Identification of the molecular requirements for an RARα-mediated cell cycle arrest during granulocytic differentiation

Retinoids are potent inducers of cell cycle arrest and differentiation of numerous cell types, notably granulocytes. However the mechanisms by which retinoids mediate cell cycle arrest during differentiation remain unclear. We have used myeloid differentiation to characterize the molecular pathways that couple cell cycle withdrawal to terminal differentiation. Using primary cells from mice deficient for either the cyclin-dependent kinase inhibitor (CDKi) p27^Klp1, the Myc antagonist Mad1, or both Mad1 and p27 ^Kip1, we observed that signals mediated through retinoic acid receptor α (RARα), but not RARβ or γ, required both Mad1 and p27^Kip1 to induce cell cycle arrest and to accelerate terminal differentiation of granulocytes. Although RARα did not directly regulate Madl or p27^Kip1, the RARα target gene C/EBPε directly regulated transcription of Mad1. Induction of C/EBPε activity in granulocytic cells led to rapid induction of Mad1 protein and transcript, with direct binding of C/EBPε to the Mad1 promoter demonstrated through chromatin immunoprecipitation assay. These data demonstrate that cell cycle arrest in response to RARα specifically requires Mad1 and p27 ^Kip1 and that Mad1 is transcriptionally activated by CCAAT/enhancer-binding protein ε (C/EBPε). Moreover, these data demonstrate selectivity among the RARs for cell cycle arrest pathways and provide a direct mechanism to link differentiation induction and regulation of the Myc antagonist Mad1.