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Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis

Nature Cell Biology volume 11, pages 925–933 (2009)Cite this article

Abstract

Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylation of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.

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Figure 1: Histones associated with Rad53 are phosphorylated, which is required for efficient histone degradation.
Figure 2: Histones associated with Rad53 are ubiquitylated and the Tyr 99 residue of histone H3 is critical for its ubiquitylation.
Figure 3: Functional proteasomes are required for the degradation of excess histones.
Figure 4: Identification of the putative E2 and E3 enzymes involved in the degradation-related ubiquitylation of histones.
Figure 5: Yeast cells lacking the factors involved in histone degradation accumulate excess endogenous histones bound to histone chaperones Asf1 and Cac1.
Figure 6: Ubc4, Ubc5, Tom1 and Rad53 interact with each other in vivo and can ubiquitylate histones in vitro.

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Acknowledgements

We thank J. Diffley, D. Finley, M. Hochstrasser, S. Jentsch, N. Lowndes, C. Mann, H. Masumoto, M. A. Osley, R. Rothstein, D. Stern, D. Stillman, J. Svejstrup, A. Verreault and Y. Wang for strains and reagents, A. Verreault and D. Brown for critical reading of this manuscript, M. Abdul-Rauf for construction of the pYES2–HTH and pYES2–HTH–HHT2 plasmids, as well as the H4 mutants used in the Supplementary Information and undergraduate students S. Eckley and M. Gonzalez for technical assistance with the collection of many litres of yeast cultures for our experiments. We thank M. Blaber for assistance with the structural modelling of the Y 99 residue of histone H3, shown in Supplementary Information, Fig. S8. Research in AG's laboratory is supported by a Bankhead-Coley Cancer Research Program grant (07BN-02) from the Florida Department of Health and research in JP's laboratory is funded by a NIH grant (R15GM079678-01).

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  1. Department of Biomedical Sciences, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, 32306–4300, FL, USA

    Rakesh Kumar Singh, Marie-Helene Miquel Kabbaj, Johanna Paik & Akash Gunjan

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R.K.S. performed 70% of the experiments, aided in the design of several experiments and helped write parts of the manuscript; M.M.K. carried out 5% of the experiments, provided technical support and assisted R.K.S. as well as A.G. with experiments; J.P. aided in the conceptual design of some experiments and helped with manuscript preparation; A.G. performed 25% of the experiments, was in charge of the overall design of experiments and wrote the manuscript with help from R.K.S. and J.P.

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Correspondence to Akash Gunjan.

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Singh, R., Kabbaj, MH., Paik, J. et al. Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis. Nat Cell Biol 11, 925–933 (2009). https://doi.org/10.1038/ncb1903

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