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.
Embryonic stem (ES) cell lines represent a population of undifferentiated pluripotent cells capable of multilineage differentiation in vitro. Although very useful for studying developmental processes, human ES cell lines have also been suggested as a potential and unlimited source for cellular transplantation and the treatment of human disease. The proteomic basis of embryonic stemness (pluripotentiality and multilineage differentiation) and the transitions that lead to specific cell lineages however, remain to be defined. As an important first step in defining these processes, we have performed a proteomic analysis of undifferentiated mouse R1 ES cell lines using pH 3-10, 4-7 and 6-11 two-dimensional electrophoresis gels, matrix-assisted laser desorption/ionization and tandem mass spectrometry. Of the 700 gel spots analyzed, 241 distinct protein species were identified corresponding to 218 unique proteins, with a significant proportion functionally related to protein expression.
Summary Mitochondrial inheritance, the transfer of mitochondria from mother to daughter cell during cell division, is essential for daughter cell viability. The mitochore, a mitochondrial protein complex containing Mdm10p, Mdm12p and Mmm1p, is required for mitochondrial motility leading to inheritance in budding yeast. We observe a defect in cytokinesis in mitochore mutants and another mutant (mmr1Δ gem1Δ) with impaired mitochondrial inheritance. This defect is not observed in yeast that have no mitochondrial DNA or defects in mitochondrial protein import or assembly of β-barrel proteins in the mitochondrial outer membrane. Deletion of MDM10 inhibits contractile ring closure, but does not inhibit contractile ring assembly, localization of a chromosomal passenger protein to the spindle during early anaphase, spindle alignment, nucleolar segregation or nuclear migration during anaphase. Release of the mitotic exit network (MEN) component, Cdc14p, from the nucleolus during anaphase is delayed in mdm10Δ cells. Finally, hyperactivation of the MEN by deletion of BUB2 restores defects in cytokinesis in mdm10Δ and mmr1Δ gem1Δ cells, and reduces the fidelity of mitochondrial segregation between mother and daughter cells in wild-type and mdm10Δ cells. Our studies identify a novel MEN-linked regulatory system that inhibits cytokinesis in response to defects in mitochondrial inheritance in budding yeast.
Loss of mitochondrial DNA activates the DNA damage checkpoint kinase Rad53 to inhibit G1- to S-phase progression in budding yeast.
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