Cyclin D: CDK4/6 Kinase Activity, Regulated by GATA-1, Is Required for Megakaryocyte Polyploidization.

Megakaryocytes, which fragment to give rise to platelets, undergo a unique form of cell cycle, termed endomitosis, to become polyploid and terminally differentiate. During this process, cells transverse the cell cycle but the late stages of mitosis are bypassed to lead to accumulation of DNA up to 128N. While the mechanisms of polyploidization in megakaryocytes are poorly understood, a few cell cycle regulators, such as cyclin D3, have been implicated in this process. Hematopoietic transcription factors, including GATA-1 and RUNX1 are also essential for polyploidization, as both GATA1-deficient and RUNX1-null megakaryocytes undergo fewer rounds of endomitosis. Interestingly, GATA-1 deficient megakaryocytes are also smaller than their wild-type counterparts. However, the link between transcription factors and the growth and polyploidization of megakaryocytes has not been established. In our studies to identify key downstream targets of GATA-1 in the megakaryocyte lineage, we discovered that the cell cycle regulators cyclin D1 and p16 were aberrantly expressed in the absence of GATA-1: cyclin D1 expression was reduced nearly 10-fold, while that of p16^ink4a was increased 10-fold. Luciferase reporter assays revealed that GATA-1, but not the leukemic isoform GATA-1s, promotes cyclinD1 expression. Consistent with these observations, megakaryocytes that express GATA-1s in place of full-length GATA-1 are smaller than their wild-type counterparts. Chromatin immunoprecipitation studies revealed that GATA-1 is bound to the cyclin D1 promoter in vivo, in primary fetal liver derived megakaryocytes. In contrast, GATA-1 is not associated with the cyclin D1 promoter in erythroid cells, which do not become polyploid. Thus, cyclin D1 is a bona fide GATA-1 target gene in megakaryocytes. To investigate whether restoration of cyclin D1 expression could rescue the polyploidization defect in GATA-1 deficient cells, we infected fetal liver progenitors isolated from GATA-1 knock-down mice with retroviruses harboring the cyclin D1 cDNA (and GFP via an IRES element) or GFP alone. Surprisingly, expression of cyclin D1 did not increase the extent of polyploidization of the GATA-1 deficient megakaryocytes. However, co-overexpression of cyclin D1 and Cdk4 resulted in a dramatic increase in polyploidization. Consistent with the model that cyclinD:Cdk4/6 also regulates cellular metabolism, we observed that the size of the doubly infected cells was also significantly increased. Finally, in support of our model that cyclin D:Cdk4/6 kinase activity is essential for endomitosis, we discovered that introduction of wild-type p16 TAT fusion protein, but not a mutant that fails to interact with Cdk4/6, significantly blocked polyploidization of primary fetal liver derived megakaryocytes. Taken together, our data reveal that the process of endomitosis and cell growth relies heavily on cyclinD:Cdk4/6 kinase activity and that the maturation defects in GATA-1 deficient megakaryocytes are due, in part, to reduced Cyclin D1 and increase p16 expression.