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ABSTRACTExpression of the Xistgene, a key player in mammalian X inactivation, has been proposed to be controlled by the antisense Tsixtranscript. Targeted deletion of theTsixpromoter encompassing the DPXas34 locus leads to nonrandom inactivation of the mutant X, but it remains unresolved whether this phenotype is caused by loss of Tsixtranscription or by deletion of a crucial DNA element. In this study we determined the role of Tsixtranscription in random X inactivation by using mouse embryonic stem (ES) cells as a model system. Two approaches were chosen to modulate Tsixtranscription with minimal disturbance of genomic sequences. First,Tsixtranscription was functionally inhibited by introducing a transcriptional stop signal into the transcribed region of Tsix. In the second approach, an inducible system forTsixexpression was created. We found that the truncation of the Tsixtranscript led to complete nonrandom inactivation of the targeted X chromosome. Induction of Tsixtranscription during ES cell differentiation, on the other hand, caused the targeted chromosome always to be chosen as the active chromosome. These results for the first time establish a function for antisense transcription in the regulation of X inactivation.

Expression of the Xistgene, a key player in mammalian X inactivation, has been proposed to be controlled by the antisense Tsixtranscript. Targeted deletion of the Tsixpromoter encompassing the DPXas34 locus leads to nonrandom inactivation of the mutant X, but it remains unresolved whether this phenotype is caused by loss of Tsixtranscription or by deletion of a crucial DNA element. In this study we determined the role of Tsixtranscription in random X inactivation by using mouse embryonic stem (ES) cells as a model system. Two approaches were chosen to modulate Tsixtranscription with minimal disturbance of genomic sequences. First, Tsixtranscription was functionally inhibited by introducing a transcriptional stop signal into the transcribed region of Tsix. In the second approach, an inducible system forTsixexpression was created. We found that the truncation of the Tsixtranscript led to complete nonrandom inactivation of the targeted X chromosome. Induction of Tsixtranscription during ES cell differentiation, on the other hand, caused the targeted chromosome always to be chosen as the active chromosome. These results for the first time establish a function for antisense transcription in the regulation of X inactivation.

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designatedGRX1and GRX2, which share 40–52% identity and 61–76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1andGRX2were viable but lacked heat-stable oxidoreductase activity using β-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1mutant was unaffected in oxidoreductase activity, whereas the grx2mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1andGRX2. In addition, a grx1mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.