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H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability

Nature Cell Biology volume 9, pages 25–35 (2007)Cite this article

Abstract

Investigations aimed at identifying regulators of nuclear architecture in Drosophila demonstrated that cells lacking H3K9 methylation and RNA interference (RNAi) pathway components displayed disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared with wild type. We also observed a substantial increase in extrachromosomal circular (ecc) repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 and ecc DNA formation is not induced by removal of cohesin. We conclude that the structural integrity and organization of repeated DNAs and nucleoli are regulated by the H3K9 methylation and RNAi pathways, and other regulators of heterochromatin-mediated silencing. In addition, repeated DNA stability involves suppression of non-homologous end joining (NHEJ) or other recombination pathways. These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.

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Figure 1: Su(var) mutants contain multiple nucleoli.
Figure 2: Su(var) mutants have dispersed rDNA foci, each of which forms an ectopic nucleolus.
Figure 3: Satellite DNA organization is disrupted in Su(var)3-9null mutant nuclei.
Figure 4: Analysis of histone modifications in chromatin containing repeated DNA in wild-type and Su(var)3-9null cells.
Figure 5: Levels of extrachromosomal repeated DNAs are significantly increased in Su(var)3-9null mutant tissues compared with wild type.
Figure 6: The RNAi pathway is also required to maintain the structural integrity of repeated DNAs and the nucleolus.
Figure 7: Effects of Ligase 4 and cohesin mutants on ectopic nucleolus and eccDNA formation.
Figure 8: A schematic representation of a model for regulation of nuclear architecture by the H3K9 methylation and RNAi pathways.

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Acknowledgements

The authors thank: J. Birchler (for hlsΔ215), R. Carthew (for DCR2L811fsx), D. Dorsett (for SMC1 antibody), S. Hawley (for smc1exc461), F. Gao (for Ago251B), T. Jenuwein (for H3K9me2 antibody), K. M. Pollard (for fibrillarin antibody), G. Reuter (for Su(var)3-9 alleles 6 and 17) and J. Rine (for dSir217); S. Weiss for experimental contributions; A. Dernburg for help with FISH experiments; and A. Blanco (formerly Metamorph) for assistance with volumetric analysis. We thank N. Bhalla, A. Dernburg, S. Erhart, P. Heun, B. Mellone, A. Minoda and W. Zhang for helpful discussions and critical comments on the manuscript. This work was supported by a National Institutes of Health (NIH) grant, R01GM061169.

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Authors and Affiliations

  1. Department of Genomics and Bioinformatics, Lawrence Berkeley National Lab, One Cyclotron Road, Berkeley, 94720, CA, USA

    Jamy C. Peng & Gary H. Karpen

  2. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, 94720, CA, USA

    Jamy C. Peng & Gary H. Karpen

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I.P. performed all the experiments, and J.P. and G.K. collaborated on data analysis and project planning.

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Correspondence to Gary H. Karpen.

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Peng, J., Karpen, G. H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol 9, 25–35 (2007). https://doi.org/10.1038/ncb1514

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