Abstract

The transcription factor PU.1 is an important regulator of hematopoiesis, directing cell fate decision in a dosage dependent manner with higher levels driving monocytic differentiation and lower levels directing B-cell differentiation. The mechanism by which PU.1 controls this process is not fully understood. Here we show that PU.1 is involved in regulating the expression of several microRNAs. MicroRNAs regulate gene expression at a post-transcriptional level by binding target mRNA through the 3′ UTR and either repressing translation or causing degradation of the mRNA itself. We used microarray expression profiling to assess a large group of microRNAs in PUER cells, a PU.1 −/− cell line that differentiates into macrophages when PU.1 activity is restored. Several miRNAs showed changes in expression four days after restoration of PU.1 activity. We focused on a cluster of microRNAs that includes miR-23a, miR27a, and miR24-2, which we call the miR23a cluster. All three miRNAs are coded for on a single pri-miRNA transcript. Northern blot analysis verified the findings of the microarray–that expression of this microRNA cluster is enhanced by PU.1. The promoter for the cluster contains several conserved predicted binding sites for PU.1. Chromatin immunoprecipitation and EMSA has confirmed that PU.1 binds to these sites. In addition, reporter assays show that the mir23a promoter can be activated by PU.1. The 23a cluster appears to be a critical target gene for PU1 to promote myeloid development. Using in vitro hematopoietic cultures we have observed that expressing this cluster in hematopoietic progenitors promotes myeloid development over B cell development. In addition, data from bone marrow transplant assays will be presented demonstrating the role of this cluster in directing hematopoiesis in vivo. Targetscan and miRanda computer algorithms predict several B cell transcription factors as targets of the miRNAs of the 23a cluster. PU.1 potentially activates expression of the 23a cluster to downregulate B cell transcription factors in order to commit cells to the myeloid cell fate.

Disclosures: No relevant conflicts of interest to declare.

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