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

Retinoids are potent inducers of cell cycle arrest and differentiation of numerous cell types, notably granulocytes. How-ever the mechanisms by which retinoids mediate cell cycle arrest during differen-tiation remain unclear. We have used my-eloid differentiation to characterize the molecular pathways that couple cell cycle withdrawal to terminal differentiation. Us-ing primary cells from mice deficient for either the cyclin-dependent kinase inhibi-tor (CDKi) p27Kip1, the Myc antagonist Mad1, or both Mad1 and p27Kip1, we ob-served that signals mediated through reti-noic acid receptor (RAR), but not RAR or , required both Mad1 and p27Kip1 to induce cell cycle arrest and to acceler-ate terminal differentiation of granulo-cytes. Although RAR did not directly regulate Mad1 or p27Kip1, the RAR target gene C/EBP directly regulated transcrip-tion of Mad1. Induction of C/EBP activity in granulocytic cells led to rapid induc-tion of Mad1 protein and transcript, with direct binding of C/EBP to the Mad1 promoter demonstrated through chroma-tin immunoprecipitation assay. These data demonstrate that cell cycle arrest in re-sponse to RAR specifically requires Mad1 and p27Kip1 and that Mad1 is tran-scriptionally activated by CCAAT/ enhancer-binding protein (C/EBP). Moreover, these data demonstrate selec-tivity among the RARs for cell cycle ar-rest pathways and provide a direct mecha-nism to link differentiation induction and regulation of the Myc antagonist Mad1