SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span

Abstract

Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.

Figure 1. SIRT6 Interacts with RELA

(A) Co-IP of RELA with FLAG-tagged SIRT6 protein. Western blot (WB) analysis reveals RELA protein in immunoprecipitates (IP) of FLAG-tagged SIRT6, but not several other SIRTs, expressed in 293T cells. A very weak interaction with SIRT7 may be observed under these conditions. (B) Endogenous SIRT6 and RELA proteins interact. Western blot analysis of RELA following co-IP with endogenous SIRT6 or SIRT1, or control IP is shown. (C) SIRT6-RELA interaction is induced upon TNF-α treatment. Endogenous co-IP of RELA with SIRT6 from 293T cells following TNF-α (20 ng/ml) treatment. (D) In vitro translated RELA binds purified recombinant SIRT6-GST. (E) In vitro translated SIRT6 binds purified recombinant GST-RELA (aa1-431).

Figure 2. RELA Recruits SIRT6 to Target Promoters

(A) FLAG-SIRT6 is recruited to promoters of NF-κB target genes. HeLa cells stably expressing either pBabe-FLAG-SIRT6 or empty pBabe vector were treated with TNF-α (20 ng/ml) for 45 minutes. ChIP with α-FLAG antibodies was performed, and FLAG-SIRT6 occupancy (mean ± s.e) is shown relative to background signals in ChIPs from pBabe control cells. (B) Endogenous SIRT6 is recruited to the promoters of NF-κB target genes. HeLa cells were treated with TNF-α (20 ng/ml) for 45 minutes. ChIP with α-SIRT6 antibodies was performed, and SIRT6 occupancy (mean ± s.e) is shown relative to background signals in IgG negative control ChIPs. (C) Western analysis of RELA protein in HeLa cells stably expressing RELA shRNA (RELA sh). (D) RELA is required for recruitment of SIRT6 to the promoters of NF-κB target genes. Cells were transduced with either RELA shRNA or control pSR vector and treated with TNF-α (20 ng/ml) for 1 hour. ChIP with α-SIRT6 antibodies was performed and SIRT6 occupancy relative to untreated pSR control cells is shown.

Figure 3. SIRT6 Represses RELA Target Gene Expression

(A) SIRT6 and SIRT1 repress NF-κB transcriptional activity. Top: Relative luciferase units (RLU) of RELA mediated transcription of IL1-Luc (NF-κB reporter gene) activity upon transfection of FLAG-tagged SIRT proteins or vector control (mean ± s.e.). *, p<0.05, Student’s t-test. Bottom: Immunoblot showing expression of FLAG-tagged SIRT proteins. (B) Western analysis of SIRT6 protein in HeLa cells stably expressing two independent SIRT6 shRNAs (S6 sh1 and S6 sh2). (C, D) SIRT6 inhibits TNF-α-induced (C) and RELA-induced (D) reporter gene expression. RLU of IL1-Luc (NF-κB reporter gene) activity (mean ± s.e.) in control (pSR) and SIRT6-knockdown (S6 sh2) HeLa cells is shown. (E) Increased expression of NF-κB target genes in SIRT6 knock-down cells. Quantitative TaqMan real-time PCR analysis of the indicated mRNAs is shown normalized to GAPDH levels (mean ± s.e.). (F) Regulation of 18 SIRT6-dependent NF-κB target genes in six microarray data sets of human aging. Each row is a data set; each column is a gene. Significant induction (red) or repression (green) with age (p<0.05, one-sided t-test) is shown. The preponderance of age-dependent induction (81% observed vs. 50% expected) is significant (p=0.001, hypergeometric distribution).

Figure 4. SIRT6 Deacetylates Histone H3K9 at promoters of RELA target genes

(A) Western analysis of SIRT6 protein in HeLa cells stably expressing SIRT6 shRNA (sh2) (B) SIRT6 is required for H3K9 deacetylation at promoters of RELA target genes. ChIP with α-H3K9Ac and α-H3 antibodies was performed. H3K9 acetylation at RELA target gene promoters (mean ± s.e.) is shown relative to untreated control samples and normalized to total H3 levels. (C) SIRT6 is required to limit RELA occupancy at the promoter of RELA target gene promoters. ChIP with α-RELA antibodies was performed following a 30 minute TNF-α pulse; RELA occupancy (mean ± s.e.) at promoters relative to untreated control samples is shown. (D)-(E) SIRT6-mediated deacetylation of nucleosomes inhibits nucleosome binding to RELA. Western analysis of H3K9Ac levels on nucleosomes following incubation with SIRT6 in NAD-dependent deacetylation or mock reactions (D). The acetylated or SIRT6-deacetylated nucleosomes were used in nucleosome binding assays with increasing amounts of GST-RELA protein (E). Extent of nucleosome binding can be estimated by comparing levels of bound (**) and unbound (*) nucleosomes. Reduced binding is observed in the SIRT6-deacetylated samples (compare 10 ug RELA samples)

Figure 5. SIRT6 Alters NF-κB-mediated Apoptosis and Senescence

(A) SIRT6 regulates NF-κB-dependent apoptosis resistance. pSR and S6 sh2 cells were transfected with the indicated plasmids and a GFP-expression plasmid. Transfected (GFP-positive) apoptotic cells were identified by nuclear morphology (DAPI-staining; mean ± s.e.). * indicates transfected, apoptotic cells. TUNEL analysis confirmed that cells with pyknotic nuclei were undergoing apoptosis (data not shown). (B)-(D) RELA depletion reverses cellular senescence in SIRT6-depleted primary human keratinocytes. (B) Immunoblot of RELA and SIRT6 three days after siRNA nucleofection. (C) Quantification of SA-β-Gal-positive cells, as shown in (D) (mean ± s.e.). *, p<0.0001 compared to all other samples, Student’s t-test.

Figure 6. Sirt6 Deficiency Leads to Ectopic Expression of NF-κB Target Genes In Vivo

(A) Schematic for unbiased screen for candidate transcriptional regulators of gene expression changes in Sirt6-/- tissues. (B) Motif modules induced or repressed in Sirt6-/- tissues. Shown is the number of motifs that are induced or repressed in the indicated number of Sirt6-/- tissues. The motif module of NF-κB was induced in all Sirt6-/- tissues examined. Motifs labeled in gray were found to be both induced and repressed in all Sirt6-/- tissues in combination with different motifs. * indicates motifs previously implicated in a screen for transcription factor motifs responsible for driving gene expression changes in mammalian aging (Adler et al., 2007). See Table S1 for motif modules induced or repressed upon Sirt6 knockout. (C) Sirt6 knockout leads to NF-κB target gene induction. Shown is gene expression analysis in wild-type (WT) or Sirt6-/- MEFs, following TNF-α treatment (20 ng/mL) for the indicated times, of genes enriched for NF-κB motifs in promoters and known NF-κB responsive genes (Hinata et al., 2003; Hinz et al., 2001). Note the increased red (induced expression above control) genes in the SIRT6-/- columns, particularly at 24 hours. Average expression of each column is shown at the top.

Figure 7. Haploinsufficiency of RelA Attenuates the Lethality and Aging-like Phenotypes of Sirt6-deficient Mice

(A) Immunoblot showing reduced RelA protein levels in livers of Sirt6-/-RelA+/- animals compared to RelA+/+ animals. (B) Photograph of 23 day old Sirt6-/-RelA+/+ and Sirt6-/-RelA+/- littermates. (C) Kaplan-Meier curve showing the survival of wild-type (n=8), Sirt6-/-RelA+/+ (n=6), and Sirt6-/-RelA+/- (n=16) mice (p<0.0007, Cox-Mantel log-rank test). (D) Growth curve showing weight (grams; mean ± s.e.) of wild-type (n=6), Sirt6-/-RelA+/+ (n=7), and Sirt6-/-RelA+/- mice versus age in days. Sirt6-/-RelA+/- mice were categorized as survivors (n=13) or non-survivors (n=12) based on their ability to survive past 40 days. (E) Excessive activation of the NF-κB motif module in Sirt6-/- spleen is attenuated in Sirt6-/-RelA+/- spleen. Shown is the fold induction (mean ± s.e.) of genes in the NF-κB motif module, normalized to wild-type. (F) Serum glucose levels in wild-type (n=6), Sirt6-/-RelA+/+ (n=4), and Sirt6-/-RelA+/- mice (mean ± s.e.). Sirt6-/-RelA+/- mice were categorized as survivors (n=9) or non-survivors (n=6) based on their ability to survive past 40 days; p-values are indicated. (G) Gross appearance of spleen from 122-day-old wild-type and Sirt6-/-RelA+/- mice. (H) Hematoxylin and eosin (H&E) staining of spleen from wild-type and Sirt6-/-RelA+/- mice. Bar, 100μM. (I) Model. NF-κB is a stress-responsive transcription factor that induces expression of target genes involved in aging-related processes including cell senescence, apoptosis, and inflammation. NF-κB signaling is limited by SIRT6, which is recruited to NF-κB target gene promoters by physical interaction with the NF-κB subunit RELA. SIRT6 deacetylates histone H3 lysine 9 on target gene promoters, thereby altering chromatin structure to facilitate NF-κB destabilization and signal termination.