Regulation of Hematopoietic Stem and Progenitor Cell Differentiation By Mirn23a/b Micrornas

Differentiation from hematopoietic stem and progenitor cells (HSPCs) to committed blood lineages is dependent on lineage specific transcription factors that simultaneously promote gene expression that commits progenitors to specific lineages while repressing genes associated with alternative lineages. In addition to transcription factors, small non-coding microRNAs (miRNAs) also have the potential to influence cell fate decisions through negative regulation of lineage specific genes. We previously observed that germline knockout of the mirn23a miRNA cluster (which codes for mature miRNAs miR-23a, miR-27a, and miR-24-2) resulted in increased common lymphoid progenitors (CLPs) and B cells with a concomitant decrease in granulocyte/monocyte progenitors (GMPs) and their progeny. This was the first evidence of a miRNA being able to influence a lymphoid/myeloid cell fate decision using a genetic knockout model. To follow up these results, we sought to identify a detailed molecular mechanism of the mirn23a^-/- mouse phenotype. Evaluation of HSPC populations by flow cytometry revealed that while mirn23a^-/- mice have no difference s in their LT-HSC populations, they show imbalanced levels of MPP3 and MPP4 populations, suggesting that bifurcation from the MPP2 to the MPP3/MPP4 is the earliest cell type regulated by mirn23a to influence hematopoietic cell fate decisions. RNA and protein analysis of multipotent EML cell lines generated from wildtype and mirn23a^-/- mice revealed that mirn23a negatively regulated critical HSPC genes Runx1, Satb1, Ikzf1, Mef2c, Bach1, and Bach2 that are involved in committing MPPs to CLPs. Additionally, genes associated with the commitment of CLPs to B cells, EBF1 and Pax5, were also increased. We observed that miR-24-2 target, Trib3, antagonizes PI3K/AKT signaling to promote EBF1 and Pax5 expression through nuclear accumulation of FoxO1. Trib3 also agonizes the BMP/Smad pathway through negative regulation of E3-ubiquitinase Smurf1. Ex vivo OP9 cultures with primary mirn23a^-/- cells cultured with FoxO1 and BMP inhibitors revealed that both the PI3K/Akt and BMP/Smad pathway are critical for mirn23a^-/- phenotypes. Consistent with mirn23a being a critical gene for myeloid commitment of hematopoietic progenitors, we observe that B Cell factor EBF1 represses transcription of mirn23a, creating a regulatory feedback loop between mirn23a and EBF1. In addition to mirn23a, there is a homologous mirn23b miRNA cluster that is expressed at lower levels in hematopoietic cells. To investigate compound loss of mirn23a and mirn23b in adult hematopoiesis, we generated mirn23a^-/-mirn23b^f/f Mx-1 cre mice to circumvent variable embryonic/ neonatal lethality. These mice showed a further increase in B lymphopoiesis and decrease in myelopoiesis compared to mirn23a^-/- mice. Interestingly, these mice also exhibited decreased bone marrow cellularity at 3 weeks post mirn23b excision. As judged by overall numbers and percent of bone marrow, LT-HSC, MPP, and LSK populations were decreased. We are currently investigating the underlying mechanism for the decreased stem cells and overall cellularity. Overall, these results show that mirn23a/b miRNAs bias cell fate decisions at the MPP through negative regulation of critical lymphoid transcription factors. Sustained commitment to the B cell lineage is dependent on both the PI3K/Akt and BMP/Smad signaling pathways, both of which are regulated by mirn23a target Trib3. In turn, EBF1 negatively regulates mirn23a, creating a regulatory feedback loop between EBF1 and mirn23a. Compound loss of mirn23a/mirn23b results in decreased bone marrow cellularity and stem cell loss.