The regulatory mechanism of megakaryopoiesis with transcription factor c-Myb

原癌基因c-myb在造血细胞的生长、存活和分化过程中起着至关重要的作用。我们建立了c-myb基因敲除(KD)小鼠，这是由于c-myb基因上游77kb处插入了trangene，导致巨核细胞-红细胞谱系限制性祖细胞(MEPs)中c-Myb表达降低。这些小鼠表现为血小板增多症、贫血和脾肿大。C-MYB KD小鼠骨髓和脾巨核细胞生成均显著增加。此前，我们在MEPs与OP9细胞的共培养实验中显示，这些异常是可以在体外重现的，但通过逆转录病毒在MEPs中的表达消除了这些异常。通过移植突变的小鼠骨髓细胞，在小鼠体内也重建了这些异常。为了更好地了解伴随c-myb基因表达下降的转录程序，我们进行了MEPs的DNA微阵列分析。我们在我们的c-myb突变小鼠中发现了74个上调的基因和36个下调的基因。在这些基因中，有10个基因的表达水平实际上在c-myb突变小鼠的骨髓细胞中发生了变化，并且在上游区域含有c-Myb识别元件。C-myb基因突变小鼠骨髓细胞CD9和Ly6a的表达水平升高，并通过染色质免疫印迹法检测其与c-Myb的相互作用。CD9和Ly6a激动型抗体与CD9激动型抗体在数量和大小上均能刺激巨核细胞集落形成。这些结果表明，c-myb基因表达降低是巨核细胞生成的刺激因子，CD9和Ly6a都是c-myb靶基因的候选基因。阐明基于c-myb基因的转录网络对巨核细胞的作用将采取新一轮的谱系特异性调控。

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We established c-myb knock down (KD) mice due to the trangene insertion into 77-kb upstream of the c-myb gene which yielded the reduction of c-Myb expression in megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs). These mice exhibited thrombocythemia, anemia, and splenomegaly. Megakaryopoiesis of c-myb KD mice were extremely increased in both bone marrow and spleen. Previously we showed that these abnormalities were reproducible in vitro in a co-culture assay of MEPs with OP9 cells, but eliminated by the retroviral expression of c-Myb in MEPs. These abnormalities were also reconstituted in mice in vivo through the transplantation of mutant mouse bone marrow cells. To better understand the transcriptional program that accompanies the decline of c-myb gene expression, we performed DNA microarray analysis of MEPs. We identified 74 genes that are upregulated and 36 genes are downregulated in our c-myb mutant mice. Of these genes, expression levels 10 genes are actually changed in bone marrow cells of c-myb mutant mice, and harbor c-Myb recognition elements in the upstream region. The expression levels of CD9 and Ly6a were increased in bone marrow cells of c-myb mutant mice and we revealed their interaction with c-Myb using chromatin immunopretipitaion assay. Agonistic antibodies of CD9 and Ly6a stimulated megakaryocytic colony formation in number and especially in size with the agonistic CD9 antibody. These results thus demonstrate that the reduced expression of c-myb gene affects as the stimulator of megakaryopoiesis and both CD9 and Ly6a were candidates of c-Myb target gene. Elucidation of transcription network based on the c-myb gene on megakaryopoiesis will take a new turn of lineage specific regulation.