Dietary selenium (Se) requirements in rats have been based largely upon glutathione peroxidase-1 (Gpx1) enzyme activity and Gpx1 mRNA levels can also be used to determine Se requirements. The identification of the complete selenoprotein proteome suggests that we might identify additional useful molecular biomarkers for assessment of Se status. To characterize Se regulation of the entire rat selenoproteome, weanling male rats were fed a Se-deficient diet (<0.01 microg Se/g) supplemented with graded levels of Se (0-0.8 microg/g diet) for 28 d, Se status was determined by tissue Se concentration and selenoenzyme activity, and selenoprotein mRNA abundance in liver, kidney, and muscle was determined by quantitative real-time-PCR. Tissue Se and selenoenzyme biomarkers indicated that minimal Se requirements were

Synopsis Gpx (glutathione peroxidase)-1 enzyme activity and mRNA levels decrease dramatically in selenium (Se) deficiency, whereas other selenoproteins are less affected by Se deficiency. This hierarchy of Se regulation is not understood, but the position of the UGA selenocysteine codon is thought to play a major role in making selenoprotein mRNAs susceptible to nonsense-mediated decay. Thus in the present paper we studied the complete selenoproteome in the mouse to uncover additional selenoprotein mRNAs that are highly-regulated by Se status. Mice were fed Se-deficient, Se-marginal, and Se-adequate diets (0, 0.05 and 0.2 μg Se/g, respectively) for 35 days, and selenoprotein mRNA levels in liver and kidney were determined using microarray analysis and quantitative real-time PCR analysis. Se-deficient mice had liver Se concentrations and liver Gpx1 and thioredoxin reductase activities that were 4, 3 and 3%, respectively, of the levels in Se-adequate mice, indicating that the mice were Se-deficient. mRNAs for Selh (selenoprotein H) and Sepw1 (selenoprotein W) as well as Gpx1 were decreased by Se deficiency to <40% of Se-adequate levels. Five and two additional mRNAs were moderately down-regulated in Se-deficient liver and kidney, respectively. Importantly, nine selenoprotein mRNAs in liver and fifteen selenoprotein mRNAs in kidney were not significantly regulated by Se deficiency, clearly demonstrating that Se regulation of selenoprotein mRNAs is not a general phenomenon. The similarity of the response to Se deficiency suggests that there is one underlying mechanism responsible. Importantly, the position of the UGA codon did not predict susceptibility to Se regulation, clearly indicating that additional features are involved in causing selenoprotein mRNAs to be sensitive to Se status.

This review discusses progress in understanding the hierarchy of selenoprotein expression at the transcriptome level from selenium (Se) deficiency to Se toxicity. Microarray studies of the full selenoproteome have found that 5 of 24 rodent selenoprotein mRNA decrease to <40% of Se adequate levels in Se deficient liver but that the majority of selenoprotein mRNA are not regulated by Se deficiency. These differences match with the hierarchy of selenoprotein expression, helping to explain these differences and also showing that selenoprotein transcripts can be used as molecular biomarkers for assessing Se status. The similarity of the response curves for regulated selenoproteins suggests one underlying mechanism is responsible for the downregulation of selenoprotein mRNA in Se deficiency, but the heterogeneity of the UGA position in regulated and nonregulated selenoprotein transcripts now indicates that current nonsense mediated decay models cannot explain which transcripts are susceptible to mRNA decay. Microarray studies on the full liver transcriptome in rats found only <10 transcripts/treatment were significantly down- or upregulated by Se deficiency or by supernutritional Se up to 2.0 μg Se/g diet (20× requirement), suggesting that cancer prevention associated with supernutritional Se may not be mediated by transcriptional changes. Toxic dietary Se at 50× requirement (5 μg Se/g diet), however, significantly altered ∼4% of the transcriptome, suggesting number of transcriptional changes itself as a biomarker of Se toxicity. Finally, panels of Se regulated selenoprotein plus nonselenoprotein transcripts predict Se status from deficient to toxic better than conventional biomarkers, illustrating potential roles for molecular biomarkers in nutrition.

BackgroundProtein and mRNA levels for several selenoproteins, such as glutathione peroxidase-1 (Gpx1), are down-regulated dramatically by selenium (Se) deficiency. These levels in rats increase sigmoidally with increasing dietary Se and reach defined plateaus at the Se requirement, making them sensitive biomarkers for Se deficiency. These levels, however, do not further increase with super-nutritional or toxic Se status, making them ineffective for detection of high Se status. Biomarkers for high Se status are needed as super-nutritional Se intakes are associated with beneficial as well as adverse health outcomes. To characterize Se regulation of the transcriptome, we conducted 3 microarray experiments in weanling mice and rats fed Se-deficient diets supplemented with up to 5 μg Se/g diet.ResultsThere was no effect of Se status on growth of mice fed 0 to 0.2 μg Se/g diet or rats fed 0 to 2 μg Se/g diet, but rats fed 5 μg Se/g diet showed a 23% decrease in growth and elevated plasma alanine aminotransferase activity, indicating Se toxicity. Rats fed 5 μg Se/g diet had significantly altered expression of 1193 liver transcripts, whereas mice or rats fed ≤ 2 μg Se/g diet had < 10 transcripts significantly altered relative to Se-adequate animals within an experiment. Functional analysis of genes altered by Se toxicity showed enrichment in cell movement/morphogenesis, extracellular matrix, and development/angiogenesis processes. Genes up-regulated by Se deficiency were targets of the stress response transcription factor, Nrf2. Multiple regression analysis of transcripts significantly altered by 2 μg Se/g and Se-deficient diets identified an 11-transcript biomarker panel that accounted for 99% of the variation in liver Se concentration over the full range from 0 to 5 μg Se/g diet.ConclusionThis study shows that Se toxicity (5 μg Se/g diet) in rats vastly alters the liver transcriptome whereas Se-deficiency or high but non-toxic Se intake elicits relatively few changes. This is the first evidence that a vastly expanded number of transcriptional changes itself can be a biomarker of Se toxicity, and that identified transcripts can be used to develop molecular biomarker panels that accurately predict super-nutritional and toxic Se status.

SUMMARYHox genes are key regulators of development. In mammals, the study of these genes is greatly confounded by their large number, overlapping functions and interspersed shared enhancers. Here, we describe the use of a novel recombineering strategy to introduce simultaneous frameshift mutations into the flanking Hoxa9, Hoxa10 and Hoxa11 genes, as well as their paralogs on the HoxD cluster. The resulting Hoxa9,10,11 mutant mice displayed dramatic synergistic homeotic transformations of the reproductive tracts, with the uterus anteriorized towards oviduct and the vas deferens anteriorized towards epididymis. The Hoxa9,10,11 mutant mice also provided a genetic setting that allowed the discovery of Hoxd9,10,11 redundant reproductive tract patterning function. Both shared and distinct Hox functions were defined. Hoxd9,10,11 play a crucial role in the regulation of uterine immune function. Non-coding nonpolyadenylated RNAs were among the key Hox targets, with dramatic downregulation in mutants. We observed Hox cross-regulation of transcription and splicing. In addition, we observed a surprising anti-dogmatic apparent posteriorization of the uterine epithelium. In caudal regions of the uterus, the normal simple columnar epithelium flanking the lumen was replaced by a pseudostratified transitional epithelium, normally found near the more posterior cervix. These results identify novel molecular functions of Hox genes in the development of the male and female reproductive tracts.