Expression of Core-Binding Factor-β Is Regulated By Mir-143 and Mir-223 during Granulopoiesis

Granulopoiesis is a strictly regulated process governed to a large extent by an ordered temporal expression of transcription factors. One of the earliest expressed transcription factors, obligatory for correct granulopoiesis, is the Runx1:CBF-beta heterodimer that induces cell division and transcription of genes encoding azurophile granule proteins. The expression level of Runx1:CBF-beta peaks in myeloblasts (MBs) and promyelocytes (PMs) and declines when cells enter the myelocyte (MC) stage, where cell proliferation ceases and terminal differentiation commences. Runx1 binds DNA directly and provides the transcriptional activation domain whereas CBF-beta binds to Runx1 and increases its DNA-binding affinity. CBF-beta is in most cases required for Runx1-mediated transcriptional activity.

microRNAs (miRNAs) post-transcriptionally regulate protein expression and are important for proper granulopoiesis. Using a combined density gradient and immunomagnetic purification protocol, we have isolated neutrophil precursors from human bone marrow and identified 135 differentially regulated miRNAs. Two of these, miR-143 and miR-223, were predicted to target CBFB mRNA that encodes the CBF-beta subunit. Both miRNAs are expressed at low levels in MBs and PMs and increases 5-fold when the cells mature to MCs and remain high in the following stages of neutrophil differentiation. This expression pattern supports a role for miR-143 and miR-223 as translational inhibitors of CBF-beta that diminish the transcriptional capacity of Runx1 and thus aid in terminating cell proliferation in MCs.

Transient transfection of HEK293 cells, that express CBF-beta endogenously, with pre-miR-143 and pre-miR-223 demonstrated a strong suppression of CBF-beta synthesis when both miRNAs were present. A luciferase reporter assay where the 3’-UTR of CBFB mRNA is inserted in the 3’-UTR of the firefly luciferase transcript confirmed that both miRNAs affect translation. Mutation of either the miR-143 or miR-223 miRNA recognition element (MRE) partly relieved repression of the luciferase mRNA and mutation of both MREs completely abolished translational repression. shRNA against CBFB mRNA was used to detect transcriptional targets affected by downregulation of CBF-beta in a murine promyelocyte cell line (MPRO) and in the myeloblastic cell line 32Dcl3 and expression of a number of genes encoding azurophilic granule proteins were found to be significantly diminished.

Together these data demonstrate that miR-143 and miR-223 affect CBF-beta expression and indicate that correct timing of expression of these two miRNAs is a prerequisite for proper granulopoiesis.

Grant acknowledgments: The Danish Medical Research Council, Novo Nordisk Foundation, Brøchner Mortensen Foundation, and the Danish Cancer Society.

MTL and AB contributed equally to the work