Analysis of DNA damage and repair in Hypsibius exemplaris after γ-ray irradiation.

(a) Analysis of phospho-H2AX expression after exposure of H. exemplaris to IR.

Western blot analysis with in-house antibody against phosphorylated H. exemplaris H2AX (anti-phospho-H2AX) at indicated time points after irradiation of tardigrades with indicated dose of γ-ray irradiation. Phospho-H2AX levels were normalized by total α−tubulin expression levels and quantification is provided in Supp Figure 2a. (−) lanes show extracts from control tardigrades processed in parallel to irradiated tardigrades at indicated time points post-irradiation.

(b) Analysis of phospho-H2AX expression in whole mount H. exemplaris after exposure to 100 Gy. Tardigrades were exposed to 100 Gy, fixed with 4% PFA at 4 and 24h post irradiation, immunolabeled with anti-phosphoH2AX antibody and anti-rabbit IgG conjugated to Alexa488 and visualized by confocal microscopy using the Airyscan2 module. Maximum projection of confocal Z-stack are shown. Images at different time points were taken with identical settings so that signal intensity could be compared. Upper panel shows Hoechst staining of nuclei (in blue). Arrowhead indicates position of the gonad (revealed by intense Hoechst and larger nuclei signal). The gonad exhibits intense labeling phospho-H2AX at 4h which is no longer detected at 24h, showing efficient DNA repair consistent with preservation of the capacity to lay eggs and reproduce after 100 Gy IR (Beltran-Pardo et al, 2005). * indicates autofluorescence of bucco-pharyngeal apparatus. Scale bar 20µm

(c) Analysis of single-strand breaks by denaturing agarose gel electrophoresis of DNA isolated from ∼8000 H. exemplaris at indicated time points post-irradiation (100Gy or 1000Gy γ-rays from 137Cs source). (−) indicates DNA from control, non-irradiated tardigrades collected and processed in parallel to treated samples from indicated time points. MW corresponds to the Molecular Weight ladder. * indicates a discrete band of single stranded DNA detected in H. exemplaris genomic DNA. Arrowhead indicates high molecular weight single-stranded DNA that is not resolved by agarose gel electrophoresis. (−) lanes show DNA from control tardigrades processed in parallel to irradiated tardigrades at 4h or 8h30 post-irradiation as indicated.

Transcriptomic response of Hypsibius exemplaris to IR and Bleomycin.

(a) and (b) Volcano plots representing Log2 Fold Change and adjusted p-value (−log base 10) of RNA levels between H. exemplaris irradiated with 1000Gy γ-rays and untreated controls (n=3) (a) and between H. exemplaris treated with 100 µM Bleomycin for 4 days and untreated controls (n=3) (b). The vertical dotted line indicates the Log2Fold Change value of 2.

(c) Correlation between Log2 Fold Change after exposure to IR and after Bleomycin (BL) treatment for abundant transcripts (with baseMean>500 after DESeq2 analysis).

Blue dots represent transcripts with a Log2 Fold Change with an adjusted p-value p<0.05. Brown dots indicate transcripts of DNA repair genes (based on KEGG DNA repair and recombination gene group ko03400) that have a Log2 Fold Change with adjusted p-value p<0.05. Grey dots represent transcripts with a Log2 Fold Change with an adjusted p-value p>0.05. Brown labels indicate representative strongly upregulated genes of DNA repair. Blue labels indicate two tardigrade specific genes induced in response to IR: the TDR1 gene identified in this work, and the AMNP-like gene (BV898_10264), a member of the family of AMNP/g12777-like genes upregulated in response to desiccation and UVC (Yoshida et al 2022).

Changes in protein expression in Hypsibius exemplaris after exposure to IR.

(a) Western blot analysis of He-TDR1, He-XRCC5, He-XRCC6 (among the most strongly stimulated genes at the RNA level) and He-Dsup (not stimulated at the RNA level) in irradiated H. exemplaris tardigrades Control, untreated H. exemplaris (Ctrl) or H. exemplaris treated with 100µM Bleomycin for 4 days, or with 1000Gy γ-rays and extracts prepared at indicated times post-irradiation (IR4h and IR24h). Alpha-tubulin was used for normalization and phospho-H2AX for showing induction of DNA double-strand breaks. Quantification of 4 independent experiments are shown in Supp Figure 5c. Molecular weight marker present in uncropped Western blots (Supp Figure 14b) is consistent with the expected 16kDa size of TDR1.

(b) Volcano plot representing Log2 Fold Change and −Log10(limma p-value) of proteins between H. exemplaris 24h post-irradiation with 1000Gy γ-rays and untreated control animals (n=4).

Blue dots represent proteins with a Log2 Fold Change with a −Log10(limma p-value)>=2. Brown dots represent DNA repair proteins (based on KEGG DNA repair and recombination gene group ko03400) with −Log10(limma p-value)>=2. Grey points represent proteins with Log2 Fold Change with−Log10(limma p-value)<2 and the vertical grey lines delimit Log2(FC)>0.3 or <-0.3. Brown labels indicate representative strongly upregulated genes of DNA repair. Blue labels indicate two tardigrade specific genes induced in response to IR: the TDR1 gene identified in this work, and the AMNP-like gene (BV898_10264), a member of the family of AMNP/g12777-like genes upregulated in response to desiccation and UVC (Yoshida et al 2022).

(c) Correlation between Fold Changes of protein levels 24h post-irradiation with 1000 Gy (as measured in (b)) and Log2FoldChange of RNA levels 4h post-irradiation (as measured in Figure 2a).

Proteomic analysis metrics: numbers of differentially expressed (DE) proteins (with limma p-value < 0.01 and Log2FoldChange<-0.3 or >0.3) for each indicated condition in Hypsibius exemplaris.

The numbers of tardigrade specific DE proteins are also indicated. 9 tardigrade specific DE proteins were common to the 3 conditions, the corresponding list is provided in Supp Table 7.

Tardigrade specific proteins are defined as proteins that have no homolog in C. elegans, D. melanogaster or in humans and don’t have significant sequence homology to any sequence in the NCBI protein database (excluding tardigrade sequences) (see Methods section).

Transcriptomic response of Acutuncus antarcticus and Paramacrobiotus fairbanksi to IR

(a) and (b) Volcano plots representing Log2Fold Change and adjusted p-value (−log base 10) of RNA levels after irradiation with 1000 Gy γ-rays between irradiated A. antarcticus and untreated controls (n=3)(a) and between irradiated P. fairbanksi and untreated controls (n=3) (b).

Blue dots represent transcripts with an adjusted p-value p<0.05. Brown dots indicate transcripts of DNA repair genes (based on KEGG DNA repair and recombination gene group ko03400) with an adjusted p-value p<0.05. Brown labels indicate representative strongly upregulated genes of DNA repair. Blue labels indicate two tardigrade specific genes induced in response to IR: the TDR1 gene identified in this work, and the AMNP-like gene (BV898_10264), a member of the family of AMNP/g12777-like genes upregulated in response to desiccation and UVC (Yoshida et al 2020).

(c) Venn diagram showing upregulated genes with an adjusted p-value p<0.05 common to the transcriptomic response to IR in the three species analysed and to Bleomycin in H. exemplaris and A. antarcticus.

Number of differentially expressed genes (DEG with adjusted p-value < 0.05) after IR with 1000 Gy γ-rays vs untreated in 3 species (H. exemplaris, A. antarcticus, P. fairbanksi) and Bleomycin treatment for 4 or 5 days in H. exemplaris and A. antarcticus.

A heatmap of the 50 upregulated genes common to all conditions is given in Supp Figure 8.

He-TDR1 interacts directly with DNA.

(a) Sequence alignment of the conserved C-terminal domain of TDR1 proteins from H. exemplaris (He), A. antarcticus (Aa), P. fairbanksi (Pf) (identified in this work), and from P. richtersi (Pr) (NCBI transcriptome assembly GFGY00000000.1), P. metropolitanus (Pm), R.coronifer (Rc) (Kamilari et al 2019), M. philippinicus (Mp) (Mapalo et al 2020). He_BV898_10457 corresponds to a paralog of HeTDR1 in H. exemplaris with weaker sequence identity to HeTDR1 than TDR1 homologs from other species.

(b-c) Gel shift assay of recombinant He-TDR1 with circular plasmid (b) or linear plasmid (c). Mixes of plasmid DNA and recombinant He-TDR1 at indicated protein to DNA (bp) ratios were incubated at 25°C for 20 minutes and migrated, either directly or after proteinase K digestion, at room temperature on 0.75% agarose with ethidium bromide. Fluorescence was revealed with a ChemiDoc MP imager. Complexes of plasmid DNA and recombinant He-TDR1 are indicated by a bracket. High-molecular weight complexes that remained in the loading wells and did not migrate into the gel are indicated by an asterisk.

(d) Expression of He-TDR1-mNeonGreen in transient transgenic H. exemplaris tardigrades Expression plasmids of He-TDR1-mNeonGreen (mNG) and mCherry (both under control of the He-Actin promoter) were microinjected into the body fluid of H. exemplaris adults and electroporation was performed to induce delivery into cells following the protocol of Tanaka et al. 2023. Confocal microscopy was carried out on live animals immobilized in carbonated water at day 8 post-microinjection after 2 days of treatment with 20µM Hoechst 33342 to stain nuclei. Maximum projections of confocal Z-stack are shown.

(e) High resolution imaging of nuclei expressing He-TDR1-mNG and Hoechst staining of the nucleus using the Airyscan2 module (one Z-slice is shown). Nuclear He-TDR1-mNG is co-localized with Hoechst staining except for 1 big foci which was observed in some high-resolution images (e-yellow channel), likely corresponding to nucleolar accumulation of overexpressed He-TDR1-mNG.

Reduced numbers of phospho-H2AX foci after Bleomycin treatment in human U20S cells expressing TDR1-GFP from multiple tardigrade species.

(a) Violin plot of the number of phospho-H2AX foci per nucleus of cells expressing the indicated protein. Phospho-H2AX foci were counted after 10µg.ml−1 Bleomycin 1 hour-treatment of U2OS cells electroporated with a plasmid expressing either eGFP (control), RvDsup-GFP, TDR1-GFP from H. exemplaris (He), A. Antarcticus (Aa), R. coronifer (Rc) and P. richtersi (Pr), He-RNF146-GFP. Cells were fixed with a 4% PFA PBS solution for 1h, immunolabeled with chicken anti-GFP and mouse anti-phospho-H2AX antibodies and imaged by confocal microscopy.

**** indicates P<0.0001 (Kruskal-Wallis test). A minimum of 308 nuclei were counted in each experimental condition. A representative experiment is shown here. Data from independent replicates are given in Supp Figure 11.

(b) Representative confocal fluorescence imaging of experiment analyzed in (a). Images were taken with identical settings and printed with same thresholding so that signal intensity could be compared. Scale bar corresponds to 10 µM.