Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Vera Gorbunova ( vera.gorbunova@rochester.edu ).

SIRT6 KO and WT mice were acquired from Jackson laboratories (strain 129 Sirt6tm1Fwa/J, 006050) and maintained in accordance with the regulations designated and approved by the University of Rochester Committee on Animal Resources (UCAR), which adheres to FDA and NIH animal care guidelines and reviews all animal protocols prior to approval (UCAR 2017-027). Animals were housed using microisolator technology in SPF conditions in a one-way facility. All mouse-derived cell lines were isolated from these animals. All primary cells were maintained in physiological 3% O2 concentration. All cell lines were tested every month for mycoplasma contamination. Both cell line and mouse reagents were derived from mixed sex cohorts determined by anogenital distance (in the case of organ derived cell lines) or by Sry PCR genotyping (in the case of MEFs). Experimental WT and SIRT6 KO MEF cell lines were represented by both male and female-derived cell lines. The SIRT6 identity of the cell lines was authenticated by PCR genotyping. MOSES mice () were all male and provided from Bar Ilan University.

Method Details

NRTI Treatments Pregnant SIRT6 heterozygous mice were administered 2 mg/mL 3TC or d4T in drinking water starting immediately after mating. Postnatally, the pups were given 400 mg/kg/day d4T, or 600 mg/kg/day 3TC, or water (control) by a flexible pipette into the animal’s mouth once a day. For treatment of the WT mice, male and female C57BL/6 mice, after weaning, received d4T in drinking water at 2 mg/mL. Mice were euthanized for blood and tissue collection at 55 weeks of age.

Analysis of Cytokines in Mouse Plasma Following d4T Treatment 55-week-old C57BL/6 mice were maintained either with or without 2 mg/mL d4T in drinking water (neutral pH) since weaning (4-5 week old). After reaching 61 weeks of age, mice from each group were either treated or untreated with i.p.-injected poly(I:C), 25 mg/mouse, to induce interferon type I response. Control groups were injected with the vehicle (PBS). Blood was collected 6 hours post treatment using heparin tubes. Equal volumes of plasma were combined from two males and two females from each group for the detection of circulating cytokines and chemokines using Mouse Cytokine Antibody Array C3 kit (RayBiotech, Norcross, GA) according to the manufacturer’s protocol. Briefly, the membranes precoated with capture antibodies were blocked and then incubated with plasma overnight at 4°C (plasma was diluted two-fold with blocking buffer). The membranes were then washed with washing buffer and incubated with biotinylated detection antibody cocktail overnight at 4°C. Following this step, the membranes were washed once again and incubated with streptavidin-HRP for two hours and developed using detection buffers provided in the kit. The immunoblot images were captured and visualized using the BioRad Molecular Imager GelDoc and the intensity of each spot was analyzed using ImageJ software.

Cell Culture All cell lines were maintained in humidified incubators at 5% CO 2 , 5% O 2 , at 37°C. Cells were grown in Eagle’s minimum essential medium with 15% fetal bovine serum and 1x penicillin/streptomycin. Drug treated cells were supplemented with 10 μM 3TC or D4T. The cell lines are routinely tested for mycoplasma contamination.

Transfections Transfections were carried out by plating cells at a density of 500,000 cells/10 cm plate two days prior to transfection. Transfections were carried out using the Amaxa Nucleofector with Normal Human Dermal Fibroblast transfection solution.

Quantitative RT-PCR Van Meter et al., 2014 Van Meter M.

Kashyap M.

Rezazadeh S.

Geneva A.J.

Morello T.D.

Seluanov A.

Gorbunova V. SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age. Total RNA was isolated from cells at 80% confluence using Trizol Reagent and then treated with DNase. cDNA was synthesized using Superscript III (Life Technologies) cDNA kit with the Random Hexamer primer. qRT-PCR was performed on the BioRad CFX Connect Real Time machine with SYBR Green Master Mix (BioRad) using 30 ng of cDNA per reaction with 4x reactions/sample. Probes targeting a conserved region of the L1MdA1 family the first 108 bp of ORF1 coding region were used to assess full length L1 transcripts. Additonal primers targeting bp 2288-2430 of the L1MdA ORF2 reading frame were assessed, along with primers to the L1MdTf family ORF1 and ORF2 reading frames (bp 706-900 and bp 3362-3536, respectively). All primer sets were tested for specificity and efficiency. All primer sets produced comparable results and expression trends equivalent to the L1MdA ORF1 primer pair. Efficiency verified L1MdA1 (mL1) primers previously described were used to assess L1 transcript abundance standardized to QuantumRNA Actin Universal primers (). All primers can be found in Key Resources Table

L1 DNA Content Tissues were harvested from animals and immediately frozen in liquid nitrogen. Tissues were cold processed with pestle and mortar and then genomic DNA was isolated using Quiagen’s DNeasy Blood and Tissue kit. DNA concentration was measured in quadruplicate and then serially diluted to 3 picograms/μl. Two μl of diluted genomic DNA was then loaded into SYBR Green Master Mix reaction and assayed using mL1 primers on BioRad CFX Connect Real Time machine.

Cytoplasmic DNA Extraction Shen et al., 2015 Shen Y.J.

Le Bert N.

Chitre A.A.

Koo C.X.

Nga X.H.

Ho S.S.

Khatoo M.

Tan N.Y.

Ishii K.J.

Gasser S. Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells. Shen et al., 2015 Shen Y.J.

Le Bert N.

Chitre A.A.

Koo C.X.

Nga X.H.

Ho S.S.

Khatoo M.

Tan N.Y.

Ishii K.J.

Gasser S. Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells. For MEFs, cells were grown to 75% confluence, gently trypsinized from the plate, counted and collected by centrifugation. A cytoplasmic lysis solution () was used to resuspend cells, which were incubated at 4°C on a rotor for 10 min. Nuclei were removed by centrifugation and the supernatant was treated as previously described (). A solution of 3M CsCl, 1M potassium acetate, and 0.67M acetic acid was added to supernatant, incubated on ice for 15 min, then centrifuged to remove any residual cellular debris and genomic DNA before column purification using Qiaquick PCR cleanup columns. Final elutions were quantified and assayed for nuclear genomic contamination using primers for GAPDH and 5S ribosomal subunit with 10 ng of DNA via qPCR. For organs, organs were harvested from animals and mixed cell-type cultures were generated from the organs in F12 media (GIBCO). Cells were passaged once after initial colonies formed from processed tissue. At 75% confluence, cyptoplasmic fractions were isolated by direct application of cytoplasmic lysis solution direction to the plate and incubated at 4°C with gentle shaking for 10 min. Lysates were then collected and processed in the same manner as described above.

Mouse Activity Assays For flight response assays, animals were placed in an open arena in the center of a 4” radius stage and then timed until they fully exited the stage. Full exit is defined as all four limbs outside of the circle. Each animal was tested 3 times, with 30 min rest intervals in normal housing between trials. Foraging and exploration activity were assayed by placing animals in a gridded arena for 30 s at a time. A camera was used to record animal movement, which was then quantified using Tracker software. Animals were allowed to rest in 30 min intervals between trials in normal housing to prevent acclimation to the arena. Deacon (2013) Deacon R.M. Measuring the strength of mice. Mouse strength was assayed by inverted screen test as described in. A mouse was placed in the center of the wire mesh screen, the screen was rotated to an inverted position over 2 s, with the mouse’s head declining first. The screen was kept above a padded surface. The time when the mouse fell off was recorded. The following scoring was used: Falling between 1-10 s = 1; Falling between 11-25 s = 2; Falling between 26-60 s = 3; Falling after 60 s = 4. For each animal the point score was calculated as a sum of the scores for three trials.

Immunofluorescence and Apoptosis Mao et al., 2011 Mao Z.

Hine C.

Tian X.

Van Meter M.

Au M.

Vaidya A.

Seluanov A.

Gorbunova V. SIRT6 promotes DNA repair under stress by activating PARP1. γH2AX and 53PB immunostaining was carried out as previously described (). Anti- γH2AX and anti-53BP1 antibody was purchased from Abcam (ab22551 and ab36823). For ssDNA immunostaining, cells were fixed with PFA, followed by 24hr methanol incubation at −20°C. Cells were then incubated at 37°C for 4hrs with RNaseA. Antibody from Millipore (MAB3868). Apoptosis in fibroblasts was measured using the Annexin V Staining Kit (Roche). Apoptosis in tissues was measured using TUNEL method with in situ apoptosis detection kit, Abcam (ab206386).

Western Blotting Cells and tissues were collected using a 5% SDS, 100mM Tris pH = 7 solution and incubated on ice for 15 min, during which time the samples were passed through a large guage needle several times and vortexed every 5min. Samples were then spun at 14,000 RPM to remove debris and the supernatant was mixed 1:1 with 2x laemmli buffer. Samples were boiled for 20min before being centrifuged at 14,000 RPM for 1min and loaded into a BioRad Criterion 4%–20% gel. After transfer to PDVF membrane and blocked (5% dehydrated milk) for 2hr at RT, membranes were incubated overnight with 1:500 dilution of ORF1p or 1:10,000 β-tubulin in 2.5% blocking buffer. Membranes were washed 3x with TBST for 10 min each before secondary antibody in TBST was added for 2hr incubation at RT. Membranes were washed and then imaged.

ORF1p Immunostaining Liver specimens from 5 m and 25 m old animals were embedded in OCT and cyrosectioned to 8 μm. Slides were then fixed with 4% PFA and 0.5% Triton X-100 in PBS for at room temp for 20min. Slides were blocked with 4%BSA, 2% donkey serum and 0.1% Triton X-100 for 1hr at RT. Anti-ORF1p antibody diluted in blocking solution (1:200) was incubated on slides overnight at 4°C in a rocking humidified chamber. Slides were washed with PBS+0.2% Triton X-100 3x for 15min. Secondary antibody (AlexaFluor 546, Life Technologies) diluted in blocking buffer was added and incubated for 2hr at RT, followed by 3x 15min washes with PBS+Triton X-100. Slides were stained with 2ug/mL DAPI in PBS+0.2% Triton X-100 for 15 min, prior to mounting with ProLong Antifade Mountant.

Histology Mouse tissue was fixed in Bouin’s fixative prior to being embedded in paraffin. Fixed tissues were then sectioned at 6 μm and hematoxylin/eosin staining was performed by standard methods at the University of Rochester Medical Center’s HBMI core.

Synthesis of 5′-O-Methyl d4T Reaction was conducted in oven-dried glassware under an argon atmosphere. Reaction solvents were obtained from a solvent system by Innovative Technologies. Reagent grade solvents were used for all work-up procedures and extractions. D4T was purchased from Matrix-Scientific, sodium hydride (60% dispersion in mineral oil) and methyl iodide were purchased from Sigma-Aldrich. Nuclear magnetic resonance spectra were obtained on a Bruker Avance 500 MHz spectrometer. Mass spectra were obtained using a Shimadzu LCMS-2010 mass spectrometer. 3 . The organic layer was dried over magnesium sulfate, concentrated in vacuo, and then purified by silica gel chromatography (elution by 1:1 then 1:4 hexanes/EtOAc) to afford 148 mg of the final product, > 97% purity. The spectroscopic and spectrometric data were consistent with those reported in the literature ( Fowler et al., 2014 Fowler B.J.

Gelfand B.D.

Kim Y.

Kerur N.

Tarallo V.

Hirano Y.

Amarnath S.

Fowler D.H.

Radwan M.

Young M.T.

et al. Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity. 1H NMR (500 MHz, DMSO, ppm): δ 11.31 (s, 1H), 7.50 (d, 1H), 6.82 (dd, 1H), 6.42 (dd, 1H), 5.91 (dd, 1H), 4.88 (s, 1H), 3.56 (m, 2H), 3.28 (s, 3H), 1.75 (s, 3H) To a solution of d4T (224 mg, 1 mmol) in dry THF/DMF (6 mL, 5:1 v/v) was added sodium hydride (400 mg, 10 mmol). The reaction mixture was stirred for 10 min under an argon atmosphere. The reaction mixture was then cooled to 0°C, and methyl iodide (67 μL, 1 mmol) was added slowly. The reaction mixture was stirred overnight while temperature was slowly raised from 0°C to room temperature. The reaction was quenched by methanol, neutralized by acetic acid, then volatiles were removed by evaporation. DMF was removed by azeotropic distillation with toluene. The resulting solid was suspended in DCM, and the mixture was washed with aq. NaHSO. The organic layer was dried over magnesium sulfate, concentrated in vacuo, and then purified by silica gel chromatography (elution by 1:1 then 1:4 hexanes/EtOAc) to afford 148 mg of the final product, > 97% purity. The spectroscopic and spectrometric data were consistent with those reported in the literature ().H NMR (500 MHz, DMSO, ppm): δ 11.31 (s, 1H), 7.50 (d, 1H), 6.82 (dd, 1H), 6.42 (dd, 1H), 5.91 (dd, 1H), 4.88 (s, 1H), 3.56 (m, 2H), 3.28 (s, 3H), 1.75 (s, 3H)

Comet Assays Mouse embryonic fibroblasts were maintained in control or drug treated media for 15 population doublings and then analyzed for DNA damage using the Trevigen CometAssay system. In brief, non-confluent cells were embedded in agarose, fixed, and then subjected to electrophoresis in a neutral buffer solution. Cells were then stained using SYBR Gold and analyzed by microscopy. Quantitative analysis was performed using CASP software.

Blood Glucose Analysis Blood was collected from tail bleeds at the indicated age, and serum glucose was measured. Serum glucose was measured using One-Touch Ultra-2 blood glucose glucometer kit, per the manufacturer’s instructions.

Bone Marrow Stem Cell Counting Bone marrow nucleated cells were isolated from 2 femurs and 2 tibias from each mouse, stained with trypan blue and counted. Two million bone marrow cells were stained with DAPI and antibodies (BioLegend): anti-mouse lineage-Pacific blue, anti-mouse Sca1-APC, anti-mouse c-Kit-PE/Cy7, anti-mouse CD48-APC/Cy7, anti-mouse CD150-PerCP/Cy5.5, followed by flow cytometry analysis. In methylcellulose assay, 50,000 cells per ml were plated onto ultra-low attachment 35mm plates, incubated in a humidified 37°C incubator with 5% CO 2 and 20% O 2 . Colonies were scored 12-14 days later.

LINE1 RNAi Vector Systems The ABM Good iLenti siRNA vector system was used to generate the siRNA cassette using the conserved L1 sequence, TGGACCAGAAAAGAAATTCCTC. The BLOCK-iT Pol II miR RNAi Expression Vector was used to generate the shRNA expressing vector. Five shRNAs were combined into the final vector. The targeting sequences are as follows; shRNA 1: TCCAAATAGACTGGACCAGAA, shRNA 2: AGAGCCTGGACAGATGTTATA, shRNA 3: GAGGAGTAGACGGCAGGAAAT, shRNA 4: TGGGATTAGTGCAGAGTTCTA, shRNA5: CCATACTTATCTCCTTGTACT

DNA FISH Cells were rinsed with PBS and then fixed with 4% paraformaldehyde for 10 min at 37°C, followed by a wash with ice-cold PBS and cold 100% methanol for 10 min. 70% ethanol was added and incubated at RT for 10 min. Cells were incubated with 1mg/mL RNase A for 30min at RT. 1M Tris pH 8.0 was added for 5 min prior to addition 2 ng/μl of 5′ AlexaFluor 288 labeled probe in hybridization buffer (1mg/mL yeast tRNA, 0.005% BSA, 10% dextran sulfate, 25% dionized formamide, 2X SCC). Slides were incubated overnight in a humidity box at 37°C. Cell were then washed once with 4x SCC for 5min, followed by 3 washes with 2x SCC for 5min. Mounting media with DAPI was added and the slides sealed. For controls, slides were incubated with PureLink DNase for 15min prior to probe hybridization.

Immunoprecipitation of cGAS Cultured MEFs were washed with PBS prior to crosslinking and fixation. 200mJ of UV radiation was applied using a Stratolinker UV system, followed by cell harvest and counting. Cells were then fixed for 10 min in 4% PFA and washed 3x with PBS. Cells were then lysed for 30 min at 4°C while rotating using a lysis buffer containing 20 mM Tris-HCl [pH 7.5], 0.5 mM EDTA, 150 mM NaCl, 10 mM KCl, 0.5% Triton, 1.5 mM MgCl2, 10% glycerol, 0.2 mM PMSF, 10 mM β-mercaptoethanol. Lysates were cleared by centrifugation at 12,000 g for 30 min at 4°C. cGAS was bound using either 5ug cGAS D3080 (Cell Signaling) or ABF124 (Millipore) for an overnight incubation, followed by the addition of 30 μL of Agarose A beads with salmon sperm DNA and 2h incubation while rotating. Beads were washed 5x with lysis buffer before DNA isolation using QIAGEN DNeasy Blood and Tissue kit.

p16 Luciferase Measurement in d4T Treated Mice Forty six weeks old male and female p16(LUC) mice were imaged to estimate the luminescence and randomized based on the cumulative luciferase signal between groups maintained on regular water or on water with 2 mg/mL of d4T. Level of luminescence of each individual mouse was assessed on week 9 and 22 following placement on d4T (55 and 68 weeks of age, respectively). Burd et al., 2013 Burd C.E.

Sorrentino J.A.

Clark K.S.

Darr D.B.

Krishnamurthy J.

Deal A.M.

Bardeesy N.

Castrillon D.H.

Beach D.H.

Sharpless N.E. Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model. Male and female C57BL/6J mice with hemizygous p16(Ink4a) knock-in of firefly luciferase (p16Ink4a/Luc) were obtained from our breeding colony, originally obtained from Dr. Norman Sharpless (). To assess accumulation of p16(LUC)-positive cells, mice were injected intraperitoneally with a 200 μL solution of 15 mg/mL D-luciferin potassium salt (Syd Labs; Boston, MA) in D-PBS without calcium and magnesium. At 10 min post-injection, isoflurane-anesthetized mice were placed into the IVIS Spectrum in vivo bioluminescent imaging system (PerkinElmer; Waltham, MA) for detection of luciferase activity (60 s exposure). Bioluminescence in p16(LUC) mice was quantified as total flux (p/s) of luminescent signal from the whole body using via Living Image software.