c-Myb Regulates CD9 Gene Expression during Megakaryopoiesis.

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We previously generated through insertion mutagenesis a c-myb gene knockdown (KD) line of mice. In the mice transgene was inserted 77-kb upstream of the c-myb gene and c-Myb expression was markedly decreased in megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs) of the homozygous knockdown mutant mice (c-myb KD mice). The c-myb KD mice exhibited anemia, thrombocythemia, and splenomegaly and these abnormalities were reproducible in a co-culture assay of MEPs with OP9 cells, but abrogated by the retroviral expression of c-Myb in MEPs. To understand the transcriptional program that accompanies the decline of c-myb gene expression, we performed DNA microarray analysis with MEPs and identified 74 genes that are upregulated and 36 genes that are downregulated in the c-myb KD mice. Of these genes, expression levels 15 genes are actually changed significantly in bone marrow cells of the c-myb KD mice. These genes harbor c-Myb recognition elements in their regulatory regions. Especially, we found that the CD9 expression was upregulated in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified using a luciferase reporter assay and chromatin immunoprecipitation assay. Agonistic antibody of CD9 stimulated megakaryocytic colony formation. On the contrary, upon the bone marrow suppression with 5-fluorouracil recovery of platelet number was delayed in the CD9-null mice. Furthermore, proplatelet formation was impaired when we used CD9-null mouse megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. These results thus demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryopoiesis CD9 is derepressed and acts to induce the megakaryopoiesis. Elucidation of c-myb-based transcription network seems to be of important to understand the megakaryocytic differentiation.