBPα and Gfi1. PU.1-induced upregulation of c-Fos, Egr-1 and Egr-2 in PUER cells was abolished upon inhibition of Erk1/2 signaling with the Mek1/2 inhibitors U0126 or PD032901. Interestingly, treatment of PUER cells with either Mek1/2 inhibitor redirected PUER cells to develop along the neutrophil lineage at the expense of monocyte fate in response to PU.1 activation. Together, these results reveal an important mechanism by which PU.1 promotes monocyte development, i.e., through transcriptional activation of c-Fos, Egr-1, and Egr-2.