Genome Maintenance by Selenoprotein H in the Nucleolus
Schilling, M. Wes
Selenoprotein H (SELENOH) is a nucleolar oxidoreductase with DNA binding properties whose function is not well understood. To determine the functional and physiological roles of SELENOH, a knockout of SELENOH was generated in cell lines using CRISPR/Cas9-mediated genomic deletion and in mice by targeted disruption. Based on the sequenced genome, the results of deduced protein sequences indicated various forms of mutants in the CRISPR/Cas9-mediated knockout, including a frame-shift by aberrant splicing and truncated SELENOH by early termination of the translation process. Loss of SELENOH in HeLa cells induced slow cell proliferation, the formation of giant multinucleated cells, accumulation of unrepaired DNA damage and oxidative stress, and cellular senescence. SELENOH cells were enlarged and possessed a single large nucleolus. Atomic force microscope showed increased stiffness in the nucleoli of SELENOH knockout cells, which suggests that SELENOH maintains the flexible structure of the nucleolus. Furthermore, the knockout of SELENOH led to a large-scale reorganization of the nucleolar architecture with the movement of nucleolar protein into nucleolar cap regions in response to oxidative stress. The nucleolar reorganization is dependent on ATM signaling. Altogether, results suggest that SELENOH appears to be a sensor of oxidative stress that plays critical roles in redox regulation and genome maintenance within the nucleolus. To determine the physiological role of SELENOH in vivo, Selenoh knockout mice were generated by targeted deletion through homologous recombination. Selenoh+/− mice were fertile and phenotypically indistinguishable from wild-type littermates. Results from matings of Selenoh+/− mice showed a significantly reduced fraction of Selenoh−/− offspring on the basis of Mendelian segregation. Since some Selenoh−/− were born, it is likely that Selenoh is a partially essential gene in mice. Live-born Selenoh−/− mice were viable and born without apparent phenotypes. Selenoh−/− mice at 2-month of age showed increased GPX activity in the lung but not in the brain and liver. Furthermore, loss of Selenoh resulted in the aggravated formation of aberrant crypt foci in the colon of Selenoh+/− mice that were injected with azoxymethane. Altogether, SELENOH has critical roles in embryogenesis and colorectal carcinogenesis.