SUMMARY Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify novel genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose down-regulation led to elevated levels of H2AX phosphorylation (γH2AX) and revealed new links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased γH2AX levels to the neurological disorder, Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.
A requisite component of nervous system development is the achievement of cellular recognition and spatial segregation through competition-based refinement mechanisms. Competition for available axon space by myelinating oligodendrocytes ensures that all relevant CNS axons are myelinated properly. To ascertain the nature of this competition, we generated a transgenic mouse with sparsely labeled oligodendrocytes and establish that individual oligodendrocytes occupying similar axon tracts can greatly vary the number and lengths of their myelin internodes. Here we show that intercellular interactions between competing oligodendroglia influence the number and length of myelin internodes, referred to as myelinogenic potential, and identify the amino-terminal region of Nogo-A, expressed by oligodendroglia, as necessary and sufficient to inhibit this process. Exuberant and expansive myelination/ remyelination is detected in the absence of Nogo during development and after demyelination, suggesting that spatial segregation and myelin extent is limited by microenvironmental inhibition. We demonstrate a unique physiological role for Nogo-A in the precise myelination of the developing CNS. Maximizing the myelinogenic potential of oligodendrocytes may offer an effective strategy for repair in future therapies for demyelination.T he spatial alignment of myelin internodes along axons facilitates the process of saltatory conduction, maximizing the speed and efficacy of action potential propagation throughout the nervous system. In the CNS, myelin is formed by oligodendrocytes, cells with the capacity to form multiple myelin internodes (1). What developmental mechanisms control the generation and coordination of the precise number and length of myelin internodes? Though recent studies have revealed extrinsic cues and transcriptional and epigenetic determinants that regulate oligodendrocyte differentiation (2-7), achievement of the precise spatial organization of myelin internodes necessitates a mechanism whereby neighboring oligodendroglial cells coordinate the appropriate number of myelin internodes generated. This coordination could be achieved through oligodendroglial competition for inductive cues and/or available axonal space (8). Variations in the number and length of myelin internodes formed by individual oligodendrocytes support the likelihood of environmental regulation of myelination. However, providing evidence of this variation requires a strategy that facilitates the resolution and analysis of individual myelinating oligodendrocytes in vivo. Results Individual Oligodendrocytes Exhibit Great Variability in the Numberand Lengths of Myelin Internodes Throughout the CNS. Though the heterogeneity of oligodendrocyte morphology has been recognized for the past century (9, 10), efforts to accurately examine oligodendrocytes have been hindered by the high density of myelinating cells, limiting the ability to confidently attribute specific myelin internodes to any particular oligodendrocyte. Existing methods approximate the n...
Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.
ABBREvIATIOns CMV ABsTRAcTHigh content cell-based genetic and small molecule library screens are powerful strategies in drug discovery and investigations of disease mechanisms. We report that primary cells derived from a transgenic mouse model expressing a fluorescence mitosis biosensor provide unambiguous phenotype readouts without the need for transfection or immunocytochemistry. Phenotype profiles of cell cycle disruption and of apoptosis are easily detectable at a single time point selected from time-lapse live fluorescence microscopy. Most importantly, this transgenic mouse model may be crossed with cancer mouse models to derive biosensor-expressing primary cancer cells for use in high content screening strategies targeting discovery of tumor-specific chemotherapeutic compounds.
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