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Regulation of wee1(+) expression during meiosis in fission yeast.

Murakami-Tonami Y, Ohtsuka H, Aiba H, Murakami H - Cell Cycle (2014)

Bottom Line: Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene.In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene.These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.

View Article: PubMed Central - PubMed

Affiliation: a Aichi Cancer Center Research Institute ; Division of Molecular Oncology ; Nagoya , Japan.

ABSTRACT
In eukaryotes, the cyclin-dependent kinase Cdk1p (Cdc2p) plays a central role in entry into and progression through nuclear division during mitosis and meiosis. Cdk1p is activated during meiotic nuclear divisions by dephosphorylation of its tyrosine-15 residue. The phosphorylation status of this residue is largely determined by the Wee1p kinase and the Cdc25p phosphatase. In fission yeast, the forkhead-type transcription factor Mei4p is essential for entry into the first meiotic nuclear division. We recently identified cdc25(+) as an essential target of Mei4p in the control of entry into meiosis I. Here, we show that wee1(+) is another important target of Mei4p in the control of entry into meiosis I. Mei4p bound to the upstream region of wee1(+) in vivo and in vitro and inhibited expression of wee1(+), whereas Mei4p positively regulated expression of the adjacent pseudogene. Overexpression of Mei4p inhibited expression of wee1(+) and induced that of the pseudogene. Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene. In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene. These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.

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Expression of wee1+ and SPCC18B5.02c during meiosis and the effects of mei4+ deletion or overexpression. Cells of the indicated genotypes (wild-type (WT), HM1307; mei4, HM2163; mei4+-OP, HM4582; WT wee1+-HA, HM4732; wee1+-HA, mei4, HM4833; and mei4+-OP wee1+-HA, HM4735) were subjected to synchronous induction of meiosis as described in the Materials and Methods. Samples were collected at the indicated time points thereafter and subjected to Northern blot analysis of wee1+ and SPCC18B5.02c mRNA (rRNA (rRNA) was stained with ethidium bromide as the loading control) (A) or Western blot analysis with anti-HA and anti-α-tubulin (loading control) antibodies (B).
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f0001: Expression of wee1+ and SPCC18B5.02c during meiosis and the effects of mei4+ deletion or overexpression. Cells of the indicated genotypes (wild-type (WT), HM1307; mei4, HM2163; mei4+-OP, HM4582; WT wee1+-HA, HM4732; wee1+-HA, mei4, HM4833; and mei4+-OP wee1+-HA, HM4735) were subjected to synchronous induction of meiosis as described in the Materials and Methods. Samples were collected at the indicated time points thereafter and subjected to Northern blot analysis of wee1+ and SPCC18B5.02c mRNA (rRNA (rRNA) was stained with ethidium bromide as the loading control) (A) or Western blot analysis with anti-HA and anti-α-tubulin (loading control) antibodies (B).

Mentions: We previously reported that Mei4p controls phosphorylation of Cdc2p on Tyr15.20 This suggests that wee1+ is also regulated by Mei4p, in addition to cdc25+. We examined the expression of wee1+ mRNA during meiosis by Northern blot analysis (Fig. 1A). To induce meiosis synchronously, we used a temperature-sensitive pat1 strain of Schizosaccharomyces pombe.21 Cells were synchronized in G1 phase by nitrogen deprivation and then shifted to the restrictive temperature to induce meiosis. The amount of wee1+ mRNA decreased around the onset of MI (5 h) in wild-type (WT) cells. By contrast, the level of wee1+ mRNA was almost constant in mei4+-deleted (mei4) cells. Furthermore, the level of wee1+ mRNA was decreased earlier (4 h) in mei4+-overproducing (mei4+-OP) cells. These results suggest that Mei4p negatively regulates wee1+ mRNA.Figure 1.


Regulation of wee1(+) expression during meiosis in fission yeast.

Murakami-Tonami Y, Ohtsuka H, Aiba H, Murakami H - Cell Cycle (2014)

Expression of wee1+ and SPCC18B5.02c during meiosis and the effects of mei4+ deletion or overexpression. Cells of the indicated genotypes (wild-type (WT), HM1307; mei4, HM2163; mei4+-OP, HM4582; WT wee1+-HA, HM4732; wee1+-HA, mei4, HM4833; and mei4+-OP wee1+-HA, HM4735) were subjected to synchronous induction of meiosis as described in the Materials and Methods. Samples were collected at the indicated time points thereafter and subjected to Northern blot analysis of wee1+ and SPCC18B5.02c mRNA (rRNA (rRNA) was stained with ethidium bromide as the loading control) (A) or Western blot analysis with anti-HA and anti-α-tubulin (loading control) antibodies (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612672&req=5

f0001: Expression of wee1+ and SPCC18B5.02c during meiosis and the effects of mei4+ deletion or overexpression. Cells of the indicated genotypes (wild-type (WT), HM1307; mei4, HM2163; mei4+-OP, HM4582; WT wee1+-HA, HM4732; wee1+-HA, mei4, HM4833; and mei4+-OP wee1+-HA, HM4735) were subjected to synchronous induction of meiosis as described in the Materials and Methods. Samples were collected at the indicated time points thereafter and subjected to Northern blot analysis of wee1+ and SPCC18B5.02c mRNA (rRNA (rRNA) was stained with ethidium bromide as the loading control) (A) or Western blot analysis with anti-HA and anti-α-tubulin (loading control) antibodies (B).
Mentions: We previously reported that Mei4p controls phosphorylation of Cdc2p on Tyr15.20 This suggests that wee1+ is also regulated by Mei4p, in addition to cdc25+. We examined the expression of wee1+ mRNA during meiosis by Northern blot analysis (Fig. 1A). To induce meiosis synchronously, we used a temperature-sensitive pat1 strain of Schizosaccharomyces pombe.21 Cells were synchronized in G1 phase by nitrogen deprivation and then shifted to the restrictive temperature to induce meiosis. The amount of wee1+ mRNA decreased around the onset of MI (5 h) in wild-type (WT) cells. By contrast, the level of wee1+ mRNA was almost constant in mei4+-deleted (mei4) cells. Furthermore, the level of wee1+ mRNA was decreased earlier (4 h) in mei4+-overproducing (mei4+-OP) cells. These results suggest that Mei4p negatively regulates wee1+ mRNA.Figure 1.

Bottom Line: Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene.In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene.These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.

View Article: PubMed Central - PubMed

Affiliation: a Aichi Cancer Center Research Institute ; Division of Molecular Oncology ; Nagoya , Japan.

ABSTRACT
In eukaryotes, the cyclin-dependent kinase Cdk1p (Cdc2p) plays a central role in entry into and progression through nuclear division during mitosis and meiosis. Cdk1p is activated during meiotic nuclear divisions by dephosphorylation of its tyrosine-15 residue. The phosphorylation status of this residue is largely determined by the Wee1p kinase and the Cdc25p phosphatase. In fission yeast, the forkhead-type transcription factor Mei4p is essential for entry into the first meiotic nuclear division. We recently identified cdc25(+) as an essential target of Mei4p in the control of entry into meiosis I. Here, we show that wee1(+) is another important target of Mei4p in the control of entry into meiosis I. Mei4p bound to the upstream region of wee1(+) in vivo and in vitro and inhibited expression of wee1(+), whereas Mei4p positively regulated expression of the adjacent pseudogene. Overexpression of Mei4p inhibited expression of wee1(+) and induced that of the pseudogene. Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene. In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene. These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.

Show MeSH
Related in: MedlinePlus