A Screen In Primary Erythroblasts Reveals a MicroRNA-Centered Homeostatic Mechanism for Regulating Cyclin E Activity.

Abstract 1543

A growing body of evidence highlights the importance of microRNAs in regulating the expression of mediators of cell cycle progression. A theme emerging from these studies is that microRNAs participate in feedback or feed-forward circuits to provide bistability for key transition points in the cell cycle. We previously have shown that proper regulation of cyclin E activity is required for normal erythroid cell maturation in vivo, using cyclin E^T74AüT393A knock-in mice, which have markedly dysregulated cyclin E due to its failure to interact with the Fbw7 ubiquitin ligase complex. We hypothesized that we could identify novel, microRNA-based molecular circuitry for maintaining appropriate levels of cyclin E activity by screening cyclin E knock-in erythroblasts for alterations in microRNA expression. We analyzed data we obtained from multiplex real-time PCR arrays comparing the expression of over 500 microRNAs in cyclin E^{T74A T393A} knock-in versus wild-type erythroblasts (Ter119+/CD71+) and found down-regulated expression of a number of microRNAs targeting CDK inhibitors. We also identified down-regulated expression of potential microRNA regulators of Fbw7 expression. We found that overexpression of miR-223, in particular, significantly reduces Fbw7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the Fbw7 3’ untranslated region. We then found that reduced miR-223 expression leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression in K562 cells is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway and that dysregulated Fbw7 expression alters the erythroid differentiation capacity of these cells. Mir-223 plays an important role in myeloid and erythroid differentiation by regulating multiple substrates involved in these maturation programs. Here, we identify Fbw7 as a novel target of miR-223. Our data also indicate that miR-223 modulates Fbw7 expression as part of a homeostatic mechanism to regulate cyclin E activity and provide the first evidence that activity of the SCF^Fbw7 ubiquitin ligase can be controlled by the microRNA pathway.