Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

  1. M. Anoud
  2. E. Delagoutte
  3. Q. Helleu
  4. A. Brion
  5. E. Duvernois-Berthet
  6. M. As
  7. X. Marques
  8. K. Lamribet
  9. C. Senamaud
  10. L. Jourdren
  11. A. Adrait
  12. S. Heinrich
  13. G. Toutirais
  14. S. Hamlaoui
  15. G. Gropplero
  16. I. Giovannini
  17. L. Ponger
  18. M. Gèze
  19. C. Blugeon
  20. Y. Coute
  21. R. Guidetti
  22. L Rebecchi
  23. C. Giovannangeli
  24. A. De Cian
  25. J-P. Concordet

  • Curated by eLife

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    eLife assessment

    This study offers valuable insights into the remarkable resistance of tardigrades to ionizing radiation by showing that radiation treatment induces a suite of DNA repair proteins. They identify a strongly induced tardigrade-specific DNA-binding protein that can reduce the number of double-strand breaks in human cancer-derived cells. The evidence of upregulation of repair proteins is compelling and the case for a role of the newly identified protein in repair can be strengthened as genetic tools for tardigrades become better developed. The results will be of interest the fields of DNA repair and radiobiology as well as tardigrade biologists.

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Abstract

Tardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicals in vitro . However, Dsup has only been found in two species within the Hypsibioidea superfamily.

To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model species Hypsibius exemplaris . By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species, H. exemplaris , Acutuncus antarcticus and Paramacrobiotus fairbanksi , the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, named T ardigrade D NA damage R esponse protein 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.

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  1. Version published to 10.1101/2023.09.08.556854v2 on bioRxiv
  2. Version published to 10.7554/elife.92621 on eLife
  3. Version published to 10.7554/elife.92621.1 on eLife
  4. eLife

    eLife assessment

    This study offers valuable insights into the remarkable resistance of tardigrades to ionizing radiation by showing that radiation treatment induces a suite of DNA repair proteins. They identify a strongly induced tardigrade-specific DNA-binding protein that can reduce the number of double-strand breaks in human cancer-derived cells. The evidence of upregulation of repair proteins is compelling and the case for a role of the newly identified protein in repair can be strengthened as genetic tools for tardigrades become better developed. The results will be of interest the fields of DNA repair and radiobiology as well as tardigrade biologists.

  5. eLife

    Reviewer #1 (Public Review):

    Summary:
    The manuscript "comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation" aims to provide insights into the mediators and mechanisms underlying tardigrade radiation tolerance. The authors start by assessing the effect of ionizing radiation (IR) on the tardigrade lab species, H. exemplaris, as well as the ability of this organism to recover from this stress - specifically, they look at DNA double and single-strand breaks. They go on to characterize the response of H. exemplaris and two other tardigrade species to IR at the transcriptomic level. Excitingly, the authors identify a novel gene/protein called TDR1 (tardigrade DNA damage response protein 1). They carefully assess the induction of expression/enrichment of this gene/protein using a combination of transcriptomics and biochemistry - even going so far as to use a translational inhibitor to confirm the de novo production of this protein. TDR1 binds DNA in vitro and co-localizes with DNA in tardigrades.

    Reverse genetics in tardigrades is difficult, thus the authors use a heterologous system (human cells) to express TDR1 in. They find that when transiently expressed TDR1 helps improve human cell resistance to IR.

    This work is a masterclass in integrative biology incorporating a holistic set of approaches spanning next-gen sequencing, organismal biology, biochemistry, and cell biology. I find very little to critique in their experimental approaches.

    Strengths:
    1. Use of trans/interdisciplinary approaches ('omics, molecular biology, biochemistry, organismal biology)
    2. Careful probing of TDR1 expression/enrichment
    3. Identification of a completely novel protein seemingly involved in tardigrade radio-tolerance.
    4. Use of multiple, diverse, tardigrade species of 'omics comparison.

    Weaknesses:
    1. No reverse genetics in tardigrades - all insights into TDR1 function from heterologous cell culture system.
    2. Weak discussion of Dsup's role in preventing DNA damage in light of DNA damage levels measured in this manuscript.
    3. Missing sequence data which is essential for making a complete review of the work.

    Overall, I find this to be one of the more compelling papers on tardigrade stress-tolerance I have read. I believe there are points still that the authors should address, but I think the editor would do well to give the authors a chance to address these points as I find this manuscript highly insightful and novel.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:
    This paper describes transcriptomes from three tardigrade species with or without treatment with ionizing radiation (IR). The authors show that IR produces numerous single-strand and double-strand breaks as expected and that these are substantially repaired within 4-8 hours. Treatment with IR induces strong upregulation of transcripts from numerous DNA repair proteins including Dsup specific to the Hypsobioidea superfamily. Transcripts from the newly described protein TDR1 with homologs in both Hypsibioidea and Macrobiotoidea supefamilies are also strongly upregulated. They show that TDR1 transcription produces newly translated TDR1 protein, which can bind DNA and co-localizes with DNA in the nucleus. At higher concentrations, TDR appears to form aggregates with DNA, which might be relevant to a possible function in DNA damage repair. When introduced into human U2OS cells treated with bleomycin, TDR1 reduces the number of double-strand breaks as detected by gamma H2A spots. This paper will be of interest to the DNA repair field and to radiobiologists.

    Strengths:
    The paper is well-written and provides solid evidence of the upregulation of DNA repair enzymes after irradiation of tardigrades, as well as upregulation of the TRD1 protein. The reduction of gamma-H2A.X spots in U2OS cells after expression of TRD1 supports a role in DNA damage.

    Weaknesses:
    Genetic tools are still being developed in tardigrades, so there is no mutant phenotype to support a DNA repair function for TRD1, but this may be available soon.

  7. eLife

    Reviewer #4 (Public Review):

    The manuscript brings convincing results regarding genes involved in the radio-resistance of tardigrades. It is nicely written and the authors used different techniques to study these genes. There are sometimes problems with the structure of the manuscript but these could be easily solved. According to me, there are also some points which should be clarified in the result sections. The discussion section is clear but could be more detailed, although some results were actually discussed in the results section. I wish that the authors would go deeper in the comparison with other IR-resistant eucaryotes. Overall, this is a very nice study and of interest to researchers studying molecular mechanisms of ionizing radiation resistance.

    I have two small suggestions regarding the content of the study itself.

    1. I think the study would benefit from the analyses of a gene tree (if feasible) in order to verify if TDR1 is indeed tardigrade-specific.
    2. It would be appreciated to indicate the expression level of the different genes discussed in the study, using, for example, transcript per millions (TPMs).
  8. Version published to 10.1101/2023.09.08.556854v1 on bioRxiv