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. 2008 Mar 27;452(7186):492-6.
doi: 10.1038/nature06736. Epub 2008 Mar 12.

SIRT6 Is a Histone H3 Lysine 9 Deacetylase That Modulates Telomeric Chromatin

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Free PMC article

SIRT6 Is a Histone H3 Lysine 9 Deacetylase That Modulates Telomeric Chromatin

Eriko Michishita et al. Nature. .
Free PMC article

Abstract

The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

Figures

Figure 1
Figure 1. SIRT6 knockdown leads to premature cellular senescence and telomere dysfunction
a, Western analysis of SIRT6 expression in WI-38 SIRT6 knockdown (S6KD) or control (pSR) cells. b, Serial passaging experiments revealing premature replicative senescence of S6KD WI-38 cells. Passaging was begun at population doubling 39 and cumulative population doublings were calculated after the indicated days. c, Increased SA-β-gal+ staining in S6KD cultures at day 11 of serial passaging. d, e, Increased telomere dysfunction in S6KD WI-38 cells. TIFs were detected (d) by co-localization of γ-H2AX and telomeres, and cells with at least five TIFs were scored (e). Data represent the average of 20 fields. Error bars indicate s.e.m.; n = 158 (pSR); n=110 (S6KD). The P value was calculated with the two-tailed Student’s t-test. f, Increased chromosome end-to-end fusions in S6KD cells observed by spectral karyotype (SKY) analysis. Values represent the numbers of fused chromosomes as a percentage of total metaphases. S6KD1 and S6KD2 are knockdown cells generated with two independent SIRT6-specific shRNAs. g, Stabilizing telomeres by means of hTERT expression reverses the premature senescence of S6KD cells, whereas augmenting BER activity, by expression of the DNA polymerase-β (Pol-β)-dRP lyase domain, does not. hTERT, Pol-β or empty vector were ectopically expressed in S6KD WI-38 cells and passaged in physiological (2%) oxygen conditions. Cells were stained with SA-β-gal at population doubling 36.5. Error bars indicate s.e.m.; n=8 (pSR); n = 16 (S6KD1); n = 20 (S6KD2).
Figure 2
Figure 2. SIRT6 associates with telomeric chromatin
a, Western analysis of U2OS cells transduced with pBabe-Flag-SIRT6 or empty virus (pBabe) control. b, T-ChIP analysis with anti-Flag antibodies from the cells shown in a. c, T-ChIP assays in U2OS cells with antibodies specific for SIRT6,WRNor IgG negative control. d, T-ChIP assays with SIRT6 antibodies in S6KD cells or in control pSR IMR90 cells. In bd, slot-blots are shown of telomere or Alu repeat sequences in ChIP and input DNA. Input loaded represents 5% (b, c) or 2.5% (d) of total.
Figure 3
Figure 3. SIRT6 deacetylates lysine 9 of histone H3 at telomeric chromatin
a, Summary of in vitro deacetylation assays on acetylated histone tail peptides, analysed by mass spectrometry. b, Western analysis showing SIRT6 deacetylation of H3K9 on full-length histone H3 in vitro. Reactions with NAD+, SIRT6 or the catalytic H133Y SIRT6 mutant protein (SIRT6-HY) are indicated. c, SIRT6 deacetylates H3K9 in cells. Western analysis of 293T cells overexpressing SIRT6, SIRT6-HY or empty vector. d, Western analysis of SIRT6 knockdown in U2OS cells used for T-ChIP in e and f. e, T-ChIP analysis showing hyperacetylation of H3K9 at telomeric chromatin in S6KD U2OS cells. f, Quantification of multiple independent T-ChIP experiments as shown in e. Values represent the T-ChIP signal normalized to input signal, and subtracted for background signal in an IgG control. Error bars indicate s.e.m.; n = 4. P values were determined by two-tailed Student’s t-test.
Figure 4
Figure 4. SIRT6 stabilizes WRN at telomeric chromatin and prevents replication-associated telomere defects
a, b, T-ChIP experiments showing reduced association of WRN with telomeric chromatin in SIRT6 knockdown cells. a, Representative T-ChIP analysis of WRN occupancy in S6KD and control cells. b, Quantification of three independent experiments as shown in a. c, Representative chromosome end-to-end fusions observed in S6KD cells. Telomere FISH signals are yellow, and DAPI-stained chromosomes are blue. Weak or no telomere signals are detected at the sites of fusion (arrows). dic(13;17)(pter;qter) and dic(7,14)(qter;qter) indicate dicentric chromosomal fusions between the p and q arms of chromosomes 13 and 17, and the q arms of chromosomes 7 and 14, respectively. d, Representative S6KD metaphases showing aberrant telomere signals. Red arrows, sister telomere loss; blue arrows, telomere doublets. e, f, Quantification of sister telomere loss (e) and telomere doublets (f) in S6KD cells and in pSR control cells. pSR, n=15; S6KD1 and S6KD2, n=22. Error bars in b, e and f indicate the s.e.m., and P values were determined by two-tailed Student’s t-test.

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