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SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence

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Abstract

Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing has been observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover an essential role of the human SIRT6 enzyme in pericentric transcriptional silencing, and we show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, residue K18 of histone H3 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, depletion of these transcripts through RNA interference rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and regulation of acetylated H3K18 at heterochromatin, and demonstrate the pathogenic role of deregulated pericentric transcription in aging- and cancer-related cellular dysfunction.

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Figure 1: H3K18ac is a physiologic SIRT6 substrate.
Figure 2: SIRT6 selectively regulates H3K18 deacetylation at pericentric chromatin.
Figure 3: SIRT6 depletion disrupts pericentric chromatin silencing and leads to aberrant accumulation of satellite transcripts.
Figure 4: Aberrant accumulation of pericentric satellite transcripts in SIRT6-deficient cells causes mitotic defects and cellular senescence.

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  • 15 April 2009

    In the version of this article initially published online, acknowledgement of funding support for K.F.C. by a sponsored research agreement with Daiichi Sankyo Co., Inc. had been omitted, and a positive competing financial interest statement had not been included. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank O. Gozani and members of the laboratories of K.F.C. and O. Gozani for useful discussions, and S. Paredes, T. Hong, and L.D. Boxer for technical assistance. We thank X. Shi (University of Texas M.D. Anderson Cancer Center) for providing bacterial expression vectors for the AF9 YEATS domain and Z. Yang (Stanford University) for KAP1 expression vectors. This work was supported by grants from the US National Institutes of Health (NIH) to K.F.C. (R01 AG028867, R56AG050997), the Department of Veterans Affairs to K.F.C. (Merit Award), the Paul F. Glenn Laboratories for the Biology of Aging (K.F.C.), and fellowship awards to L.T. (Italian Foundation for Cancer Research fellowship abroad, American Italian Cancer Foundation postdoctoral research fellowship, and Stanford Dean's fellowship) and to Z.O. (Walter and Idun Berry postdoctoral fellowship). Work in the laboratory of W.L. was funded in part by grants from the Cancer Prevention Research Institute of Texas (RP150292) and the NIH (R01HG007538 and R01CA193466). Research of K.F.C. is partly funded by Daiichi Sankyo Co., Inc.

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Authors and Affiliations

Authors

Contributions

L.T. and K.F.C. conceived the project, designed the experiments and wrote the manuscript. L.T. performed in vitro and cellular deacetylation assays, ChIP and ChIP–seq experiments, RNA expression analysis, microscopy, and cell biology experiments. Y.X. and W.L. performed bioinformatic analyses for the ChIP–seq experiments and contributed to the corresponding manuscript sections. W.Z. performed ChIP experiments in KAP1-depleted cells and contributed to analysis of KAP1 in SIRT6-depleted cells. R.I.T. contributed to the deacetylation assay on H3K18ac peptides, analysis of satellite transcripts, and manuscript editing. Z.O. performed ChIP experiments in SIRT6-overexpressing cells. F.S. purified nucleosomes for deacetylation assays.

Corresponding authors

Correspondence to Wei Li or Katrin F Chua.

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Competing interests

Research of K.F.C. is partly funded by Daiichi Sankyo Co., Inc.

Integrated supplementary information

Supplementary Figure 1 H3K18ac is a physiologic SIRT6 substrate.

(a) Western analysis showing H3K18Ac levels on purified calf thymus histone H3 after in vitro deacetylation assay. Reactions with or without NAD+, control GSTprotein, wild type GST-SIRT6 (GST-SIRT6 WT) or the catalytically inactive GST-SIRT6 H133Y mutant protein (GST-SIRT6 HY) are indicated. Total H3 is shown as loading control. Results are representative of 3 independent experiments. (b) Additional exposures of western blots showing H3K18Ac, H3K9Ac and H3K56Ac levels in SIRT6 KO MEFs compared to WT littermate control MEFs presented in Figure 3d (litter1), and western analysis on lysates from a second set of WT and SIRT6 KO littermate MEF lines (litter 2).

Supplementary Figure 2 SIRT6 selectively regulates H3K18 deacetylation at pericentric chromatin.

(a) Genome-wide peak profile analysis of H3K18Ac ChIP-seq reads within ±1Kb flanking TSS’s in SIRT6 knockdown (KD1) and control cells (two-tailed Student’s t-test, n=22198 peaks). (b) Genome-wide peak profiles of H3K9Ac and H3K56Ac ChIP-seq reads showing average occupancy within 100Kb windows from centromeric gaps (Pericentric) (two-tailed Student’s t-test, n=488 peaks for H3K9Ac, n=500 peaks for H3K56Ac). (c) H3K18Ac ChIP-seq enrichment at families of repetitive DNA elements annotated in Repbase in SIRT6 KD1 versus control cells. (d) ChIP-seq H3K18Ac levels at several centric consensus (CTcons1, CTcons2) or the indicated chromosome-specific centric (CT) sequences. Graph shows values of forward and reverse paired-end reads (r1 and r2) normalized to control. (e) Western blot showing Flag-SIRT6 levels in U2OS cells transfected with SIRT6 wild type (WT), catalytic mutant (SIRT6 HY), or empty control vectors for ChIP experiments in (f). β-tubulin, loading control. (f, g) H3K9Ac and H3K56Ac ChIP-qPCR in U2OS cells after SIRT6 overexpression (f) or knock-down (g) at pericentric repeats (Sat II, Sat III) or chromosome-specific centric α-satellite repeats (17a, 21a, 21b, Xa). Control, 5S ribosomal DNA (5SR) repeats. In (f), data represent mean +/- s.e.m. of n=3 independent cell cultures. In (g), H3K9Ac data represent mean +/- s.e.m. of independent knockdown experiments (n=6 for Sat II, Sat III, Xa, and n=5 for 17a, 21a, 21b), for H3K56Ac (mean +/- s.e.m. of n=4 independent knockdown experiments for all sequences, except Xa where n=5). *p<0.05, when not indicated p>0.05 (one-tailed Student’s t-test).

Supplementary Figure 3 SIRT6 depletion disrupts silencing of pericentric chromatin and leads to aberrant accumulation of satellite transcripts.

(a) ChIP-qPCR for SIRT6 at pericentric satellite repeats (Sat II, Sat III), compared to positive control sequences (LINE1, RPL23 promoter DNA) and negative control sequence (Neg, Myosin-1 promoter) (mean +/- s.e.m. of n=3 technical replicates). Similar results were observed in 2 independent experiments. (b) Detection of Sat III transcripts from cytoplasmic (C) and nuclear (N) RNA by northern blot. HeLa cells heat shocked (H.S.) were used as positive control. Ethidium bromide staining is provided as loading control. Similar results were observed in 3 independent knockdown experiments. (c) Top, western analysis of SIRT7 levels in SIRT7-depleted (SIRT7 KD) U2OS cells. β-tubulin (β-tub), loading control. Bottom, qRT-PCR of Sat III transcripts (mean +/- s.e.m. of n=3 independent knockdown experiments). (d) ChIP-qPCR for H3K36me3 at pericentric repeats or control 5S ribosomal DNA (5SR) (mean +/- s.e.m. of n=3 technical replicates). Similar results were observed in 3 independent experiments. (e) ChIP-seq H3K9me3 levels at two pericentric consensus sequences (PCT cons1, PCT cons2) or at control 18S ribosomal DNA (18S). Graph shows average value of paired-end reads. (f) ChIP-qPCR showing HP1a enrichment (mean +/- s.e.m. of n=3 independent knockdown experiments). (g) Western analysis showing SIRT6 depletion in SIRT6 knock-out (KO) U2OS cells. H3, loading control. (h) Western blot showing KAP1 levels in U2OS KAP1 knockdown (KAP1 KD) cells. β-tub, loading control. (i) ChIP-qPCR for KAP1 and IgG control at pericentric satellite repeats and LINE1 sequences upon KAP1 knockdown. Actin promoter is shown as negative control (mean +/- s.e.m. of n=3 technical replicates). (j) ChIP-qPCR for H3K9me3 at pericentric repeats, and control sequences (LINE1), in KAP1 KD cells (mean +/- s.e.m. of n=3 independent knockdown experiments). In (a), (c), (d), (f), (i) and (j): *p<0.05; **p<0.01; ***p<0.001; when not indicated, p>0.05 (one-tailed Student’s t-test).

Supplementary Figure 4 Aberrant accumulation of pericentric satellite transcripts in SIRT6-deficient cells causes mitotic defects and cellular senescence.

(a) Representative image showing asymmetric mitosis in SIRT6 depleted (SIRT6 KD) cell. DNA was stained with DAPI (blue), microtubules with α-tubulin (green) and centrosomes with γ-tubulin (magenta). Bar, 5 μm. (b) Immunofluorescence showing SIRT6 KD cells with micronuclei (arrows), and micronuclei containing centromeres (detected by anticentromere CREST antibodies). Bar, 10 μm. (c) Quantitative RT–PCR of Satellite III transcript and SIRT6 mRNA levels in cells used in Figure 4e,h. U2OS cells were transiently transfected with combinations of siRNAs specific for SIRT6 (siSIRT6), Sat III transcripts (siSat III), and negative control siRNAs (–) as indicated (mean +/- s.d. of n=3 technical replicates). The results are representative of 3 independent knockdown experiments. (d) Senescence-associated β-galactosidase (SA-β-gal) activity assay. Representative image of U2OS cells quantified in Figure 4h. Bar, 25 μm. (e) Left, representative images of SA-β-galactosidase assays in Sat III overexpressing (Sat III OE) or control cells. Right, quantification of SA-β-gal positive cells (mean +/- s.e.m. of n=5 quantifications, for a total of > 2000 cells scored per sample). Experiment is representative of 2 independent biological replicates. (f) Left, SA-β-galactosidase assay images of SIRT6 depleted HeLa and A549 cells. Percentages (%) of SA-β-gal positive cells (mean +/- s.e.m. of n=5 quantifications) are indicated on the representative images. Bar, 25 μm. Right, levels of Sat III transcripts determined by qRT–PCR (mean +/- s.e.m. of n=3 independent knockdown experiments). (g) Top, qRT–PCR of Satellite III transcripts in SIRT6-deficient A549 cells co-depleted of Sat III transcripts. Bottom, quantification of SA-β-gal positive cells (mean +/- s.e.m. of n=5 quantifications). The results are representative of 2 independent knockdown experiments. In all panels, *p<0.05; **p<0.01; ***p<0.001; and when not indicated, p>0.05 (two-tailed Student’s t-test).

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Tasselli, L., Xi, Y., Zheng, W. et al. SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence. Nat Struct Mol Biol 23, 434–440 (2016). https://doi.org/10.1038/nsmb.3202

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