The identification of a class of noncoding RNAs of small size (micro-RNAs) that control the translation and stability of specific target messenger RNAs (mRNA) has set the stage for the discovery of new regulatory territories of hematopoiesis. In this paper, a group of Italian investigators describe an elegant series of experiments leading to the detection of a circuit involving a miRNA (i.e., miR-223) with a critical role in granulopoiesis.
miR-223 had previously been shown to be expressed in murine hematopoietic tissues. The investigators show that mRNA of miR-223 is preferentially expressed in neutrophilic cells in marrow and blood, as well as in the blasts from patients with acute promyelocytic leukemia (APL). miR-223 was strongly induced in vitro in APL cells following exposure to retinoic acid (RA). miR-223 also increased in vivo in patients undergoing treatment with RA. Using a variety of cell lines, Fazi et al. sorted out that upregulation of miR-223 was critically dependent on RA responsiveness. Only the RA sensitive cell lines, not the RA resistant variants, showed increases of miR-223 expression. C/EBPα is an established key transcription factor in granulopoiesis, and it is rapidly induced by RA in APL cells (NB4 cells). The investigators identified two putative C/EBPα binding elements in the promoter of miR-223. This suggested that the C/EBPα sites might be involved in induction of miR-223 by RA. Using various reporter constructs, the investigators demonstrated that the increase of miR-223 in response to RA was absolutely dependent on the C/EBPα binding sites. In subsequent chromatin immunoprecipitation experiments, they further showed that C/EBPα is also physically recruited to the miR-223 promotor upon RA treatment. NFI-A is another protein that may bind to C/EBPα binding sites. A NFI-A site was noted in the miR-223 promoter. The physical association of NFI-A with the miR-223 promoter (chromatin IP) was demonstrated, but in this instance in undifferentiated cells, i.e., in the absence of RA. Following treatment with RA, a progressive dissociation of NFI-A from the promoter region was seen. The dissociation in response to RA was opposite to the enhanced association of C/EBPα. These findings clarified the interrelations between the three molecules, MiR-223, C/EBPα, and NFI-A. Apparently, RA activation results in the physical replacement of NFI-A by C/EBPα at the miR-223 promoter. miR-223 was stably expressed in NB4 cells and elicited cellular alterations (granulocytic surface makers, G-CSF- transcripts, granulocytic morphology) in APL cells that indicated enhanced granulocytic cell development. Conversely, knockdown of miR-223 blocked the granulocytic differentiation response to RA entirely. RNA-interference (RNAi) against C/EBPα (siRNA) in APL cells suppressed miR-223 in spite of RA treatment. On the other hand, knockdown of NFI-A enhanced miR-223. These observations demonstrate the impact of high levels of C/EBPα and suppression of NFI-A for activation of miR-223 and subsequent granulocytic differentiation.
Altogether these findings establish an important novel circuitry of regulation of two well-known transcription factors, C/EBPα and NFI-A, and a recently discovered micro-RNA in granulopoiesis (see Figure). It elucidates some of the molecular mechanisms of the therapeutic response in APL to RA. RA first induces the transcription factor CEBPα that will physically displace NFI-A from the promoter of miR-223. The displacement of the NFI-A block by C/EBPα and the binding of C/EBPα to the promoter activates miR223, which will induce cellular granulocytic differentiation.
