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Thioredoxin reductase-dependent inhibition of MCB cell cycle box activity in Saccharomyces cerevisiae
A K Machado1, B A Morgan, G F Merrill
1Department of Biochemistry and Biophysics and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331, USA.
Abstract:
Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Deltaswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
Insights
Mutations in thioredoxin reductase (TRR1) activate cell cycle gene transcription. Oxidizing thioredoxin, rather than its level, is linked to Mlu1 cell cycle box (MCB) element activation in yeast.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Cell Cycle Regulation
Background:
- Mlu1 cell cycle box (MCB) elements regulate G1/S-specific gene expression in yeast.
- Swi6 is a transcription factor essential for MCB-dependent promoter activity.
Purpose of the Study:
- To identify factors that regulate MCB-dependent transcription in the absence of Swi6.
- To investigate the role of thioredoxin reductase (TRR1) and thioredoxin oxidation in cell cycle gene regulation.
Main Methods:
- Screening for yeast mutations activating MCB reporter genes.
- Functional cloning to identify mutated genes.
- Reporter gene assays and quantitative PCR (qPCR) to measure gene expression.
- Deletion and overexpression of TRR1, TRX1, and TRX2 genes.
- Exposure to hydrogen peroxide (H2O2) and hydroxyurea (HU) to modulate thioredoxin redox state.
Main Results:
- Mutations in TRR1 were identified that activate MCB reporter genes.
- TRR1 mutations strongly stimulate MCB- and SCB-containing reporter genes, and weakly stimulate other reporter genes.
- Deletion of TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations.
- Oxidizing conditions (H2O2) induced MCB gene expression, while reducing conditions (HU) inhibited it.
- TRR1 mutations did not alter the mRNA levels or periodicity of endogenous MCB genes.
Conclusions:
- Thioredoxin oxidation, not just its level, contributes to MCB element activation.
- A link exists between thioredoxin-oxidizing processes, such as ribonucleotide reduction, and cell cycle-specific gene transcription.