Engineering Radioprotective Human Cells Using the Tardigrade Damage Suppressor Protein, DSUP

Abstract

Spaceflight has been documented to produce detrimental effects to physiology and genomic stability, partly a result of Galactic Cosmic Radiation (GCR). In recent years, extensive research into extremotolerant organisms has begun to reveal how they survive harsh conditions, such as ionizing radiation. One such organism is the tardigrade (Ramazzottius varieornatus) which can survive up to 5kGy of ionizing radiation and the vacuum of space. In addition to their extensive network of DNA damage response mechanisms, the tardigrade also possesses a unique damage suppressor protein (Dsup) that co-localizes with chromatin in both tardigrade and transduced human cells to protect against DNA damage from reactive oxygen species induced by ionizing radiation. While Dsup has been shown to confer human cells with increased radiotolerance; much of the mechanism of how it does this in the context of human cells remains unknown. Until now there is no knowledge yet of how introduction of Dsup into human cells can perturb molecular networks and if there are any systemic risks associated with foreign gene introduction. Here, we created a stable HEK293 cell line expressing Dsup, validated its radioprotective phenotype, and performed multi-omic analyses across different time points and doses of radiation to delineate molecular mechanism of the radioprotection and assess molecular network pertubations. Dsup expressing human cells showed an enrichment for pathways seen in cells overexpressing HMGN1, a chromosomal architectural protein that has a highly similar nucleosome binding motif. As HMGN1 binding to nucleosomes promotes a less transcriptionally repressed chromatin state, we further explored the hypothesis that Dsup could behave similarly via ATAC-seq analysis and discovered overall selective differential opening and closing of the chromatin landscape. Cut&Run analysis further revealed global increases in histone post translational modifications indicative of open chromatin and global decreases in repressive marks, with Dsup binding preferentially towards promoter regions marked by H3K27ac and H3K4me3. We further validated some of the enriched pathways via in-vitro assays and revealed novel phenotypes that Dsup confers to human cells such as reduction in apoptosis, increased cell proliferation, and increased cell adhesion properties. Our analysis provides evidence that the Dsup protein in the context of HEK293 cells may behave as a chromatin architectural protein and that in addition to its nucleosome shielding effect, may confer radio-resistance via chromatin modulation. These results provide future insight into mitigating some of the major challenges involved with long term spaceflight as well as understanding some of the molecular architectural underpinnings that lead to radioresistant cancer phenotypes back home.