Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase

Abstract

Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci1, telomeres2 and the ribosomal DNA3,4, and that also suppresses recombination in the rDNA5 and extends replicative life span6. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions7,8,9. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones9,10, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3 (ref. 8). Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.

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Figure 1: In vitro deacetylation assays of the H3 peptide (residues 1–20) di-acetylated at lysines 9 and 14 by recombinant yeast Sir2.
Figure 2: Amino-terminal sequencing of peaks 4 and 5 of the Sir2 deacetylase reaction at 1 mM NAD as determined by Edman degradation.
Figure 3: The deacetylation activity of yeast Sir2 on the H4 peptide (residues 2–19) tetra-acetylated at lysines 5, 8, 12 and 16.
Figure 4: Effects of inhibitors on the deacetylase and ADP-ribosyltransferase activities of recombinant Sir2 (rSir2).
Figure 5: Deacetylation activity of Sir2 is essential for silencing, recombination suppression and life-span extension in vivo.

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Acknowledgements

We thank R. Cook, A. Park, and H. Amoroso at the MIT Biopolymers lab for the HPLC and electron-spray mass spectroscopy, and P. Matsudaira for the MALDI mass spectroscopy. We also thank S. Inamoto for bacterial strains, and H. Tissenbaum and E. Ford for comments on the paper. This work was funded by The Human Frontier Science Program Organization Long-Term Fellowship to S. I., a NIH predoctoral grant to C.A. and M.K., and grants from the NIH, Seaver Foundation, Ellison Medical Foundation, and Howard and Linda Stern Fund to L.G.

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Correspondence to Leonard Guarente.

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Imai, S., Armstrong, C., Kaeberlein, M. et al. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800 (2000). https://doi.org/10.1038/35001622

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