A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A

Abstract

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD⁺ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which was correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

Keywords: SIRT6; centenarians; lamin; longevity.

Figure 1. CentSIRT6 missense variant identified in AJ centenarians demonstrates lower deacetylation activity

1. A

   Age, gender, and BMI information of the Ashkenazi Jewish Centenarian cohort. Table showing the age, gender, and BMI information in control and centenarian groups. Pie chart showing the percentage of male/female individuals in control and centenarian groups. Right panel showing the age and BMI distribution of all individuals recruited in targeted capture‐seq. Data were presented as mean and SD. Student's t‐test, two‐tailed, ***P < 0.001.

2. B, C

   Histograms of normalized allele counts in individuals 75+ years old compared with all age groups (N = 125,748) across (B) all chromosome 19 SNPs and small insertions/deletions, and (C) missense SNPs and indels only. Dotted vertical lines show centSIRT6.

3. D

   The structure of the SIRT6 gene. centSIRT6 contains two missense mutations in the C‐terminus N308K and A313S. White boxes represent UTRs, colored boxes represent protein‐coding regions, and blue boxes represent enzymatic domains.

4. E

   Michaelis–Menten kinetic parameters as calculated by saturation curves using the differential concentrations of myristoylated peptide or NAD⁺. n = 3 technical replicates (TR); error bars = SD.

Figure EV1. rs350845 (chr19:4174953:A:G) is a cis‐eQTL for SIRT6 upregulation across multiple tissues

1. GTEx tissue types are shown sorted by m‐value, which is the posterior probability that an eQTL exists for SIRT6 in a specific tissue from a cross‐tissue meta‐analysis. NES = Normalized Effect Size based on single‐tissue analysis; P‐value is from a t‐test comparing observed NES to a null within a single tissue.

2. Single‐tissue eQTL P‐value vs. m‐values.

3. Violin plots showing SIRT6 expression differences across three example tissues with m‐value = 1. Reference allele is G and alternate allele is A, and each panel shows the normalized expression of SIRT6 in a different tissue. Values beneath genotypes represent a number of individuals.

Figure EV2. Turnover rate and biochemical properties of purified SIRT6 proteins

1. Protein turnover rate. SILAC analysis on HEK293 cells expressing SIRT6 variants.

2. Coomassie staining of SIRT6 purified from Rosetta‐Gami E. coli cells. Proteins were purified at the University of Rochester (Roc) and the Ichor Therapeutics facility in Ithaca. All in vitro experiments were performed with both protein preparations and the data was consistent between the two preps.

3. Thermostability of purified SIRT6 proteins. Data represent two replicates with two technical replicates each using SIRT6 from the Roc and Ichor preps. n = 4 TR; error bars = SD. Student's t‐test, two‐tailed.

4. Tryptophan fluorescence curves for SIRT6 variants with titrated concentrations of NAD⁺.

Figure 2. CentSIRT6 possesses reduced deacetylase activity

1. A, B

   Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones were incubated with purified SIRT6 and 5 mM NAD⁺, then resolved by SDS–PAGE and analyzed by immunoblotting with acetyl‐specific histone antibodies. n = 3 TR; error bars = SD. Student's t‐test, two‐tailed. Asterisks indicate P < 0.05 compared with WT, color corresponds to allele. H3K9ac, H3K18ac, and H3 are blots, SIRT6 loading Coomassie stain HeLa histone preps were also probed as shown in Fig EV3A.

2. C

   Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9–17 compared with the summed total of all of the peptide quantitation values for the same region. Acetylation of other H3 peptides is shown in Supplementary Data 1. Histone preparation is shown in Fig EV3F. n = 3 biological replicates (BR); error bars = SD. Student's t‐test, two‐tailed.

3. D

   Whole cell histone H3 acetylation levels in cumate‐inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot and administered accordingly to respective cell lines (Fig EV3B and C). Cells were incubated with an appropriate cumate dose for 48 h prior to harvest, with PQ‐treated cells receiving PQ 24 h after initial cumate induction.

4. E–H

   Relative abundance of H3K9ac at SIRT6‐dependent NF‐κB target genes. Anti‐H3K9ac antibodies were used to perform ChIP in the absence (−Cu) or presence of (+Cu) cumate‐induced SIRT6 expression. Samples were normalized using 10% input. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed. Black asterisks indicate P < 0.05 compared with –Cu condition. Red asterisks indicate P < 0.05 compared with +Cu WT.

5. I, J

   Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity. Anti‐H3K18ac antibodies were used to perform ChIP in the absence (−Cu) or presence of (+Cu) cumate‐induced SIRT6 expression. Samples were normalized using 10% input. n = 3 TR; error bars = SD. Student's t‐test, two‐tailed. Black asterisks indicate P < 0.05 compared with –Cu condition.

Source data are available online for this figure.

Figure EV3. HeLa histone deacetylation by recombinant SIRT6, and SIRT6 expression in cumate‐inducible cell lines

1. In vitro deacetylation rates of purified SIRT6 proteins with histones purified from HeLa cells. H3K9ac, H3K18ac, and H3 are immunoblots, SIRT6 loading Coomassie stain. Control lanes, from the same gels, were repositioned for consistency. Uncut gels are shown in Appendix Fig S1. Asterisks indicate P < 0.05 compared with WT. n = 3 TR; error bars = SD. Student's t‐test, two‐tailed.

2. SIRT6 expression in cumate‐inducible telomerase‐immortalized HCA2 human fibroblast cell lines. SIRT6 alleles were integrated into the genome of SIRT6 knockout HCA2 cells using PiggyBac Transposon Vector System. Different dosages of cumate and resulting SIRT6 abundance were used to determine the dose needed to achieve equivalent SIRT6 expression for each cell line. Cells were normalized by count and total protein. Subsequent experiments utilizing cumate‐inducible SIRT6 fibroblasts were controlled for SIRT6 abundance using these data. For each cell line, the red box indicates the corresponding cumate dosage for equivalent expression (WT = 60 μg/ml, N308K = 30 μg/ml, A313S = 30 μg/ml, and Cent = 7.5 μg/ml). These concentrations were used in experiments with these cells.

3. qRT–PCR expression analysis of SIRT6 using standardized dosages of cumate (colored bars are cells treated with cumate, and empty bars are uninduced controls). Cumate dosage corresponds to red boxes in Appendix Fig S2B). Actin was used for standardization and samples were normalized to uninduced WT. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed.

4. Apoptosis assay of HCA2 cells treated with varying doses of cumate. n = 3 TR; error bars = SD. Student's t‐test, two‐tailed.

5. Population doubling rate of HCA2 cells treated with different doses of cumate to assess the impact on cell division.

6. Coomassie‐stained SDS–PAGE of histone preparations for quantitative mass spectrometry.

Source data are available online for this figure.

Figure 3. CentSIRT6 demonstrates enhanced mADPr activity

1. Self‐ribosylation of SIRT6 using biotin‐labeled NAD⁺. Recombinant SIRT6 was incubated with NAD⁺ conjugated with a biotin residue and then run on an SDS–PAGE. Each allele was assessed relative to its 0 h time point and normalized to SIRT6 total protein loading controls. n = 3 TR; error bars = SD. Student's t‐test, two‐tailed. Asterisks indicate P < 0.05. Black asterisks indicate significance over 0 h control. Red asterisks indicate significance over time‐matched WT SIRT6.

2. Self‐ribosylation of SIRT6 with titration of NAD⁺. mADPr‐specific antibody (Bonfiglio et al, 2020) was used to detect ribosylated wild‐type SIRT6 and centSIRT6 proteins. Plot fits the Michaelis–Menten best‐fit with GraphPad software. K_{m NAD} was 142 ± 29 for centSIRT6 and 106 ± 45 for wild‐type SIRT6, and the maximal signal was ~2x greater for centSIRT6 compared with the wild‐type SIRT6. Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed. n = 3 TR; error bars = SD.

3. Activation of PARP1 by SIRT6 variants. SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly‐ADPr antibody. Poly‐ADPr activity of PARP1 results in a wide range of product size. Activity was assessed by quantifying poly‐ADPr signal in whole lanes for each sample. n = 3 TR; error bars = SD. Two‐way ANOVA. Asterisks indicate P < 0.05. Black asterisks indicate significance over HPRT control. Red asterisks indicate significance over wild‐type SIRT6.

Source data are available online for this figure.

Figure 4. CentSIRT6 enhances LINE1 retrotransposon suppression and DNA repair

1. A

   qRT–PCR analysis of LINE1 expression in cumate‐inducible SIRT6 fibroblasts. Primers assessed both 5′ (ORF1) and 3′ (ORF2) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. Asterisks indicate P < 0.05. n = 3 TR; error bars = SD. Student's t‐test, two‐tailed.

2. B

   LINE1 EGFP retrotransposition assay. Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry. Asterisks indicate P < 0.05. Red asterisks indicate significance over SIRT6 KO control. Black asterisks indicate significance over WT SIRT6. n = 3 BR; error bars = SD. Two‐way ANOVA.

3. C

   In vitro KAP1 ribosylation by SIRT6. Asterisks indicate P < 0.05. Red asterisks indicate significance over 0 h, black asterisks indicate significance over compared with WT SIRT6. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed.

4. D, E

   Stimulation of NHEJ and HR by SIRT6 variants. Reporter cell lines were co‐transfected with SIRT6‐expressing plasmid, I‐Sce1 plasmid, and DsRed transfection control. After 72 h recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as the ratio of GFP⁺/DsRed⁺ positive cells. Representative FACS traces are shown in Fig EV4A. Blots demonstrate equivalent SIRT6 abundance in transfected cells. Asterisks indicate P < 0.05 Black asterisks indicate significance over control; red asterisks indicate significance over WT SIRT6. n = 3 BR; error bars = SD. Two‐way ANOVA.

5. F

   Basal γH2AX foci in cumate‐inducible SIRT6 fibroblasts. Foci were scored in at least 80 cells per condition. Representative images of the foci are shown in Fig EV4B. Asterisks indicate P < 0.05. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed.

6. G

   DNA repair kinetics in cumate‐inducible SIRT6 fibroblasts. Cells were grown on slides and irradiated with 2 Gy gamma radiation, followed by immunostaining for γH2AX. Irradiation was conducted when the cells were at 75% confluency on slides. Cells were fixed and foci scored at t = 0.5 h, 2 h, 4 h, 6 h, and 24 h postirradiation. Foci were scored in at least 80 cells per genotype per time point. Representative images of the foci are shown in Fig EV4C. Asterisks indicate P < 0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed.

7. H, I

   Oxidative stress resistance. Cumate‐inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 h. Resistance was determined by apoptosis staining 48 h after exposure. Representative FACS traces are shown Fig EV4D. Asterisks indicate P < 0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed.

Source data are available online for this figure.

Figure EV4. Quantification of SIRT6 expression and FACS gating for DNA repair assays

1. Representative images of FACS analysis for DNA DSB repair assays (Fig 4D and E).

2. Representative images of basal γH2AX immunostaining (Fig 4F). Dashed outline denotes nucleus borders. Scale bar 10 μm.

3. Representative images of γH2AX immunostaining after γ‐irradiation (Fig 4G). Scale bar 10 μm.

4. Representative images of FACS traces for paraquat sensitivity assay (Fig 4H and I).

Figure EV5. Analysis of CRISPR/Cas9‐edited human MSC cell lines

1. A, B

   Double strand repair efficiency in wild‐type and centSIRT6 hMSCs. DSB repair reporter constructs were integrated into hMSCs. After 72 h recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as the ratio of GFP⁺/DsRed⁺ positive cells. Asterisks indicate P < 0.05. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed.

2. C

   Cell viability in MMS‐treated wild‐type and centSIRT6 hMSCs. hMSCs were treated with MMS for 48 h and cell viability was evaluated by MTS assay. Data were normalized to the control group (0 mM). Asterisks indicate P < 0.05. n = 6 BR; error bars = SD. Student's t‐test, two‐tailed.

3. D

   Immunofluorescence staining of 53BP1 in wild‐type and centSIRT6 hMSCs under MMS treatment. Numbers of 53BP1 foci in the nuclei of wild‐type and centSIRT6 hMSCs with or without MMS (0.25 mM) treatment were quantified. >600 nuclei from 10 TR images were scored. Asterisks indicate P < 0.05. Error bars = SD. Student's t‐test, two‐tailed.

4. E

   qRT–PCR of NRF2 stress response pathway genes in wild‐type and centSIRT6 hMSCs. n = 3 BR; error bars = SEM. Student's t‐test, two‐tailed.

5. F

   RNAseq expression of NRF2 stress response pathway genes in wild‐type and centSIRT6 hMSCs. n = 4 BR; error bars = SEM. Student's t‐test, two‐tailed.

6. G

   Western blot analysis of SIRT6 in wild‐type and centSIRT6 hMSCs. β‐Actin was used as a loading control and used to normalize quantification. Asterisks indicate P < 0.05. n = 2 BR; error bars = SD. Student's t‐test, two‐tailed.

Source data are available online for this figure.

Figure 5. CentSIRT6 induces increased cell death in cancer cells

1. A

   Number of adherent cells after transfection with SIRT6 variants. Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 h. HCA2 are normal human foreskin fibroblasts. Asterisks indicate P < 0.05. n = 3 BR; error bars = SD. Student's t‐test, two‐tailed.

2. B, C

   Apoptosis staining of cancer cell lines 48 h after transfection. Cells were stained with Annexin V/PI and analyzed by flow cytometry. Asterisks indicate P < 0.05. Red asterisks indicate significance over control, black asterisks indicate significance over WT SIRT6.

n = 3 BR; error bars = SD. Two‐way ANOVA.

Figure 6. CentSIRT6 allele shows enhanced interaction with LMNA

1. Proteins showing stronger interaction with centSIRT6 compared with the wild‐type SIRT6. Proteins were prepared from cumate‐induced SIRT6 cell lines and immuno‐precipitated with SIRT6 antibody; rabbit preimmune serum was used as a control. Prior to analysis by mass spectrometry, samples were labeled with tandem mass tags.

2. IP experiments on lysates from cumate‐induced fibroblasts expressing wild‐type or centSIRT6 alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6 expression was induced 48 h prior to IP. n = 3 BR. One representative set of IPs is shown.

3. CentSIRT6 shows stronger interaction with LMNA compared with the wild‐type SIRT6. Quantification of the IP experiment shown in (B). SIRT6 IP followed by Western blot with antibodies to LMNA.

4. LMNA shows enhanced interaction with centSIRT6 compared with wild type. Quantification of the IP experiment shown in (B). LMNA IP from cumate‐inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6.

5. centSIRT6 shows enhanced mADPr. Quantification of the IP experiment shown in (B). SIRT6 IP from cumate‐inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues (Bonfiglio et al, 2020).

6. LMNA shows an enhanced mADPr signal in cells expressing centSIRT6. Quantification of the IP experiment shown in (B). IP with mADPr antibody (Bonfiglio et al, 2020) using extract from cumate‐induced SIRT6 fibroblasts, followed by Western blot with antibodies to LMNA.

7. SIRT6 and LMNA are colored red and featured as central points in two opposing nodes of interactions. A third node (upper middle) shows interaction partners that are shared by SIRT6 and LMNA. Proteins whose interactions are enhanced by the centSIRT6 allele are colored yellow. Proteins that interacted equally with wild‐type and centSIRT6 alleles are uncolored. H1.5 is a special case and colored orange because it showed increased interaction with the centSIRT6 and decreased interaction with LMNA in the presence of the centSIRT6 allele. Proteins known to be ribosylated in previous reports are shown as hexagons.

Data information: For C‐F, n = 3 BR; error bars = SD. Student's t‐test, two‐tailed. Asterisks indicate P < 0.05. Source data are available online for this figure.

Figure 7. Altered molecular functions of centSIRT6

CentSIRT6 allele shows reduced deacetylation activity and enhanced mADPr activity. This results in enhanced DNA repair, improved LINE1 suppression, and cancer cell killing. centSIRT6 shows stronger interaction with LMNA and enhances LMNA interactions with its partners.