Transcription Factors PU.1 and EGR2 Inhibits the Oncogenic Microrna Cluster Mir-17-92 during Macrophage Differentiation

PU.1 (Sfpi1) is an ets family transcription factor required for the proper generation of both myeloid and lymphoid lineages. Distinct nuclear levels of PU.1 are required for macrophage differentiation (DeKoter 2000). MicroRNAs are a novel class of 22nt long non-coding post-transcriptional regulators, associated with differentiation, development and carcinogenesis. To identify miRNAs involved in PU.1 dependent hematopoietic differentiation we used PU.1-null hematopoietic progenitor cells stably expressing PU.1 fused to the estrogen receptor ligand-binding domain inducible by tamoxifen (PUER) (Walsh 2002). PU.1 affected expression approximately 100 mature miRNA. Among them, we have observed that PU.1 downregulates members of miR-17~92 cluster and its paralog miR-106b~25 previously associated with macrophage differentiation, apoptosis and hematologic and other malignancies (Ota 2004, Hayashita 2005, Fontana 2008). During macrophage differentiation miR-17~92 becomes downregulated and its sustained expression is associated with the differentiation block (Fontana 2007). In tamoxifen-stimulated PUER cells, PU.1 efficiently downregulates mature miRNAs of the miR-17~92 (-17.5p, -20a, -92) cluster and its paralog miR-106b~25 (but not miR-106a~363) within 96 hours and in dose dependent manner. PU.1 in the same cells activates expression of Egr2, myeloid transcription factor promoting macrophage transcriptional program a putative 8-mer-seed sequence-containing target of miR-17~92 cluster (predicted by TargetScan). We tested whether PU.1 represses expression of miR-17~92 cluster by inducing Egr2. We demonstrate that ectopic expression of Egr2 in unstimulated PUER cells inhibited expression of miR-17~92 cluster, both at the levels of mature miRs and pri-miRNAs, and induced expression of macrophage differentiation markers (Cd14, Csf1r) coupled with cell cycle arrest. Trichostatin-dependent derepression of miR-17~92 indicated that its expression is regulated at the chromatin level. Chromatin immunoprecipitation revealed specific Egr2 occupancy within the regulatory region upstream of the miR-17~92 cluster. Our data indicate that in differentiating cells, a mechanism exists involving direct repression of the miR-17~92 cluster that in turn has the potential to inhibit Egr2 expression. We have tested this mechanisms in peripheral blood mononuclear cells isolated from acute myeloid leukemia (AML) patients (N=9) where levels of PU.1 and Egr2 are significantly lower that in normal controls. 5 out of 9 AML patients significantly overexpressed miR- 17.5p, miR-20a, miR-92 coinciding with downregulation of its established targets p21 and BIM. Rescue of Egr2 in AML cells (N=2) led to downregulation of miR-17~92 cluster and upregulation of BIM mRNA levels coincident with induction of apoptosis. We conclude that PU.1 regulates transcription of the miR-17~92 cluster by inducing Egr2 and demonstrate this mechanism in differentiating macrophages and its disruption in AML cells.