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

造血干细胞的细胞周期调控是人们感兴趣的，因为对这一机制的理解似乎有助于开发出离体方法来操纵各种干细胞。GATA2是调节造血干细胞增殖和静止不可缺少的转录因子。我们发现GATA-2和细胞周期蛋白依赖性激酶(Cdk)/细胞周期蛋白复合物双向控制它们的表达。在白血病和正常未成熟造血细胞中，GATA-2在G0/G1和S/G2期高表达，在S和G2/M期早期低表达。免疫沉淀/免疫印迹分析显示GATA-2在Cdk-consensus基序上磷酸化，S/T_0P_<+1>，并且GATA-2与Cdk2/Cyclin A2相互作用。Cdk2/Cyclin A2、Cdk2/Cyclin El或Cdk4/Cyclin D1直接磷酸化免疫沉淀的GATA-2。用蛋白酶体抑制剂MG132或Cdk抑制剂罗斯科维汀处理可调节细胞周期依赖性的GATA-2表达振荡。GATA-2的更多共识基序显示绿色荧光蛋白- GATA-2结构域融合蛋白的表达谱发生改变。因此，Cdk/Cyclin磷酸化GATA-2并调节其蛋白酶体依赖性降解。另一方面，虽然GATA-2、CyclinEl和CyclinD2在胚胎10.5 d野生型胎盘中表达，但在GATA-2基因座的胎盘中，CyclinEl和CyclinD2 mRNA的表达量显著降低。同步白血病细胞的ChIP实验显示，GATA-2在G1期晚期与CyclinEl和CyclinD2基因结合，在S期早期脱离。GATA-2在G1期晚期的短暂结合伴随着RNA聚合酶II与这些基因的结合，并伴随CyclinEl和CyclinD2 mRNA的表达，并且被罗斯科维汀1小时抑制。因此，GATA2结合并调节CyclinD2和CyclinEl基因的转录，而CyclinD2、CyclinEl和其他Cyclin与Cdk形成复合物，使GATA-2磷酸化并调节该因子的稳定性。这种GATA-2与Cdk/Cyclins之间的双向调控可能决定了表达GATA-2的造血细胞的细胞周期特性。少

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