Mirn23a Inhibits B Lymphopoiesis through Antagonism of the B Cell Transcription Factor Network

Transcription factors (TFs) regulate genetic networks to direct the differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature blood lineages. Historically studies have focused on identifying genes activated by TFs that act as master hematopoietic regulators. Recently, it has become appreciated that these regulatory TFs direct one distinct differentiation program while simultaneously directing the repression of genes associated with alternative blood lineages. MicroRNAs (miRNAs) are short (~22nt) non-coding RNA molecules that negatively regulate gene expression at the post-transcriptional level and are hypothesized to act downstream of lineage specific TFs to contribute to the silencing of genes associated with alternative cell fate adoption. MiRNAs are observed to direct cell fate choice in assays utilizing overexpression and knockdown approaches; however, experiments from mouse gene targeting have yet to confirm these results. Specifically no miRNA has been shown to direct the differentiation of multipotent progenitors (MPPs) to granulocyte monocyte progenitors (GMPs) or common lymphoid progenitors (CLPs). We previously observed that overexpression of the mirn23a gene (Codes for three mature miRNAs: miR-23a, miR-24-2, and miR-27a) in hematopoietic progenitors increased myelopoiesis at the expense of B lymphopoiesis both in vitro and in vivo. To follow up this observation we generated a mirn23a germline knockout mouse. Flow cytometry characterization of hematopoiesis in mirn23a^-/- mice reveals a significant increase in B220⁺ B-lymphocytes in both the bone marrow (BM) and the spleen. Furthermore, this increase in B-lymphocytes was shown to be at the expense of myeloid cells, as CD11b⁺ cells were significantly decreased in both the BM and spleen. Analysis of the BM progenitor populations revealed a significant increase in the CLP population, due to an increase in the B-cell specific lymphoid progenitor (BLP, CLP⁺ Ly6D⁺) CLP subset. Consistent with decreased mature myeloid cells, we also observed a decrease in BM GMPs. The phenotype appears to be cell intrinsic as ex vivo culture of mirn23a^-/- progenitor cells on OP9 stroma results in a significant increase in B220⁺ B-cells, and a decrease in CD11b+ myeloid cells compared to wild type cultures. Consistent with mirn23a promoting myeloid development, we observe that expression of mirn23a in 70z/3 pre-B cells increases myeloid specific gene expression programs. The upregulation of the myeloid genes may be due to the insufficient repression by B cell transcription factors Ebf1 and Pax5, which are downregulated in mirn23a expressing cells. Interestingly Ebf1 associates with the mirn23a promoter as shown by chromatin immunoprecipitation (ChIP) assay, and Ebf1 represses mirn23a promoter activity in luciferase reporter assays. Consistent with a role in repressing mirn23a, Ebf1 knockdown in A20 B Lymphoma cells increases miR-23a expression while overexpression of Ebf1 in NIH/3t3 cells decreases miR-23a expression. Previously we observed that the myeloid transcription factor PU.1 binds to the mirn23a promoter to positively regulate transcription. Additionally we observe that myeloid transcription factor C/EBP alpha associates with the mirn23a locus in ChIP assays. Data from our knockout mouse supports a critical role for mirn23a in determining immune cells fates. Our current results support the conclusion that myeloid transcription factors PU.1 and C/EBP alpha activate mirn23a in order to antagonize B cell gene regulation and promote myeloid cell fate adoption, whereas the essential B cell transcription factor Ebf1 represses mirn23a expression to avoid this repression during B cell specification.