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A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A

Abstract

Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. Here, we show that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, we found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A.

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Figure 1: MgcRacGAP is required for CENP-A protein localization to centromeres.
Figure 2: MgcRacGAP localizes to centromeres transiently at the end of CENP-A loading.
Figure 3: MgcRacGAP is required specifically to stabilize newly incorporated CENP-A.
Figure 4: GAP-inactive MgcRagGAP mutant localizes persistently to centromeres.
Figure 5: Automated analysis of CENP-A levels following shRNA depletion of various target proteins reveals differential defects in epigenetic regulation of centromeres.
Figure 6: Cdc42 localizes to centromeres and functions to maintain CENP-A levels independent of polymeric actin.
Figure 7: A small GTPase switch maintains newly incorporated CENP-A after loading.

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Acknowledgements

The authors would like to thank J.-C. Labbé, A. Desai, K. Bloom and S. Meloche for helpful comments and discussions, the IRIC proteomics facility (specifically É. Bonneil and P. Thibault) for LC–MS/MS analysis, the IRIC genomics facility for sequencing and qPCR analyses, and A. Straight and B. Moree for the generous gift of the CENP-A SNAP-tag cell line. J.F.D. was supported by a postdoctoral fellowship from the Swiss National Science Foundation; V.D.R. was supported by a student fellowship from the CIHR. A.S.M. receives salary support from the FRSQ. P.S.M. holds the Canada Research Chair in Chromosome Organization and Mitotic Mechanisms. This work was funded by grants #018450 awarded to P.S.M. and #019162 awarded to A.S.M. from the Terry Fox Foundation and the CCSRI. V.D.R., A.S.M. and P.S.M. participate in the Systems biology option in the graduate program in Molecular Biology, Université de Montréal.

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A.L. conducted all immunoprecipitation and mass spectrometry experiments. A.L. and J.F.D. performed shRNA experiments and analysis, respectively. J.F.D. performed live-cell experiments. A.L. evaluated all shRNA experiments (qPCR, western blot). V.D.R. performed the small GTPases localization experiments. All authors performed essential tasks in generating figures. P.S.M., A.L., J.D., A.-M.L., and A.S.M. conceived the experiments. P.S.M. wrote the manuscript, assisted by all authors, in particular A.S.M. and J.F.D.

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Correspondence to Paul S. Maddox.

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Lagana, A., Dorn, J., De Rop, V. et al. A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A. Nat Cell Biol 12, 1186–1193 (2010). https://doi.org/10.1038/ncb2129

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