Abstract
The cell-cycle-regulated Myb-family transcription factor B-Myb is crucial during S phase in many diploid cell types. We have examined the expression and function of B-Myb in megakaryocytic differentiation, during which cells progress from a diploid to a polyploid state. In contrast to terminal differentiation of most haematopoietic cells, during which B-myb is rapidly downregulated, differentiation of megakaryocytes is accompanied by continued B-myb RNA and protein expression. Overexpression of B-Myb in a megakaryoblastic cell line resulted in an increase in the number of cells entering S phase and, upon induction of differentiation, the fraction of cells actively endoreplicating increased. By contrast, reduction of B-Myb levels using short interfering (si)RNA resulted in a decline in S-phase progression during both normal and endoreplicative DNA synthesis. This effect correlated with aberrant localisation of initiation of DNA replication within the nucleus and an increased fraction of cells in mitosis. Chromosomal fragmentation and other aberrations, including shorter, thicker chromatids, end-to-end fusion, and loss of a chromatid, suggest that reduced B-Myb activity is also associated with structural chromosomal instability.
Highlights
B-Myb belongs to a family of transcription factors that is implicated in regulatory decisions affecting cell proliferation, differentiation and apoptosis (Oh and Reddy, 1999; Weston, 1998)
Resulted in a decline in S-phase progression during both normal and endoreplicative DNA synthesis. This effect correlated with aberrant localisation of initiation of DNA replication within the nucleus and an increased fraction of cells in mitosis
We have shown that cyclin E is necessary for endoreplication to occur (García et al, 2000) and, consistent with this, megakaryocytes from double-knockout mice lacking both cyclin E1 and E2 have a lower degree of ploidy compared with cells derived from wild-type animals (Geng et al, 2003)
Summary
B-Myb belongs to a family of transcription factors that is implicated in regulatory decisions affecting cell proliferation, differentiation and apoptosis (Oh and Reddy, 1999; Weston, 1998). B-Myb is ubiquitously expressed in proliferating cells (Latham et al, 1996), exhibiting maximal expression in S phase of the cell cycle (Lam et al, 1992). The presumed importance of B-Myb during S phase is reflected in the mechanisms that ensure maximal expression at this stage of the cell cycle. B-myb RNA expression has been reported to be limited to proliferating cells by an E2F-dependent mechanism, whereas activity of the B-Myb protein is stimulated by the cdk2-cyclin A complex in S phase (Robinson et al, 1996). The function of B-Myb in mitosis might relate at least partly to its ability to regulate cyclin B1 gene expression
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