Vitamins and Minerals
Selenoprotein H (SELENOH) was found to localize in the nucleolus and is a member of the thioredoxin-like family of selenoproteins carrying the redox-regulatory CXXU domain. The presence of an AT-hook motif also entitles SELENOH as a member of the high-mobility group family of DNA-binding proteins. We searched for nuclear localization sequences of human selenoproteins by using the cNLS Mapper program and found only SELENOH to be nucleus-exclusive. With the use of the recently matured genome editing approaches, the objective of this study was to determine the impact of SELENOH knockout on the DNA damage responses to oxidative stress in the nucleus.
Methods : A knockout of SELENOH was generated in cell lines with the use of the CRISPR/Cas9-mediated genomic deletion. Biallelic mutants were selected by single colony screening and genomic DNA sequencing. Multiple lines of homozygous SELENOH knockout HeLa cells were treated with hydrogen peroxide, followed by various cellular and biochemical analyses to assay for proliferation, cell cycle, DNA damage, and senescence.
Results : Based on the sequenced genome, the results of deduced protein sequences indicated various forms of mutants in the CRISPR/Cas9-mediated SELENOH knockout, including a frame-shift by aberrant splicing and truncation by early termination of the translation process. Knockout of SELENOH in HeLa cells resulted in profound phenotypic changes including slow cell proliferation, decreased G1 and increased G2/M phase of the cell cycle, spontaneous activation of the ataxia-telangiectasia (ATM) DNA-damage response pathway, and cellular senescence. Treatment of mock knockout Hela cells with hydrogen peroxide induced ATM-dependent 53BP1 DNA damage focus formation, but knockout of SELENOH exacerbated such an event of genomic instability. Nucleolar stiffness and nucleoli with upstream binding factor cap, a marker of ribosomal DNA breaks and rearrangement, were increased in SELENOH knockout cells.
Conclusions : Altogether, SELENOH appears to be a stress responder in the nucleolus and plays critical roles in mitigating genome instability in HeLa cells.
Funding Sources : NIH