Abstract
BackgroundThe transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1.Methodology/Principal FindingsIn the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death.Conclusions/SignificanceAppropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.
Highlights
Avian myeloblastosis viral oncogene homolog-2 is a member of a multigene family of transcription factors involved in control of cell cycle progression, differentiation and apoptosis [1,2]
Transcripts to A-Myb and C-Myb were detected in murine embryonic stem (mES) and murine embryonic germ (mEG) cells (Figure 1C), but at levels much lower than those found in testes and Sca1+ bone marrow cells, respectively
We show that knockdown of B-MYB in mES cells decreases proliferation, cell viability and colony size, Figure 5
Summary
Avian myeloblastosis viral oncogene homolog-2 (mybl2) is a member of a multigene family of transcription factors involved in control of cell cycle progression, differentiation and apoptosis [1,2]. The trans-activation and gene regulatory potential of BMYB is regulated by cyclin A/cdk2-mediated phosphorylation [8], and B-MYB is degraded through a ubiquitin-mediated process late in S phase [9] Because it is prevalent during both late G1 and early S phases, the primary functions of B-MYB have generally been assumed to be restricted to these portions of the cell cycle [2,7,10]; studies in Drosophila [11,12] and zebrafish [13]. The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1
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