IntroductionTardigrades (also termed water bears) are an invertebrate phylum of > 1200 species with broad-reaching habitats. Many can survive desiccation, extreme temperatures, high pressure, intense irradiation, and exposure to space1. The mechanisms by which various tardigrade species resist such extreme stressors are poorly understood. Ramazzottius varieornatus is highly resistant to ionizing radiation (IR); capable of surviving > 48 h after a dose of 4000 Gy2, compared to the human LD50 of ~4.5 Gy3. The R.varieornatus Dsup (Damage suppressor) protein is chromatin associated and predicted to promote IR resistance, being absent from IR sensitive tardigrade species4. Indeed, when expressed in human cells, Dsup localizes to nuclear DNA and confers IR-resistance accompanied by reduced levels of DNA single- and double-strand breaks (SSBs and DSBs)4. Radioprotection is also conferred when Dsup is expressed in tobacco plants5, flies6 and mice7.While IR can directly induce SSBs and DSBs, much of its genotoxicity is mediated by hydroxyl radicals (OH•) generated when radiation interacts with water molecules8. Indeed high-energy OH• are the most powerful oxidant among the reactive oxygen species (ROS), reacting with DNA bases to form lesions (including 8-oxoguanine; 8-oxo-G), while oxidation of the deoxyribose backbone dissociates sugar-phosphate bonds leading to DNA breaks9. Consistent with Dsup protecting against OH•, it also reduces the number of DNA breaks in human cells exposed to hydrogen peroxide (H2O2)4,10. Throughout life, oxidative DNA damage is continually generated from aerobic metabolism, with the resulting mutations thought to contribute to the ageing process11 and the development of age-related diseases12, such as neurodegeneration13 and cancer14,15. Further, most cancer treatments cause oxidative DNA damage and strand breaks, contributing to long-term side effects in survivors16. As such, the means by which proteins such as Dsup protect the genome from oxidative damage are of extreme interest.R.varieornatus Dsup is a 445 amino acid protein17 that is intrinsically disordered17,18,19. Of note, disorder at their N- and C-termini is a feature of proteins that scan and engage DNA, consistent with a DNA-binding role for Dsup20,21. C-terminal deletion (Δ aa 208-445) abrogates Dsup binding to naked DNA or human chromatin4. Indeed, Dsup binds with higher affinity to reconstituted chromatin over free DNA, and sequences within aa 360–445 are required for the association with chromatin and protection from OH• induced DSBs22. While Dsup upregulates the expression of various DNA repair genes in HEK293 cells10, the protein also prevents DNA damage by binding chromatin in a reconstituted system lacking DNA repair factors22.Within the Dsup C-terminal region, an eight amino acid stretch (aa 363–370, RRSSRLTS) has homology to the core consensus (RRSARLSA) of the nucleosome binding region of vertebrate High Mobility Group-N (HMGN) proteins22,23,24. The chromatin binding of HMGN proteins influences a wide variety of functions (including embryogenesis, development and disease protection) across diverse cell types and species25. In the prevailing model revealed by the in vitro reconstituted system, Dsup protects the genome from DNA damage by physically shielding chromatin from hydroxyl radicals, in a manner that involves its HMGN-like motif and likely additional C-terminal sequences22. However, whether this model operates in vivo is unknown.Here, we show that when expressed in budding yeast at histone-like stoichiometry R.varieornatus Dsup uses its HMGN-like motif and adjacent C-terminal sequences to protect the genome from oxidative DNA damage in a manner dependent on chromatin engagement but independent of scavenging hydroxyl radicals or differentially regulating DNA repair pathways. Dsup expression also extends yeast replicative lifespan in the face of chronic endogenous oxidative DNA damage. A detailed analysis of [Dsup: nucleosome] engagement shows that its HMGN-like motif mediates interaction with the H2A/H2B acidic patch on the nucleosome surface, while its distal C-terminal sequences bind DNA. Of note, such multivalent binding supports the observed broad engagement with in vivo chromatin, independent of the landscape of histone post-translational modifications (PTMs). Our studies indicate that tardigrade Dsup can be introduced to a heterologous in vivo system and confer viability and longevity in the face of elevated levels of oxidative damage. This is achieved by physically coating the chromatinized genome via multivalent interactions to prevent hydroxyl radicals from damaging genomic DNA.ResultsHeterologous expression of R. varieornatus Dsup in budding yeast protects against oxidative damage and promotes longevity in the face of increased oxidative stressTo initiate this study we expressed epitope tagged 6His-Dsup-FLAG (hereafter Dsup-FLAG or Dsup (WT)) in yeast under the constitutive high output TDH3 promoter26 (Supplementary Data File 1), with the goal of achieving in vivo protein levels sufficient to coat the genome. This yielded Dsup-FLAG of similar abundance to H2B-FLAG (Fig. 1a). To investigate the response of Dsup-FLAG yeast to chronic oxidative damage, we performed serial dilution assays on plates containing H2O2, observing a ~ 25-fold increased survival relative to yeast lacking Dsup (Fig. 1b). This did not extend to general genotoxin protection, since Dsup-FLAG slightly decreased yeast survival in response to non-oxidative DNA-damaging agents such as alkylating methyl methanesulfonate (MMS), DNA-intercalating Zeocin, or ultra violet (UV) light (Fig. 1b).Fig. 1: Heterologous expression of tardigrade Dsup in budding yeast promotes survival after chronic exposure to an oxidative DNA damaging agent, reduces related DNA damage, and extends lifespan through chronic endogenous oxidative damage.a Immunoblot of Dsup-FLAG (from pTDH3-6His-Dsup-FLAG) and H2B-FLAG in yeast strains containing a single integrated copy of each tagged gene (Supplementary Data File 1B). The same protein samples were ran on a second gel for detection of GAPDH and H2B. Three dilutions of protein extracts loaded as indicated. EV, Empty vector. b Relative sensitivity of yeast strains (five-fold serial dilutions) to indicated doses of hydrogen peroxide (H2O2), methyl methanesulfonate (MMS), Zeocin (Zeo), or UV. Yap1∆ is a positive control for sensitivity to oxidative DNA damage (H2O2). Cac1∆rtt106∆ is a positive control for sensitivity to other genotoxins. WT (BY4741), wild-type yeast. Dsup (WT), Dsup1 (aa 1-455). c Yeast cells expressing Dsup (WT) show reduced levels of 8-hydroxy 2 deoxyguanosine (8-OhdG) in response to oxidative DNA damage (120 min in 10 mM H2O2; mean and standard deviation from three independent experiments; p-value determined using a two-sided Student’s t-test). d. Yeast cells expressing Dsup (WT) show increased replicative lifespan when exposed to chronic oxidative damage (sod1∆−/+ Dsup (WT); n = 30 individuals for each background). Source data are provided as a Source Data file.Full size imageIn reconstituted assays recombinant Dsup protects chromatinized plasmid DNA from DSBs caused by hydroxyl radicals22, so we asked if Dsup expression protected the in vivo yeast genome from oxidative DNA damage. 8-oxoguanine (8-OHdG) is generated when ROS react with DNA27, so we quantified this base modification after transient exposure to H2O2 and observed a significant reduction in the presence of Dsup (Fig. 1c).ROS and oxidative damage increase with age, and reducing oxidative damage extends the lifespan of multiple species (yeast, worms, fruit flies, mice28), while elevated ROS production shortens lifespan29. We thus asked if Dsup expression could extend yeast lifespan. In otherwise WT yeast Dsup had a negligible impact on chronological lifespan (the length of time a cell survives in a non-dividing state; Supplementary Fig. 1a), while replicative lifespan (the maximum number of times a cell can divide) was slightly reduced (Supplementary Fig. 1b). Cells lacking the superoxide dismutase (SOD) genes are deficient in their ability to process both endogenous and exogenous ROS. As a result, they accumulate oxidative stress and damage, such that yeast lacking Sod1 have a shortened replicative lifespan30. When expressed in sod1Δ yeast, Dsup significantly increased replicative lifespan (Fig. 1d), suggesting enhanced survival and longevity in the face of chronic oxidative damage.Dsup promotes yeast survival upon oxidative damage via its C-terminal sequences, and reduces the cellular redox state via its N-terminal sequencesVertebrate High Mobility Group-N (HMGN) proteins22 contain a conserved HMGN motif (core consensus RRSARLSA31) required for chromatin binding and protein fuction25,32 (Fig. 2a). The Dsup C-terminal region contains an eight amino acid stretch with homology to this consensus22 (the HMGN-like motif: aa363-370, RRSSRLTS: Fig. 2a), suggesting physiological relevance. We thus made mutant forms of Dsup by substituting three positively charged arginines in the motif with negatively charged glutamic acid (Dsup HMGN-3R/3E: R363E/R364E/R367E), or by deleting the entire C-terminus including the HMGN-like motif (Dsup ∆HMGN ∆C: ∆360-445); alleles previously investigated in vitro22. Dsup contains a predicted nuclear localization signal (NLS)33 removed by ∆HMGN ΔC, so to this we added a repeated SV40 NLS (PKKKRKVPKKKRKV)34 to create Dsup ∆HMGN ∆C + NLS (Fig. 2a, b). By immunofluorescence Dsup (WT), Dsup HMGN-3R/3E and Dsup ∆HMGN ∆C + NLS primarily localized to the nucleus, while Dsup ∆HMGN ∆C was primarily cytoplasmic, presumably due to removal of the predicted NLS (Fig. 2c). Dsup ∆HMGN ∆C was thus omitted from further in vivo study. Importantly, the Dsup HMGN-3R/3E and Dsup ∆HMGN ∆C + NLS proteins were expressed at least as well as Dsup (WT) in yeast (Fig. 2d), and each did not significantly impact cell growth (Fig. 2e).Fig. 2: Heterologous Dsup is nuclear localized in yeast and does not negatively impact growth.a Alignment of human HMGN1-3 identifies the HMGN core consensus (RRSARLSA). Also shown the R.varieornatus Dsup HMGN-like motif (aa 363-370, RRSSRLTS [underlined]) and alleles that mutate or delete this area and/or the downstream C-terminal region (aa 371-445) for phenotypic and/or biochemical studies (adapted from22) (Supplementary Data File 1E). Residues in red, including three functionally important arginines, are identical between the HMGN core consensus and Dsup. Green indicates the duplicated SV40 nuclear localization signal (NLS: PKKKRKVPKKKRKV) added to Dsup ∆HMGN ∆C for yeast expression. Pink indicates mutated residues in the HMGN-like motif to create -3R/3E (charge reversal) or -8A (charge neutralization). *, stop codon. b Schematic of Dsup wild-type (WT) and mutant alleles stably expressed in yeast for phenotypic studies (Figs. 1–4 [Dsup ∆C + NLS (as used in Fig. 7) not depicted]). N-terminal 6xHIS and C-terminal FLAG tags on each protein are not depicted. c Immunofluorescence to examine subcellular location of Dsup alleles (anti-FLAG) in yeast. DNA stain DAPI identifies nuclei. H2A-FLAG and GAPDH are respective controls for nuclear and cytoplasmic localization. EV, Empty vector. d Immunoblot shows relative expression of Dsup alleles (anti-FLAG) in yeast. Anti-GAPDH is a loading control from samples run on the same gel. e Representative growth curves of yeast expressing Dsup alleles. Source data are provided as a Source Data file.Full size imageWe next examined the ability of Dsup mutants to enhance yeast survival through chronic or acute oxidative stress; the former by growth on plates containing H2O2 (Fig. 3a), the latter by exposing cells to H2O2 in liquid culture for 1.5 hours before recovery on plates with no oxidizing agent. (Fig. 3b). In each case Dsup HMGN-3R/3E protected cells similarly to Dsup (WT), while Dsup ΔHMGN ΔC + NLS yielded no protection, with growth indistinguishable from an Empty-vector control strain. As such, the entire C-terminus of Dsup (aa 360-445) is important for protecting yeast from oxidative DNA damage.Fig. 3: Dsup promotes survival after oxidative DNA damage in a manner that requires its C-terminus but is not due to ROS scavenging.a The Dsup HMGN-like motif (aa 363-370) and C-terminal region (aa 371-445) are required to confer yeast resistance to chronic H2O2 -mediated oxidative damage (concentrations indicated). yap1∆ is a positive control for sensitivity to H2O2. WT, wild-type. EV, Empty vector. b The Dsup HMGN-like motif and C-terminal region are required to confer yeast resistance to acute H2O2 -mediated oxidative damage. Graph plots percentage cell survival after 90-minute exposure to H2O2 (concentrations indicated). Shown are average and standard deviation of experiments using three independent colonies for each strain. p-values were determined using two-sided Student’s t-test. c Relative redox state in the cytoplasm (left; roGFP2-Grx1 [redox sensitive GFP - GlutaRedoXin 1] reporter) and nucleus (right; roGFP2-Grx1-NLS reporter) for indicated yeast strains (color key as in b) through H2O2 exposure (4 mM; time in min). T0 sample was taken immediately after H2O2 addition. Shown are average and standard deviation of experiments performed from three independent colonies. Source data are provided as a Source Data file.Full size imageFree-radical scavengers are effective at protecting yeast from oxidative stress and extending lifespan35, so we investigated if Dsup mediates such a role. Redox-sensitive GFPs (roGFPs) are excited at 405 nm in oxidizing but 488 nm in reducing conditions, so relative emissions after excitation at [405/488 nm] report on changes in redox state. In this manner, H2O2 treatment of yeast cells caused the redox state to increase in the cytoplasm and nucleus (as determined with compartment specific reporters: respectively roGFP2-Grx136 or roGFP2-Grx1-NLS) (Supplementary Fig. 2 and Fig. 3c). Of note, Dsup (WT), Dsup HMGN 3 R/3E and Dsup ΔHMGN ΔC + NLS each reduced the initial and post-H2O2 treatment redox state in the cytoplasm and nucleus (all relative to EV; Empty vector) (Fig. 3c). Importantly, these data show that while sequences within the Dsup N-terminal region (aa 1-359) reduce free-radical levels, this is insufficient to confer enhanced survival in response to H2O2 since Dsup ΔHMGN ΔC + NLS cells were not resistant to this genotoxin (Fig. 3a, b).Dsup binds chromatin throughout the yeast genome, in a manner dependent on sequences within the C-terminusDsup was first isolated from the chromatin fraction of Tardigrade cells4, and shown to bind preferentially to nucleosomes over free DNA in vitro22. Therefore, we investigated if Dsup binds yeast chromatin in vivo. After cellular fractionation to separate chromatin-bound from soluble proteins, Dsup (WT) and Dsup HMGN-3R/3E both enriched in the chromatin fraction (Fig. 4a). By contrast, Dsup ΔHMGN ΔC + NLS was entirely soluble (Fig. 4a), suggesting that despite nuclear localization (Fig. 2c), it does not bind chromatin. Of note, the chromatin association of Dsup (WT) and Dsup HMGN-3R/3E, but not Dsup ΔHMGN ΔC + NLS, paralleled their ability to promote cell survival in the face of oxidative damage (Fig. 3a, b), suggesting that chromatin binding is key.Fig. 4: Dsup fractionates with yeast chromatin and associates across the yeast genome without apparent bias.a The Dsup HMGN-like motif (aa 363-370) and C-terminal region (aa 371-445) are required for the association with yeast chromatin. Yeast spheroblasts (Input) from indicated strains were resolved to Soluble and Chromatin fractions and immunoblotted as indicated. Confirming effective fractionation: GAPDH is a soluble protein, H2A is chromatin bound. EV, Empty vector. The samples detected for FLAG were resolved on one gel and the same samples were ran on a second gel to detect GAPDH and H2A. b CUT&RUN to examine Dsup allele interactions across the yeast genome (anti-FLAG). For each strain IgG (assay background) and anti-H3K4me3 (active gene promoters) were respectively included as negative and positive controls. Each target is group-scaled (after normalization to E.coli spike-in) to the highest signal in the depicted IGV window (IgG (82); H3K4me3 (1356) or Dsup-FLAG (311)). Data is from a representative CUT&RUN of biological replicates (sequence statistics in Supplementary Data File 2). Source data are provided as a Source Data file.Full size imageThe presence of tardigrade Dsup in human and plant cells alters transcription factor binding and gene expression in response to DNA damage5,10. This is suggestive that Dsup binds certain areas of the genome to influence transcription. Alternatively, to have the greatest physically protective effect from oxidative DNA damage, Dsup might be expected to uniformly coat chromatin. To investigate these possibilities, we used Cleavage Under Targets & Release Using Nuclease (CUT&RUN)37 to map 6His-Dsup-FLAG localization (by anti-FLAG), and observed that Dsup (WT) enriched across the yeast genome, without bias or selectivity (i.e., not showing gaps, peaks or domains; Fig. 4b). Of note, the ability of CUT&RUN to map transcriptionally active promoters with anti-H3K4me3 was unaffected by Dsup (compare Empty vector (EV) and Dsup (WT)), indicating minimal impact on local chromatin structure (Fig. 4b). This was confirmed by micrococcal nuclease (MNase) digestion of chromatin from yeast −/+ Dsup, where we observed only minor differences in efficiency (Supplementary Fig. 3).We next compared CUT&RUN across Dsup alleles, first noting that relative DNA yields post MNase digestion (prior to adapter ligation) were consistently Dsup (WT) » Dsup HMGN-3R/3E > Dsup ΔHMGN ΔC + NLS > EV) (Supplementary Fig. 4). This is mirrored in the CUT&RUN data, where Dsup HMGN-3R/3E showed less enrichment than Dsup (WT) across all genomic regions, while Dsup ΔHMGN ΔC + NLS resembled Empty vector (Fig. 4b; all data group scaled after normalizing to E. coli spike-in to allow comparisons of global changes in factor binding).Taken together, these data indicate that Dsup binds without obvious bias across the genome in a manner dependent on its C-terminus (which includes the HMGN-like motif). Further, while mutation of conserved arginines in the Dsup HMGN-like motif (-3R/3E) reduced chromatin binding and/or increased its turnover (revealed by the long incubation steps of CUT&RUN but not direct chromatin fractionation (Fig. 4a)), the allele still conferred protection from oxidative DNA damage (Fig. 3a, b). These data indicate that both the HMGN-like motif and distal C-terminal sequences contribute to global chromatin binding.Cells expressing Dsup are not transcriptionally primed for DNA repair nor do they have an enhanced transcriptional response to oxidative damageGiven that Dsup binds to yeast chromatin (Fig. 4), and this engagement is important for protection from H2O2 (Fig. 3), we next asked if Dsup also alters transcription in a manner that promotes the response to oxidative stress or DNA repair. Within the well characterized “environmental stress response” (ESR), genes involved in RNA processing and protein synthesis are transcriptionally repressed, while those involved in ROS detoxification and maintaining redox balance are activated38,39,40. To determine any Dsup influence on yeast gene expression we performed RNA-seq analysis. Here we observed transcriptional changes characteristic of the ESR 30 min after exposure of yeast cells −/+ Dsup to H2O2 (4 mM or 8 mM: Fig. 5 and Supplementary Data File 3). This included induction of genes involved in the removal of superoxide radicals (Group 1; 224 genes), and detoxification of cellular oxidants and oxidoreductases (Group 4; 43 genes). We also observed the characteristic transcriptional repression of transcription factors and proteins involved in amino acid and carbohydrate transport and transmembrane transport; halting these metabolic activities until the stress has been removed (Group 3; 220 genes). Notably, we did not observe an elevated ESR in cells expressing Dsup −/+H2O2 exposure (compare Dsup (WT) to Empty vector (EV) control: Fig. 5 and Supplementary Data File 3).Fig. 5: Dsup expressing yeast do not show a poised or elevated transcriptional response to oxidative stress.Yeast +/− Dsup were exposed to H2O2 (0, 4 or 8 mM for 30 min; T0 is untreated) and RNA-seq performed. Indicated comparisons show differentially expressed genes (Supplementary Data File 3) from the most significantly enriched functional annotation categories and their false discovery rates (FDR; as determined by DAVID). Group 2 Dsup-upregulated genes included a set involved in amino acid biosynthesis. However, this did not extend to activation of the ‘General Amino Acid Response’, including elevated protein levels of Gcn4 transcriptional activator80 (Supplementary Fig. 5). Data are the average of three independent repeats. EV, Empty vector. Source data are provided as a Source Data file.Full size imageOf interest, Dsup expression per se led to transcriptional changes for 220 genes relative to Empty vector control (Group 2: Fig. 5 and Supplementary Data File 3). This included a subset of genes involved in amino acid biosynthesis, though it did not extend to increased abundance of Gcn4 transcriptional activator (and so did not represent a ‘General Amino Acid Response’; Supplementary Fig. 5). However, Group 2 also included genes proximal to telomeres, or encoded from Ty transposable elements. Both are usually actively repressed locations in budding yeast41, suggesting Dsup expression may lead to some opening of repressive chromatin; in line with the minor increase in MNase digestion efficiency also observed in these cells (Supplementary Fig. 3).Together, this suggests that cells expressing Dsup are not transcriptionally poised to respond to oxidative stress, nor do they have an enhanced transcriptional response, including of DNA damage response genes, when it is encountered (Fig. 5). As such transcriptional profiling provides no explanation for the ability of Dsup to promote yeast cell survival in the face of oxidative damage.The Dsup HMGN-like motif mediates interactions with histones while the adjacent C-terminal region binds DNA, with each needed to fully protect yeast from oxidative damageHaving ruled out a transcriptional role for Dsup in protecting yeast from oxidative damage (Fig. 5), we set out to further characterize its interaction with chromatin (Fig. 4). To interrogate potential mode(s) of engagement, we used the Captify in vitro chemiluminescent assay42. Here the biotinylated target (e.g., free DNA or fully defined mononucleosome: Supplementary Data File 1D) couples to streptavidin-donor beads while epitope-tagged Query (e.g., various forms of 6His-Dsup-FLAG; Fig. 2a, Supplementary Fig. 6 and Supplementary Data File 1E22) couples to anti-tag acceptor beads. After mixing potential reactants the donor beads are excited at 680 nm, releasing a singlet oxygen that causes emission (520–620 nm) in proximal acceptor beads: this luminescent signal is directly proportional to the amount of [Donor - Acceptor] bridged by the [Target: Query] interaction (Supplementary Fig. 7). Binding is quantified by plotting Alpha Counts (fluorescence) as a function of protein concentration and expressed as relative EC50 (for all EC50rel from this study see Supplementary Data File 4).We first titrated salt (NaCl) to examine the potential impact of non-specific ionic interactions. At 150 mM Dsup (WT) similarly bound unmodified nucleosomes (rNuc) and naked DNA, but at 250 mM showed a distinct preference for nucleosomes (Fig. 6a). Choosing the ionic strength closest to normal saline (150 mM; ~0.9% NaCl), we next tested the impact of adding salmon sperm DNA (salDNA) competitor42,43. This identified an optimized condition where nucleosome binding was retained over free-DNA (Fig. 6b and Supplementary Data File 4), confirming that the Dsup association with DNA is a significant, but not exclusive, element of its interaction with chromatin22.Fig. 6: The Dsup HMGN-like motif and C-terminal region each contribute to nucleosome binding.a Impact of ionic strength (150 mM (normal saline, 0.9%) or 250 mM NaCl) on binding of Dsup (WT: 1 - 0 μM in two-fold serial dilutions) to unmodified nucleosome (rNuc on 147 bp DNA; 10 nM) or free DNA (147 x 601: 2.5 nM). b salDNA (1μg/ml in 150 mM NaCl) was a more effective competitor of Dsup binding to free-DNA vs. to nucleosomes, suggesting the latter involves multivalent engagement. c Mutation of the Dsup HMGN-like motif (aa 363-370; charge reversing -3R/3E, or charge neutralizing -8A) or deletion of the adjacent C-terminal region (Δ371-455; ΔC) each compromised DNA / nucleosome binding compared to Dsup (WT) (see also Supplementary Fig. 8). Note the relative binding of each allele to free DNA or nucleosome targets, where Dsup HMGN mutants preferred free DNA while Dsup ΔC preferred nucleosomes. Key and reaction conditions as in (b). Data are the average and standard deviation of two technical repeats. Source data are provided as a Source Data file.Full size imageWe next used Captify to examine the contribution of various Dsup regions for nucleosome / DNA engagement. Here Dsup ∆HMGN ∆C showed no binding (Supplementary Fig. 8); not unexpected given our findings from cell fractionation and CUT&RUN (Fig. 4 and Supplementary Fig. 4). We thus created mutants to isolate the contributions of the HMGN-like motif (Dsup HMGN -3R/3E and Dsup HMGN-8A) or adjacent C-terminal region (Dsup ∆C) (Fig. 2a and Supplementary Data File 1E). Relative to Dsup (WT), each mutant had reduced binding to free DNA and nucleosomes that was undetectable in the presence of salDNA (compare Figs. 6b and 6c). Interestingly Dsup HMGN-3R/3E and -8A each preferred free DNA while Dsup ∆C preferred nucleosomes, indicating the mutated regions differentially contribute to chromatin engagement. Of note, while both HMGN mutants showed the same binding profile, charge reversing -3R/E bound nucleosomes ~two-fold weaker than charge neutralizing -8A (EC50rel 61.53 vs. 32.47 nM: Supplementary Data File 4), as might be expected given residue charge is central to how the HMGN-like motif engages the nucleosome acidic patch32.This data indicates the Dsup C-terminal domain (aa 208-445) previously defined as responsible for chromatin binding4 actually contains at least two functional elements: the nucleosome binding HMGN-like motif (aa 363-370) and DNA binding C-terminal region (aa 371-445). Our yeast analyses (Figs. 1–4) had not explored any specific contribution of the C-terminal region so we created a new deletion mutant (Dsup ΔC + NLS: Δ371-445 + NLS) for phenotypic testing. This was expressed in yeast at reduced levels relative to other forms of Dsup (Fig. 7a) but conferred some resistance to chronic H2O2 exposure (Fig. 7b: compare Dsup ΔC + NLS to BY4741 or EV). Thus, an intact HMGN-like motif or C-terminal sequences are each sufficient to grant nucleosome binding and protection from oxidative damage (albeit each weaker than Dsup (WT)), but loss of both regions yields a non-chromatin binding and non-protective Dsup allele.Fig. 7: The Dsup HMGN-like motif and C-terminal region each contribute to yeast survival during oxidative damage.a Immunoblot shows relative expression of Dsup alleles (anti-FLAG) that delete or mutate (-3R/3E) the HMGN-like motif (aa 363-370) and/or C-terminal region (aa 372-455) in yeast (each N-terminal 6xHIS and C-terminal FLAG tagged; see also Fig. 2a–d). Anti-GAPDH is a loading control resolved on the same gel. EV, Empty vector. Dsup (WT), (aa 1-455). b Relative sensitivity of yeast strains (five-fold serial dilutions) to oxidative DNA damage by H2O2 (see also Figs. 1b and 3a). yap1∆ is a positive control for sensitivity to H2O2. WT (BY4741), wild-type yeast. Source data are provided as a Source Data file.Full size imageDsup binds nucleosomes via multivalent interactions with the histone tails, acidic patch, and DNAWe next employed Captify with conditions optimized for multivalent engagement (150 mM NaCl, 1 µg/ml salDNA) and a diversity of fully defined nucleosomes (Supplementary Data File 1D) to determine which surfaces are engaged by Dsup. Here (and relative to rNuc) Dsup showed ever-decreasing binding to a H3 tail delete (H3.1 NΔ32 or H3.3 NΔ32), H4 tail delete (H4NΔ15), or ‘tail-less’ nucleosome (trypsin digested of all histone tails) (Fig. 8a). Supporting that Dsup may have a particular preference for the H4 tail, and further that this might be charge-state mediated (where acetylation neutralizes lysine charge), binding was reduced to H4tetraAc relative to H2AtetraAc or H3tetraAc nucleosomes (Fig. 8b). As predicted by CUT&RUN (Fig. 4b), [Dsup: Nucleosome] binding was not impacted by H3K4me3, nor by other lysine tri-methyl states (at H3K9, H3K27, H3K36 or H4K20: Fig. 8c). Finally mutations (H2AE61A, H2AE92K and H2BE105A/E113A) within the acidic patch, a hub of interaction for nucleosome binding proteins44,45, abolished [Dsup: nucleosome] binding (Fig. 8d). Together, this indicates that the Dsup interaction with chromatin is mediated by DNA, the histone N-terminal tails, and the H2A/H2B nucleosome acidic patch (Fig. 9).Fig. 8: Dsup binds nucleosomal DNA, histone tails and the acidic patch.Relative interaction of Dsup (WT) with fully defined nucleosomes (Supplementary Data File 1d) containing histone tail truncations (a), lysine acetylations (b), lysine methylations (c), or acid patch mutations (d). All assays performed under optimized conditions (from Fig. 6b): Dsup (WT: 1 - 0 μM in two-fold serial dilutions), nucleosome (each as indicated; 10 nM), free DNA (147 × 601; 2.5 nM), 150 mM NaCl, 1 mg/ml salDNA. Data are the average and standard deviation of two technical repeats. Source data are provided as a Source Data file.Full size imageFig. 9: Model for multivalent association of Dsup with the genome to protect from oxidative DNA damage.Dsup chromatin-interacting regions include the histone tails / acid-patch binding HMGN-like motif (aa 363-370) and DNA-binding C-terminus (aa 372-455). Multivalent binding of tardigrade Dsup to the chromatinized yeast genome protects against oxidative DNA damage (as induced by H2O2 or elevated when SOD1 (Superoxide dismutase 1) is deleted (e.g., Figs. 1, 3 and 7). Independent mutations of the Dsup HMGN-like motif or C-terminal region weaken its association with nucleosomes, but these alleles still protect from oxidative DNA damage (e.g., Figs. 3, 4, 6 and 7). However simultaneous deletion of both regions (as in Dsup ΔHMGN ΔC or Dsup ΔHMGN ΔC + NLS) eliminates chromatin binding and genome protection (e.g., Fig. 4 and Supplementary Figs. 4 and 8). Created in BioRender. Khan, L.F. (2025) https://BioRender.com/16wiptg.Full size imageDiscussionTo explore the molecular basis of the extreme radioresistance of tardigrades, we investigated how R.varieornatus Dsup protects against oxidative damage in vivo. When expressed in budding yeast, Dsup coats the genome without apparent bias. This reduced oxidative DNA damage in a manner independent of ROS scavenging or amplifying transcriptional responses that protect from oxidative stress / enhance DNA repair. Rather, our data supports a model where Dsup uses at least two C-terminal regions to mediate multivalent interaction with several nucleosome surfaces and protect the underlying genome from oxidative DNA damage. Functionally this promotes yeast survival and longevity after exposure to elevated levels of hydroxyl radicals (Fig. 9).Dsup is intrinsically disordered17, which may allow it to dynamically wrap multiple nucleosome surfaces to shield DNA from damage. This includes a C-terminus enriched in positively charged amino acids to facilitate ionic interactions with negatively charged DNA17 (Fig. 6), and an HMGN-like motif 22 for potential nucleosome binding. HMGN proteins were originally defined in vertebrates46, where human HMGN2 uses its eponymous motif (RRSARLSA) to bind nucleosomes at the H2A/H2B acidic patch32,47. Dsup also binds the acidic patch (Fig. 8d), with this interaction lost in mutants that reverse or neutralize positively charged arginines in its HMGN-like motif (respectively Dsup HMGN-3R/3E or -8A: Fig. 6c). Individual disruption of the Dsup HMGN-like motif (HMGN-3R/3E) or adjacent C-terminal region (ΔC), yields mutants that retain some chromatin binding and improve yeast survival on exposure to oxidative damage (Figs. 3, 6 and 7). However, Dsup mutants lacking both regions (ΔHMGN ΔC) show no nucleosome / DNA binding and no protection (Supplementary Fig. 8 and Fig. 3). Redundancy in the multiple interactions between Dsup and chromatin would facilitate engagement even when certain surfaces are otherwise occupied by endogenous binding proteins, potentiating its ability to coat the genome. Binding studies with fully defined nucleosomes containing histone PTMs, tail deletions or acidic patch mutants show each surface contributes to Dsup engagement, with most PTMs of minimal impact (Fig. 8). Dsup could engage each of these surfaces independently or co-operatively: despite popular conception that the histone tails extend from the globular nucleosomal core48, their default high affinity interaction is with nucleosomal DNA49, which would co-localize at least two Dsup binding partners to promote engagement.A recent cryo-EM [Dsup: nucleosome] structure supports a model where the HMGN-like motif effectively engages the acidic patch, while other Dsup regions transiently adopt multiple conformations around the nucleosome19: a binding mechanism in high concordance with this study. Further, we note R. varieomatus histones are highly conserved with those from human (as used in Captify) and yeast (with which Dsup associates in vivo) (Supplementary Data File 1E). We thus propose that the interactions dissected in this study represent the endogenous situation in tardigrades. Successful gene editing of R.varieornatus has recently been reported50 so it is now possible to test this directly.There is precedent for proteins binding the genome to protect from irradiation and H2O2. Chromatin compaction protects DNA from free radical-mediated damage caused by ionizing radiation or iron in vivo and in vitro51,52,53, suggesting a direct mechanism rather than a particular feature of the cellular environment. Compacted chromatin also protects from ROS damage after direct incubation with H2O254. Additionally, deleting proteins involved in chromatin assembly and disassembly, including the ISWI, Chd1, and INO80 remodelers, renders chromatin more sensitive to DNA damage55. Dsup can now be added to the list of proteins that protect the DNA from H2O2-induced damage by direct chromatin binding, though the precise mechanism remains to be determined.It is notable that Dsup protected yeast from oxidative damage, but increased their sensitivity to MMS, bleomycin or UV (Fig. 1b). Meanwhile, Dsup confers protection from UV in humans and plants5,10,56. The reason for this difference is unknown, but it is possible that the stoichiometry of Dsup to nucleosomes matters (see below). Future studies should examine if Dsup expression in yeast delays the repair of DNA lesions generated by various genotoxins, potentially due to hindering access of their repair machineries. We note, however, that the growth rate of Dsup expressing yeast was not impacted (Fig. 2d), indicating they are fully competent for transcriptional regulation, DNA replication and mitosis – events one could imagine might be compromised on coating the genome with a heterologous protein. That this is tolerated is almost certainly due to [Dsup: nucleosome] complexation being highly dynamic19 (Fig. 4).Dsup expression in other species induces transcriptional changes that could grant improved DNA repair and thus contribute to their genotoxin resistance5,10,56. This includes a heightened transcriptional response to oxidative stress in rice57, and induced expression of DNA repair genes after UV or H2O2 exposure in human cells10. However, flies expressing Dsup show a decreased transcriptional response to irradiation, despite their increased resistance and fewer DNA double-strand breaks6. We found no evidence that Dsup expressing yeast primed or hyperactivated the known transcriptional responses that protect from environmental (including oxidative) stress or enhance DNA repair (Fig. 5). The transcriptional effects of Dsup expression thus appear quite distinct in different systems. Dsup expression in flies downregulated 733 genes (with only two upregulated), including those involved in transcription, chromatin assembly and DNA replication, suggesting the protein acts as a non-specific repressor6. By contrast, Dsup expression in yeast led to increased expression of 220 genes, most of which were normally repressed transposons or telomere-proximal genes (Fig. 5) suggesting that chromatin at these regions was made more accessible to the transcription machinery. How this could occur warrants further investigation.As noted above, it is possible that species-specific effects of Dsup expression are due to the expression levels in different organisms, suggesting titration is needed to receive beneficial effects. Indeed, in initial testing we expressed Dsup from yeast promoters of various strengths, but only that of highly expressed TDH358 protected from oxidative damage (Fig. 1b and not shown). This yielded a Dsup expression level equivalent to that of histone H2B (Fig. 1a), suggesting the availability of at least two molecules per nucleosome. Given this Dsup expression level protects the genome from oxidative DNA damage (Fig. 1c), is bound to chromatin genome-wide (Fig. 4), and redundantly interacts with multiple nucleosome surfaces (Figs. 6 and 8), it likely non-specifically coats the in vivo genome to physically protect from oxidative damage, as proposed from in vitro studies22. It may be relevant to note that when we yeast-codon optimized R.varieornatus Dsup in an attempt to promote still higher expression levels, the resulting yeast were inviable, suggesting that too much Dsup is deleterious. In agreement, the expression of codon-optimized Dsup had detrimental effects in cultured rat neurons59.Despite not having an enhanced environmental stress response (ESR), Dsup expressing yeast had a lowered redox state (Fig. 3). However, this does not explain their resistance to oxidative stress, since non-protective Dsup ΔHMGN ΔC + NLS also effectively lowered redox potential (Fig. 3). This suggests sequences within the N-terminal portion of Dsup (aa 1-359) reduce the redox state, though the mode of action is unclear. Dsup does not include any cysteine residues so it is unlikely to directly act as an antioxidant by donating hydrogens from thiol groups60. The effect could be indirect, since Dsup expression in tobacco pollen leads to increased levels of antioxidants, polyphenol and flavonoids61. Future studies should thus examine if the ability of the Dsup N-terminus to reduce the redox state in yeast is via increased levels of endogenous antioxidants.It is intriguing that the Dsup protein appears to have evolved multiple independent mechanisms to enable tardigrades to withstand oxidative damage. This is transferrable by heterologous expression to other species and appears to manifest by a mix of reduced redox state, transcriptional changes that enhance repair, and multivalent chromatin binding for physical protection. Our studies in budding yeast suggest the last is of greatest relevance but this may be due to tightly controlled expression that achieves a stoichiometry comparable to core-histones. The effective therapeutic harnessing of Dsup will require careful optimization of its expression in whichever context it is employed.MethodsYeast strains, primers and plasmidsPlasmid pRS306-PTDH3-Dsup was created by Gibson cloning of amplified DNA fragments following kit directions (NEB Gibson Assembly® Cloning Kit). In brief, pRS30662 was digested with SacI and BglII. The TDH3 promoter was PCR amplified (primers in Supplementary Data File 1A) from yeast genomic DNA with pTDH3_SacI_F (giving homology to Sac1 digested end of pRS306) and pTDH3_R (giving homology to 5’ end of Dsup gene). The R. varieornatus Dsup gene (aa1-445; encoding protein accession P0DOW4, Supplementary Data File 1E) including N-terminal 6xHis and C-terminal FLAG epitope tags was amplified from plasmid pET21b-nHis6-Rvar-DSUP-cFLAG (kind gift from James Kadonaga22), with primers Rvar_Dsup_F and Rvar_Dsup_R, respectively giving homology to the TDH3 promoter 3’ end and ADH1 terminator 5’ end. The ADH1 terminator was amplified from yeast genomic DNA using primers tADH1_F and tADH1_BglII_R, respectively giving homology to the Dsup gene 3’ end and BglII digested pRS306 5’ end.pRS306-PTDH3-Dsup was digested with MfeI and integrated to BY474163 at the endogenous TDH3 promoter to make yeast strain RGY002 (pTDH3-6His_Dsup_FLAG: all yeast strains and their phenotypes in Supplementary Data File 1B). Mutations to integrated Dsup, including insertion of a stop codon to derive the ‘Empty vector’ strain (RAY149), were by CRISPR-Cas9 mediated genome editing64. Primer sequences to generate guide RNAs61 and HDR template DNA are in Supplementary Data File 1A. Yeast were handled using standard methods. Dsup expressing strains were grown in SC-ura media (unless otherwise indicated).P415TEF cyto roGFP2-Grx1-NLS was made from p415TEF cyto roGFP2-Grx1 (kind gift from Tobias Dick; Addgene plasmid # 65004)36 by traditional cloning. First, the roGFP2-Grx1 sequence was PCR-amplified from plasmid p415TEF cyto roGFP2-Grx1 using primers that added a 2xNLS sequence (PKKKRKVPKKKRKV) to the Grx1 C-terminus. The resulting PCR product was digested with BamHI and HindIII and ligated to similarly digested plasmid p415TEF cyto roGFP2-Grx1 (Supplementary Data File 1B). All yeast strains and plasmids are available on request.Immunoblot analysisExponentially growing yeast cells ( ~ 107 at ODλ600 0.8–1.0) were collected by centrifugation, washed once with water, and flash frozen in liquid nitrogen. Pellets were resuspended in 100 μL modified Laemmli buffer65, boiled for five minutes (mins), and clarified by centrifugation. Proteins in the supernatant were resolved by 10% SDS-PAGE, membrane transferred, and immunoblotted with antibodies to FLAG (Sigma F1804; 1:1,000), GAPDH (Sigma A9521; 1:10,000), H2B (Abcam ab1790; 1:5000), H2A (Active Motif 39235; 1:2000), GCN4 (Absolute Antibody Ab00436-1.1; 1:1000), or H3 (Abcam ab1791; 1:1000). Uncropped unprocessed blots are provided in the Source Data.ELISA for 8-OhdG30 mL yeast cultures were grown at 30 °C in shaking flasks to ODλ600 0.6. Cells were harvested by centrifugation, and half of each culture resuspended in 15 mL of fresh SC-ura media −/+10 mM H2O2. After two hours at 30 °C cells were harvested by centrifugation, and genomic DNA isolated (Thermo Scientific Yeast DNA extraction kit) and resuspended in 50 μL nuclease-free water. DNA was quantified (NanoDrop spectrometer), samples diluted to 2 mg/mL, and sequentially incubated at 95 °C for five mins and on ice for 10 min. 50 μg DNA was then sequentially incubated with nuclease P1 (NEB; 1 U at 37 °C for two hours in provided buffer), alkaline phosphatase (NEB Quick CIP; 10 U at 37 °C for one hour in provided buffer supplemented with 100 mM Tris pH 8), at 95 °C for 10 min, and centrifuged at 6000 x g for five mins. Samples were then DNA quantified (NanoDrop spectrometer) and normalized.ELISA to measure 8-hydroxy 2-deoxyguanosine (8-OhdG) was performed as per kit instructions (Abcam ab201734). For each sample 15 μg DNA was assayed in triplicate (three independent cultures for each condition) and absorbance at 450 nm measured by plate reader.Growth curve analysisYeast cultures were grown to saturation overnight in YPD at 30 °C and diluted to ODλ600 0.1–0.2. The ODλ600 of cultures from three independent colonies per genotype were measured every 30 min, and growth curves fitted with an exponential regression in Microsoft Excel. Doubling times were calculated as the slope of the curve during exponential phase and compared across independent cultures using a Student’s t-test.Immunofluorescence analysisIndirect immunofluorescence of yeast cells was carried out as previously66. 2.5 OD of early-mid log phase cells (ODλ600 0.5–0.6) were crosslinked in 4% formaldehyde for 20 min at room temperature, then spheroplasted with 500 μg/mL Zymolyase 100 T for 30 min at 30 °C with rotation. Spheroplasted cells were applied to a 10-chamber poly-lysine coated microscope slide and permeabilized by incubation in methanol at −20 °C for six min, immediately followed by incubation in acetone at −20 °C for 30 seconds. After blocking in 5% BSA, slides were incubated with anti-FLAG (Sigma F1804; 1:1,000), anti-H2A (Abcam ab18255; 1:1.000) or anti-GAPDH (Sigma A9521; 1:5,000). After washing, slides were incubated with Alexa Fluor® 594 or 488 secondary antibodies (BioLegend), and coverslips mounted using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen). Images were taken using an Olympus BX63 Fluorescence Microscope with a DP80 Camera and 60X objective.Acute and chronic damage sensitivity analysisTo measure the response to acute hydrogen peroxide (H2O2) exposure, yeast cells were grown in YPD media to mid-log (ODλ600 0.5−1.0), harvested by centrifugation, and resuspended to ODλ600 0.6 in fresh media containing H2O2 (0, 4, 6, or 8 mM). After 90 min incubation (30 °C with shaking), cultures were washed and serial dilutions spread on SC-ura agar plates. After two days at 30 °C, colonies were counted and averaged across three technical replicates. Three independent experiments were performed from separate starting colonies, and statistical analyses performed using a Student’s t-test.To measure the response to chronic H2O2 exposure, yeast cells were grown in liquid culture until mid-log, harvested by centrifugation, and resuspended in sterile water to ODλ600 1.0. Five-fold serial dilutions were made in a 96-well plate and spotted with a sterile 6x8-prong manifold onto YPD agar plates containing indicated concentrations of H2O2. Similar methods were used to evaluate sensitivity to methyl methanesulfonate (MMS; at indicated concentrations in YPD) and Zeocin (at indicated concentrations in YPD). To measure sensitivity to ultraviolet light, yeast serial dilutions on YPD plates were exposed to UV (doses (J/cm2) indicated in figures) using a crosslinker (Stratalinker). Plates were incubated for three days at 30 °C.Replicative lifespan analysisYeast cells were grown overnight to early-mid log (ODλ600 0.2−0.6) and diluted to OD 0.1 in freshly-filtered YPD. This inoculum was added to an automated dissection chip (iBiochips) as per manufacturer’s instructions to achieve single cell loading. Light microscopy images of cells were acquired every 20 min over four days using an Evos FL Auto two-cell imaging microscope and associated software (ImageJ). At least 50 cells were counted per condition, with survival curves calculated in Graphpad Prism 9, and statistical analyses performed with a log-rank test.Chronological lifespan analysisYeast chronological lifespan was measured as previously67. Data is presented as average and standard deviation across three independent cultures (each an average of two technical replicates).Redox analysisYeast cells expressing cytoplasmic or nuclear localized (Supplementary Fig. 2) roGFP (redox sensing GFP) from respective plasmids p415TEF cyto roGFP2-Grx1 (GlutaRedoXin 1)36 or roGFP2-Grx1-NLS were grown in SC-LEU media to mid-log (ODλ600 0.6−0.8) and diluted to ODλ600 0.6 in 5 mL flow cytometry tubes. Fluorescence at 405 nm and 488 nm was measured on a flow cytometer (BD Biosciences BD® LSR II) immediately before addition of H2O2 (to 4 mM), and at indicated time points. Data is presented as the mean and standard deviation of 405/488 nm values for each timepoint (independent triplicates) using FlowJo.Chromatin fractionation analysisExponentially growing yeast cells ( ~ 4 × 108 at ODλ600 0.8−1.0) were collected by centrifugation, washed once with ice cold 10% glycerol, and flash frozen. After thawing on ice, the cell pellet was washed (100 mM Tris pH 9.4, 10 mM DTT), resuspended in the same buffer, and rested on ice for 10 min. Cells were pelleted by centrifugation, washed in spheroplasting buffer (10 mM HEPES, 1.2 M Sorbitol, 0.5 mM PMSF), resuspended in spheroplasting buffer containing 56 μg/mL Zymolyase 100 T (US Biological), and incubated at 30 °C for one hour with rotation (until a 50 μL aliquot mixed with 1 mL 10% SDS had an ODλ600 ~ 10% of the starting value). Spheroplasts were collected by centrifugation (1500 g for 2 min) and sequentially washed with spheroplasting buffer and wash buffer (1 M sorbitol, 20 mM Tris pH 7.5, 20 mM KCL, 2 mM EDTA, 0.5 mM PMSF, 0.1 μM spermine, 0.25 μM spermidine, 1:100 Calbiochem Protease Inhibitor Cocktail Set IV). Cells were gently resuspended and lysed in 250 μL Lysis Buffer (wash buffer with 400 mM sorbitol) for 10 min on ice.Half of the volume after lysis (Input) was mixed with 5x Laemmli buffer, while the other half was pelleted at 14,000 x g for 15 min. The supernatant was collected (non-chromatin fraction) and mixed with 5x Laemmli buffer, and the pellet (chromatin fraction) resuspended in 1x Laemmli buffer. All fractions were boiled for 5 min, clarified by centrifugation, 7.5% of the total volume resolved by 12.5% SDS-PAGE, membrane transferred, and immunoblotted with anti-FLAG (Sigma F1804; 1:1,000) to detect Dsup. Successful fractionation was confirmed with anti-H2A (Abcam ab18255; 1:5,000) as a chromatin bound protein, and anti-GAPDH (Sigma A9521; 1:20,000) as a non-chromatin bound protein.CUT&RUN analysisNuclei from yeast cells expressing Dsup alleles (Supplementary Data File 1B) were purified as previously68 with minor modifications. In brief, yeast cells were grown to mid-log (ODλ600 0.6−0.8) in 500 mL SC-ura and collected by centrifugation. Cells were resuspended to 500 μL, spheroplasted with Zymolyase 100 T (2 mg/mL; 37 °C for 30 min), nuclei isolated as previously68, and 1 mL aliquots (5 × 107 nuclei) slow-frozen overnight in an isopropanol chamber at −80 °C.For CUT&RUN69, nuclei were rapidly thawed (2–3 min at 37 °C), and 100 μL of suspension used per reaction with the CUTANA™ ChIC/CUT&RUN Kit (EpiCypher). In brief, after immunotethering (of pAG-MNase to Rabbit IgG, anti-H3K4me3, or anti-FLAG: Supplementary Data File 1C), MNase digestion was performed (4 °C for 2 h) and DNA eluted in 12 μL final volume. 5 ng of DNA was used to prepare sequencing libraries with the Ultra II DNA Library Prep Kit (NEB #E7645L). Libraries were sequenced on an Illumina NextSeq 2000 platform, obtaining an average of ~1.1 million paired-end (PE) reads per reaction (Supplementary Data File 2).PE fastq files were aligned to the sacCer3 reference genome (Bowtie270), filtered from duplicate (SAMtools71), multi-aligned (broadinstitute.github.io/picard), and exclusion list reads72, and the resulting unique reads for comparable reactions normalized by a spike-in scaling factor (1/ % E.coli reads) (BEDTools v2.30.073) to further RPKM (Reads Per Kilobase per Million mapped reads) -normalized bigwig files (DeepTools). Peak visualization from bigwig files was by Integrative Genomics Viewer (IGV). CUT&RUN sequence data is available from NCBI Gene Expression Omnibus (accession number GSE237436). The CUT&RUN analyses were performed independently three times with consistent results.RNA-seq analysisOvernight cultures of yeast strains −/+Dsup (RAY149 and RGY002 respectively: Supplementary Data File 1B) were grown at 30 °C in SC-URA medium. Cultures were diluted to ODλ600 0.2 in 30 ml SC-URA media, grown to mid-log (ODλ600 0.6–0.8), standardized to ODλ600 0.5 and split to 12 × 2 mL aliquots. An untreated triplicate (T0) was collected by centrifugation, washed in water and flash frozen in liquid nitrogen. To the remaining aliquots in triplicate H2O2 (Fisher Scientific BP2633-500) was added (0, 4, or 8 mM) and cultures incubated at 30 °C for 30 min. Cells were collected by centrifugation, washed in water, and flash frozen.RNA extraction was performed with the MasterPure Yeast RNA Purification Kit (Epicentre MPY03010) using the standard protocol including DNA removal step. RNA quality, concentration and purity was determined by Tapestation (Agilent 5067-5576) and nanodrop (Fisher Scientific), and material stored at −80 °C until library preparation. For each biological replicate, three technical replicates of non-strand specific libraries were prepared from 200 ng mRNA (Fast RNA-seq Lib Prep Kit v2; Abclonal RK20306) and sequenced to an average depth of 25 million paired-end 150 x 150 bp. Library preparation and sequencing services were provided by Novogene.Sequencing read and base-calling quality were assessed by FastQC v0.11.9 (www.bioinformatics.babraham.ac.uk/projects/fastqc/), and paired-end fastq files aligned to the sacCer3 reference genome (Bowtie270). Files were triple filtered from duplicate (SAMtools71), multi-aligned (broadinstitute.github.io/picard), and exclusion list reads72 before further analysis. Gene FPKM (Fragment Per gene-length Kilobase per Million filtered reads) were computed with Homer v4.1174. 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We are grateful to Andres Mansisidor for help generating plasmids and yeast strains, and the Weill Cornell Flow Cytometry Core for technical support. EpiCypher is supported by National Institutes of Health (NIH) grants R44 HG010640, R44 GM117683, R44 GM136172 and R44 CA212733. CKC is supported by NIH training grant T32 GM135128 and BDS by NIH grant R35 GM126900. JKT is supported by NIH grants R35 GM139816 and R01 CA95641. RRA was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32 GM007739 to the Weill Cornell / Rockefeller / Sloan Kettering Tri-Institutional MD-PhD Program.Author informationAuthor notesThese authors contributed equally: Rhiannon R. Aguilar, Laiba F. Khan.These authors jointly supervised this work: Michael-Christopher Keogh, Jessica K. Tyler.Authors and AffiliationsDepartment of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USARhiannon R. Aguilar, Nina Arslanovic, Thea Grauer, Kaylah Birmingham, Kritika Kasliwal, Spike D. L. Posnikoff, Ujani Chakraborty, Richard Garner, Abraham Shim, J. Ignacio Gutierrez & Jessica K. TylerWeill Cornell / Rockefeller / Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY, USARhiannon R. AguilarEpiCypher Inc., Durham, NC, USALaiba F. Khan, Allison R. Hickman, Rachel Watson, Ryan J. Ezell, Sabrina R. Hunt, Laylo Mukhsinova, Hannah E. Willis, Martis W. Cowles, Bryan J. Venters, Matthew R. Marunde & Michael-Christopher KeoghDepartment of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAChristopher K. Cummins & Brian D. StrahlPharmacology Graduate Program, Weill Cornell Medicine, New York, NY, USAKaylah BirminghamBiochemistry, Cellular, and Molecular Biology Graduate Program, Weill Cornell Medicine, New York, NY, USAKritika Kasliwal, Richard Garner & Abraham ShimAuthorsRhiannon R. AguilarView author publicationsSearch author on:PubMed Google ScholarLaiba F. KhanView author publicationsSearch author on:PubMed Google ScholarChristopher K. CumminsView author publicationsSearch author on:PubMed Google ScholarNina ArslanovicView author publicationsSearch author on:PubMed Google ScholarThea GrauerView author publicationsSearch author on:PubMed Google ScholarKaylah BirminghamView author publicationsSearch author on:PubMed Google ScholarKritika KasliwalView author publicationsSearch author on:PubMed Google ScholarSpike D. L. PosnikoffView author publicationsSearch author on:PubMed Google ScholarUjani ChakrabortyView author publicationsSearch author on:PubMed Google ScholarAllison R. HickmanView author publicationsSearch author on:PubMed Google ScholarRachel WatsonView author publicationsSearch author on:PubMed Google ScholarRyan J. EzellView author publicationsSearch author on:PubMed Google ScholarSabrina R. HuntView author publicationsSearch author on:PubMed Google ScholarLaylo MukhsinovaView author publicationsSearch author on:PubMed Google ScholarHannah E. WillisView author publicationsSearch author on:PubMed Google ScholarMartis W. CowlesView author publicationsSearch author on:PubMed Google ScholarRichard GarnerView author publicationsSearch author on:PubMed Google ScholarAbraham ShimView author publicationsSearch author on:PubMed Google ScholarJ. Ignacio GutierrezView author publicationsSearch author on:PubMed Google ScholarBryan J. VentersView author publicationsSearch author on:PubMed Google ScholarMatthew R. MarundeView author publicationsSearch author on:PubMed Google ScholarBrian D. StrahlView author publicationsSearch author on:PubMed Google ScholarMichael-Christopher KeoghView author publicationsSearch author on:PubMed Google ScholarJessica K. TylerView author publicationsSearch author on:PubMed Google ScholarContributionsR.A. constructed all strains, and performed western blotting, immunofluorescence, serial dilution, ROS and DNA damage analyses, with assistance of N.S., A.S., K.B., R.G. and K.K.; L.F.K. performed Captify and CUT&RUN assays under supervision of MRM. CKC performed RNA-seq studies under supervision of B.D.S.; N.A. performed replicative aging analyses under supervision of I.G.; S.D.L.P. performed chronological aging analyses. T.G. performed Redox State assays under supervision of I.G.; U.C. performed the MNase assays. A.R.H. and B.J.V. provided data analysis and interpretation for CUT&RUN and RNAseq. R.W., R.J.E. and HEW created fully defined histones, octamers, and/or nucleosomes. S.R.H. and L.M. provided recombinant Dsup proteins. M.W.C. was responsible for approach conception. M.C.K. and J.K.T. were responsible for program conception, project supervision and support in data analysis and interpretation. R.A. and J.K.T. drafted the original manuscript with support from L.F.K., B.D.S., M.R.M. and M.C.K.; All authors contributed to and are responsible for subsequent versions.Corresponding authorsCorrespondence to Michael-Christopher Keogh or Jessica K. Tyler.Ethics declarationsCompeting interestsEpiCypher is a commercial developer and supplier of reagents (e.g., fully defined semi-synthetic nucleosomes (dNucs) and SNAP-CUTANA® K-MetStat spike-ins) and platforms (e.g., Captify™ and CUTANA™ CUT&RUN) used in this study. LFK, ARH, RW, SRH, LM, MWC, MRM and MCK are currently employed by (and own shares in) EpiCypher. RJE and HEW were previously employed by (and own shares in) EpiCypher. MWC, BDS and MCK are board members of EpiCypher. The remaining authors declare no competing interests.Peer reviewPeer review informationNature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. 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