Selenium (Se) is an essential trace element exerting its biological functions mainly through selenoproteins. Our lab has recently shown a paradoxical role of dietary Se at nutritional levels of intake in the delay of age-related degeneration while reducing longevity in mice carrying humanized telomeres. The first aim of this dissertation was to evaluate the effects of long-term dietary Se deficiency, aging, and sex on selenotranscriptome hierarchy in tissues. Four unique patterns in selenotranscriptomic changes were summarized. First, the responses of selenotranscriptomes to dietary Se deprivation and aging were sexually dimorphic. Second, a few selenoproteins responded to dietary Se deficiency and aging in parallel. Third, there were selenoproteins up-regulated by aging or dietary Se deprivation. Forth, some selenoproteins, especially those in testis, were upregulated by aging in mice on a Se-deficient diet. Selenoprotein H (SELH) is ranked low in selenoprotein hierarchy and its expression is tissue-specific and abundant during embryogenesis. SELH is a nucleolar DNA-binding protein with thioredoxin (Txn)-like fold and glutathione peroxidase activity. The known biological functions of SELH include redox regulation and suppression of cellular senescence and tumorigenesis. The second aim of this dissertation was to study the functional interactions of SELH with other selenoproteins and its protective effects against oxidative stress. A FLAG-tagged plasmid expressing wild-type SELH was constructed. Compared to standard cell culture conditions, additional sodium selenite (Na2SeO3) increased SELH expression at protein but not mRNA level when FLAG-SELH was overexpressed. In the absence of supplemental Na2SeO3, FLAG-SELH overexpression suppressed the expression of other selenoproteins such as glutathione peroxidase 1 (GPX1) and thioredoxin reductase 1 (TrxR1) at protein but not mRNA level. FLAG-SELH overexpression protected the cells against oxidative stress only when additional Na2SeO3 was added. Identification of FLAG-SELH-associated proteins confirmed its nucleolar location. Altogether, a distinctive set of selenoproteins is maintained under dietary Se deficiency in a tissue- and sex-specific manner during the aging process. While SELH is ranked low in selenoprotein hierarchy implying that it is dispensable, this nucleolar selenoprotein competes with other selenoproteins for Se and protects the cells against oxidative stress.

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.

Low-hierarchy selenoproteins are sensitive to selenium (Se) deficiency and are proposed to confer the protection of body Se against age- and development-related diseases. Selenoprotein H (SELENOH), a low-hierarchy selenoprotein, is greatly downregulated by both dietary Se deficiency and age. To explore physiological roles of SELENOH, Selenoh knockout mice were employed. Segregation analyses demonstrated reduced frequencies of homozygotes and heterozygotes among the neonates of the breeding combinations of Selenoh+/- males vs. Selenoh+/- or Selenoh-/- females, demonstrating essential roles of SELENOH in embryogenesis. Litter sizes from these two breeding groups were comparable with control, suggesting a role of SELENOH in zygotic but not somatic embryogenesis. By contrast, SELENOH was dispensable for longevity in mice. Selenoh-/- males showed azoospermia and a 34%-63% reduction in testis mass in the mice. Histological examination and the analyses of stage-specific markers by qRT-PCR indicated that the Selenoh-/- spermatocytes were arrested in the pachytene stage. Furthermore, GPX4 plausibly accounted for some of the Se loss in the testes because the knockout resulted in a 90% decrease in GPX4 mRNA level. Interestingly, the knockout increased the Selenop and Gpx1 mRNA levels by over 2.3- and 1.9-folds, respectively, but decreased the mRNA amount of Selenov by 99.9% in the testes. As a key component of the meiotic cohesion complex to promote sister chromatid cohesion, Rec8 mRNA amount was reduced by 91.1% in the Selenoh-/- testes aged 42 days. SELENOH knockout did not affect body weight or food intake but resulted in glucose intolerance and insulin resistance in the mice. Additionally, SELENOH knockout reduced the mRNA amounts of Gpx1, Gpx4, and Txnrd1 in the liver and Gpx4 in the skeletal muscle. These findings suggest that SELENOH plays a role in glucose metabolism and regulates body Se metabolism in mice. In summary, results of these three studies unveil essential roles of SELENOH in embryogenesis, spermatogenesis, and glucose tolerance in mice. SELENOH plausibly plays upstream roles to optimize these physiological events in association with the transcriptional regulation of several selenoproteins and meiotic proteins. Future studies are warranted to understand the mechanisms by which SELENOH maintains such developmental and metabolic processes.