The Oncogenic Mir-17-92 MicroRNA Cluster Is Inhibited by EGR2 During Macrophage Differentiation Via JARID1b-Mediated Histone 3 Lysine 4 Demethylation

Abstract 390

The miR-17-92 cluster (Oncomir1) encodes seven related microRNAs associated with cell proliferation, apoptosis and development and is overexpressed in number of malignancies including myeloid leukemias. The miR-17-92 cluster is highly expressed in myeloid progenitors, while it becomes downregulated upon the onset of macrophage differentiation. Conversely, sustained expression of miR-17-92 is associated with a differentiation blockade (Fontana 2007). Here we report a novel mechanism of the regulation of miR-17-92 cluster within differentiating myeloid progenitors. During macrophage differentiation, the myeloid transcription factor PU.1 transcriptionally induces the secondary determinant Early growth factor 2 (Egr2). Subsequently, Egr2 binds to the miR-17-92 cluster promoter and recruits histone demethylase Jarid1b resulting in histone H3 lysine K4 (H3K4) demethylation within the CpG island upstream miR-17-92 cluster leading to the repression of miR-17-92 expression. Reporter assays using deletion constructs of the miR-17-92 promoter region revealed that Egr2 is required for targeting the Jarid1b onto critical minimal region within CpG island of upstream miR-17-92 locus and its decreased H3K4 methylation. In addition, functional assays identified the 3'UTR of Egr2 as the target of multiple miRNAs of the miR-17-92 cluster, indicating existence of a mutual regulation between miR-17-92 cluster and Egr2, putatively involved in macrophage differentiation characterized by a bistable state, where Egr2 negatively regulates miR-17-92 cluster in differentiating cells and, in turn, miR-17-92 cluster negatively regulates Egr2 in highly proliferating progenitor cells to achieve homeostatic regulation. Ectopic expression of miR-17-92 cluster within myeloid progenitors blocked macrophage differentiation indicating that leukemogenesis may involve miR-17-92 mediated differentiation blockade. To determine if the newly identified negative regulatory mechanism of miR-17-92 is involved in leukemogenesis, we tested peripheral blood mononuclear cells isolated from acute myeloid leukemia (AML) patients (N=27). 14 of 27 AML patients exhibited significantly downregulated transcriptional factors PU.1 and Egr2 and high levels of the miR-17-92 cluster expression. Ectopic expression of Egr2 within AML blast cells (N=2) led to decreased levels of miR-17-92 and restored the expression of p21 and BIM, two established miR-17-92 targets downregulated in AML. We conclude that PU.1 represses miR-17-92 cluster during macrophage differentiation by Egr2 mediated recruitment of the Jarid1b demethylase and that dysregulation in the miR-17-92 repression mechanism may contribute to the pathogenesis of AML. (Grants # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507).