The regulatory mechanism of megakaryopoiesis with transcription factor c-Myb

核原癌基因c-myb在造血细胞的生长、存活和分化中起着至关重要的作用。我们建立了c-myb基因敲低（KD）小鼠，由于转基因插入到77 kb的c-myb基因上游，产生的巨核细胞-红细胞谱系限制性祖细胞（MEP）的c-Myb表达减少。这些小鼠表现出血小板增多症、贫血和脾肿大。c-myb KD小鼠骨髓和脾脏巨核细胞生成均显著增加。以前，我们表明，这些异常是可重复的，在体外与OP 9细胞的MEP的共培养试验，但消除了逆转录病毒表达的c-Myb在MEP。这些异常也通过移植突变小鼠骨髓细胞在小鼠体内重建。为了更好地理解伴随c-myb基因表达下降的转录程序，我们对MEP进行了DNA微阵列分析。我们在c-myb突变小鼠中鉴定了74个上调基因和36个下调基因。在这些基因中，10个基因的表达水平在c-myb突变小鼠的骨髓细胞中实际上发生了变化，并且在上游区域具有c-Myb识别元件。c-myb突变小鼠骨髓细胞中CD 9和Ly 6a的表达水平增加，我们用染色质免疫沉淀法揭示了它们与c-Myb的相互作用。CD 9和Ly 6a的激动性抗体刺激巨核细胞集落形成的数量，特别是在大小与激动性CD 9抗体。这些结果表明，c-myb基因的表达减少作为巨核细胞生成的刺激因子而起作用，并且CD 9和Ly 6a都是c-Myb靶基因的候选者。基于c-myb基因的巨核细胞发生转录调控网络的阐明，将为研究巨核细胞发生的谱系特异性调控提供新的思路。

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.