Growth control of hemaotpoietic stem cells by the collaboration of transcription factors, cell-cycle regulators, and tumor suppressors

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Cell-cycle regulation of hematopoietic stem cells is of interest because understanding of this mechanism appears to facilitate the development of ex-vivo methods to manipulate various stem cells. GATA2 is an indispensable transcription factor regulating proliferation and quiescence of hematopoietic stem cells. Here we show that GATA-2 and cyclin dependent kinase (Cdk)/Cyclin complexes bidirectionally control their expressions. In leukemic and normal immature hematopoietic cells, GATA-2 expression is high in G0/G1 and S/G2 phase, and low in early S and G2/M phase. Immunoprecipitation/immunoblotting analysis demonstrated phosphorylation of GATA-2 at Cdk-consensus motifs, S/T_0P_<+1>, and interaction of GATA2 with Cdk2/Cyclin A2. Cdk2/Cyclin A2, Cdk2/Cyclin El, or Cdk4/Cyclin D1 directly phosphorylated immunoprecipitated GATA-2 in vitro. Treatment with proteasome inhibitor MG132, or Cdk inhibitor Roscovitine modulated cell cycle-dependent oscillation of GATA-2 expression. Mutations in Cdk … More -consensus motifs of GATA-2 exhibited altered expression profiles of green fluorescent protein-GATA2 domain fusion proteins. Therefore, Cdk/Cyclin phosphorylated GATA-2 and regulated its proteasome-dependent degradation. On the other hand, while GATA-2, Cyclin El and Cyclin D2 were expressed in embryonic day 10.5 wild type placenta, CyclinEl and CyclinD2 mRNA expressions were significantly lower in placentas with hypomorphic Gata2 loci. ChIP experiments of synchronized leukemic cells revealed GATA-2 binding to CyclinEl and CyclinD2 gene at late G1 phase, and detachment of GATA-2 in early S phase. Transient GATA-2 binding in late G1 phase was accompanied by RNA polymerase II binding to these genes and concomitant CyclinEl and CyclinD2 mRNA expressions, and was suppressed by 1-hour treatment with Roscovitine. Consequently, GATA2 bound and regulated transcription of CyclinD2 and CyclinEl gene, while Cyclin D2, Cyclin El and other Cyclin, forming complexes with Cdk, phohsphorylated GATA-2 and modulate stability of this factor. This bidirectional regulation between GATA-2 and Cdk/Cyclins might determine the cell-cycle characteristics of GATA-2 expressing hematopoietic cells. Less