Myb Family Transcription Factors Contribute to G2/M Cell Cycle Transition in Normal and Malignant Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression.

Myb family transcription factors are ubiquitously expressed, and are known to play a critical role in regulating G1/S cell cycle transition. Recently, Myb-like proteins have been found to regulate G2/M transit in plants, yeast, and Drosophila. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, but not c-Myb, regulated cyclin B1 expression directly. However, in hematopoietic cells Myb’s role in regulating cell cycle check points other than G1/S is less well defined. Herein we report that c-Myb, as well as B-Myb, up-regulates cyclin B1 expression in normal and malignant human hematopoietic cells, thereby contributing to G2/M cell cycle progression. Our initial experiments revealed a direct relationship between Myb and cyclin B1 expression. We then attempted to show causality using a variety of experimental approaches. First, ChIP assays demonstrated that c-Myb protein directly bound the cyclin B1 promoter in K562 and Mo7e cells. Second, a cycle 3 GFP reporter construct, driven by the cyclin B1 promoter, was upregulated in cells co-tranfected with a c-myb expression vector. Third, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide in 6 hours. All these assays strongly suggest that c-myb directly regulates cyclin B1. Finally, cyclin B1 expression decreased by 85–90 % in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A. The biologic significance of this relationship was revealed by two independent lines of experimentation. First, silencing B-myb resulted in a delay of cell cycle progression from S to G2/M, and an accumulation of cells in M phase, in HCT116 cells and K562 cells respectively. These abnormalities could be rescued, at least partially, by expression of exogenous c-myb. This observation conflicts with the report that c-Myb does not regulate cyclin B1 or G2/M progression in T98G cells suggesting that Myb functions could well be cell type specific. Additional analysis using PCR array showed that the absence of B-myb decreased the expression of 19 of 84 cell cycle related genes. Exogenous c-myb expression partially rescued 11 genes including cyclin B1, cyclin B2, cdc2, cdc20, CKS1B, p15INK4b and Ki-67, but not cyclin D1. In another experiment an inducible dominant negative c-Myb protein decreased cyclin B1 expression in K562 human leukemia cells, and the expected consequence of this, accelerated exit from the M phase, was observed. In activated primary human T-lymphocytes with IL-2 and CD34+ bone marrow cells, expression of c-Myb and cyclin B1 increased concordantly and silencing c-myb expression resulted in decreased cyclin B1 expression. We conclude from these studies that c-Myb in addition to B-myb plays a heretofore unappreciated role in G2/M cell cycle transition in normal and malignant human hematopoietic cells by directly regulating cyclin B1 expression.