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The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

Ivanova T, Alves-Rodrigues I, Gómez-Escoda B, Dutta C, DeCaprio JA, Rhind N, Hidalgo E, Ayté J - Mol. Biol. Cell (2013)

Bottom Line: We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription.This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin.This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona 08003, Spain Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.

ABSTRACT
In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

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Chk1 effect on Cdc10 depends on MMS concentration. (A) Loading of Cdc10, Yox1, and Nrm1-HA on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated cultures with the indicated concentrations (1 h at 30ºC). Cdc10 and Nrm1 are HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA, whereas Yox1 is determined with anti-Yox1polyclonal antibodies. (B) Total RNA was prepared from untreated (–) or MMS-treated cultures of wild-type cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of Δyox1Δnrm1 cells and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) Cell viability is unaffected at the used range of MMS concentrations. Viability test of wild-type cells treated with different concentrations of MMS, using propidium iodide or phloxine to measure viable cells, was performed by fluorescence-activated cell sorting analysis.
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Figure 3: Chk1 effect on Cdc10 depends on MMS concentration. (A) Loading of Cdc10, Yox1, and Nrm1-HA on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated cultures with the indicated concentrations (1 h at 30ºC). Cdc10 and Nrm1 are HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA, whereas Yox1 is determined with anti-Yox1polyclonal antibodies. (B) Total RNA was prepared from untreated (–) or MMS-treated cultures of wild-type cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of Δyox1Δnrm1 cells and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) Cell viability is unaffected at the used range of MMS concentrations. Viability test of wild-type cells treated with different concentrations of MMS, using propidium iodide or phloxine to measure viable cells, was performed by fluorescence-activated cell sorting analysis.

Mentions: To further characterize the response to MMS, we treated cells with increasing concentrations of the drug (from 0.002 to 0.1%) for 60 min. At the lower doses, we could not observe any noticeable effect on Cdc10, since it remained bound to the canonical promoters that we tested. In fact, Cdc10 was not released from chromatin unless cells were treated with higher MMS concentrations (≥0.05%; Figure 3A). On the contrary, when we measured the effect of MMS on the repressor system (Nrm1 and Yox1), we clearly observed that both proteins were released from chromatin (and consequently from the MBF complex) already at the lower MMS concentrations (Figure 3A). This effect on Nrm1/Yox1 paralleled a noticeable induction of the transcription of the MBF genes at low MMS concentrations (Figure 3B). We detected further release of both Yox1 and Nrm1 when we treated cells with higher MMS concentrations, which paralleled the described release of Cdc10. This second wave of Nrm1 and Yox1 release at higher concentrations correlates with repression of MBF-dependent transcription at higher MMS doses (Figure 3B). To separate both events (Cdc10 release at higher concentrations and Nrm1/Yox1 release at lower concentrations) and determine whether the DNA damage checkpoint was indeed able to induce release of the MBF complex from chromatin (and its consequent down-regulation of the MBF-dependent transcription), we decided to repeat the MMS treatment in a strain lacking the repressor system (Δnrm1Δyox1 background strain). These cells, which have induced transcription of the MBF-dependent genes as their basal steady state, were exposed to increasing MMS concentration for 1 h. As shown in Figure 3C, clear repression of the two MBF-dependent genes (cdc18 and cdc22) was observed. To determine whether release of the MBF complex from chromatin was due to cell death, we measured the viability of the cells during the time of treatment. As shown in Figure 3D and Supplemental Figure S1, the MMS concentrations used (and even higher concentrations) barely affect cell viability during the time of treatment.


The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

Ivanova T, Alves-Rodrigues I, Gómez-Escoda B, Dutta C, DeCaprio JA, Rhind N, Hidalgo E, Ayté J - Mol. Biol. Cell (2013)

Chk1 effect on Cdc10 depends on MMS concentration. (A) Loading of Cdc10, Yox1, and Nrm1-HA on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated cultures with the indicated concentrations (1 h at 30ºC). Cdc10 and Nrm1 are HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA, whereas Yox1 is determined with anti-Yox1polyclonal antibodies. (B) Total RNA was prepared from untreated (–) or MMS-treated cultures of wild-type cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of Δyox1Δnrm1 cells and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) Cell viability is unaffected at the used range of MMS concentrations. Viability test of wild-type cells treated with different concentrations of MMS, using propidium iodide or phloxine to measure viable cells, was performed by fluorescence-activated cell sorting analysis.
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Related In: Results  -  Collection

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Figure 3: Chk1 effect on Cdc10 depends on MMS concentration. (A) Loading of Cdc10, Yox1, and Nrm1-HA on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated cultures with the indicated concentrations (1 h at 30ºC). Cdc10 and Nrm1 are HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA, whereas Yox1 is determined with anti-Yox1polyclonal antibodies. (B) Total RNA was prepared from untreated (–) or MMS-treated cultures of wild-type cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of Δyox1Δnrm1 cells and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) Cell viability is unaffected at the used range of MMS concentrations. Viability test of wild-type cells treated with different concentrations of MMS, using propidium iodide or phloxine to measure viable cells, was performed by fluorescence-activated cell sorting analysis.
Mentions: To further characterize the response to MMS, we treated cells with increasing concentrations of the drug (from 0.002 to 0.1%) for 60 min. At the lower doses, we could not observe any noticeable effect on Cdc10, since it remained bound to the canonical promoters that we tested. In fact, Cdc10 was not released from chromatin unless cells were treated with higher MMS concentrations (≥0.05%; Figure 3A). On the contrary, when we measured the effect of MMS on the repressor system (Nrm1 and Yox1), we clearly observed that both proteins were released from chromatin (and consequently from the MBF complex) already at the lower MMS concentrations (Figure 3A). This effect on Nrm1/Yox1 paralleled a noticeable induction of the transcription of the MBF genes at low MMS concentrations (Figure 3B). We detected further release of both Yox1 and Nrm1 when we treated cells with higher MMS concentrations, which paralleled the described release of Cdc10. This second wave of Nrm1 and Yox1 release at higher concentrations correlates with repression of MBF-dependent transcription at higher MMS doses (Figure 3B). To separate both events (Cdc10 release at higher concentrations and Nrm1/Yox1 release at lower concentrations) and determine whether the DNA damage checkpoint was indeed able to induce release of the MBF complex from chromatin (and its consequent down-regulation of the MBF-dependent transcription), we decided to repeat the MMS treatment in a strain lacking the repressor system (Δnrm1Δyox1 background strain). These cells, which have induced transcription of the MBF-dependent genes as their basal steady state, were exposed to increasing MMS concentration for 1 h. As shown in Figure 3C, clear repression of the two MBF-dependent genes (cdc18 and cdc22) was observed. To determine whether release of the MBF complex from chromatin was due to cell death, we measured the viability of the cells during the time of treatment. As shown in Figure 3D and Supplemental Figure S1, the MMS concentrations used (and even higher concentrations) barely affect cell viability during the time of treatment.

Bottom Line: We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription.This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin.This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona 08003, Spain Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.

ABSTRACT
In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

Show MeSH
Related in: MedlinePlus