P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1

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Abstract
The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34+ cells, we observed that p19INK4D expression was increased both at the mRNA and protein levels during ploidization. p19INK4D knockdown led to a moderate increase (31.7% ± 5%) in the mean ploidy of MKs suggesting a role of p19INK4D in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41highCD42high) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19INK4D overexpression in CD34+ cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19INK4D KO mice exhibited an increase in mean ploidy level from 18.7N (± 0.58N) to 52.7N (± 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19INK4D promoter. Moreover, AML-1 inhibition led to the p19INK4D down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.