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The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

Sartagul W, Zhou X, Yamada Y, Ma N, Tanaka K, Furuyashiki T, Ma Y - PLoS ONE (2014)

Bottom Line: Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways.Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+.Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT
DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

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rhp51+ and cdc18+ transcription in synchronized cdc25-22 cells treated with HU.Wild-type cells transformed with the full-length rhp51+ (A) or cdc18+ (B) reporter were cultured to mid-log phase at 25°C in EMM and shifted to 36°C for 4 h. The cells were then maintained at 36°C continuously for G2 block (“36°C→36°C”) or shifted to 25°C for block and release (“36°C→25°C”). The cells were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in Figure 2B. The reporter activity at 300 min was analyzed and plotted as described in Figure 2B. n = 8 and 4 for each group in the block condition and the block and release condition, respectively. ***P<0.001 compared with the vehicle condition for the respective genotype and temperature condition using one-way ANOVA. ##P<0.01 and ###P<0.001 compared with G2 block at the same HU concentration using one-way ANOVA.
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pone-0111936-g005: rhp51+ and cdc18+ transcription in synchronized cdc25-22 cells treated with HU.Wild-type cells transformed with the full-length rhp51+ (A) or cdc18+ (B) reporter were cultured to mid-log phase at 25°C in EMM and shifted to 36°C for 4 h. The cells were then maintained at 36°C continuously for G2 block (“36°C→36°C”) or shifted to 25°C for block and release (“36°C→25°C”). The cells were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in Figure 2B. The reporter activity at 300 min was analyzed and plotted as described in Figure 2B. n = 8 and 4 for each group in the block condition and the block and release condition, respectively. ***P<0.001 compared with the vehicle condition for the respective genotype and temperature condition using one-way ANOVA. ##P<0.01 and ###P<0.001 compared with G2 block at the same HU concentration using one-way ANOVA.

Mentions: Because MBF and its co-repressors are critical for G1/S transcription, we examined the cell cycle regulation of rhp51+ transcription and its modulation by DNA replication stress. To this end, we used cdc25-22 cells, which arrested at G2 phase at a restrictive temperature (36°C) and progressed through the cell cycle when shifted to a permissive temperature (25°C). We examined rhp51+ reporter activity in cdc25-22 cells during G2 block (continuous 36°C culture) or after block and release (36°C culture for 4 h followed by a shift to 25°C). We also examined the activity of the cdc18+ promoter using a luciferase reporter vector because cdc18+ is regulated by both G1/S transcription and DNA replication stress. The rhp51+ reporter showed HU-induced activation in wild-type cells under nominal G2 block and block and release conditions (Figure 5A, left panel). In contrast, in cdc25-22 cells, the HU-induced increase in rhp51+ transcription was abolished under G2 block conditions, but was observed under block and release conditions (Figure 5A, right panel). We also examined the activity of Rhp51MCB and Rhp51DRE in synchronized cdc25-22 cells. Rhp51MCB, but not Rhp51DRE, showed HU-induced increases in reporter activity, similar to the full-length promoter (Figure S1). The cycle dependency of HU-induced rhp51+ and cdc18+ transcription is reasonable because HU-induced DNA replication stress should occur, in principle, only during S phase. Similar to the activity of the full-length rhp51+ promoter, cdc18+ reporter activity increased in response to HU treatment under block and release conditions, but not under G2 block conditions (Figure 5B).


The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

Sartagul W, Zhou X, Yamada Y, Ma N, Tanaka K, Furuyashiki T, Ma Y - PLoS ONE (2014)

rhp51+ and cdc18+ transcription in synchronized cdc25-22 cells treated with HU.Wild-type cells transformed with the full-length rhp51+ (A) or cdc18+ (B) reporter were cultured to mid-log phase at 25°C in EMM and shifted to 36°C for 4 h. The cells were then maintained at 36°C continuously for G2 block (“36°C→36°C”) or shifted to 25°C for block and release (“36°C→25°C”). The cells were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in Figure 2B. The reporter activity at 300 min was analyzed and plotted as described in Figure 2B. n = 8 and 4 for each group in the block condition and the block and release condition, respectively. ***P<0.001 compared with the vehicle condition for the respective genotype and temperature condition using one-way ANOVA. ##P<0.01 and ###P<0.001 compared with G2 block at the same HU concentration using one-way ANOVA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4221157&req=5

pone-0111936-g005: rhp51+ and cdc18+ transcription in synchronized cdc25-22 cells treated with HU.Wild-type cells transformed with the full-length rhp51+ (A) or cdc18+ (B) reporter were cultured to mid-log phase at 25°C in EMM and shifted to 36°C for 4 h. The cells were then maintained at 36°C continuously for G2 block (“36°C→36°C”) or shifted to 25°C for block and release (“36°C→25°C”). The cells were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in Figure 2B. The reporter activity at 300 min was analyzed and plotted as described in Figure 2B. n = 8 and 4 for each group in the block condition and the block and release condition, respectively. ***P<0.001 compared with the vehicle condition for the respective genotype and temperature condition using one-way ANOVA. ##P<0.01 and ###P<0.001 compared with G2 block at the same HU concentration using one-way ANOVA.
Mentions: Because MBF and its co-repressors are critical for G1/S transcription, we examined the cell cycle regulation of rhp51+ transcription and its modulation by DNA replication stress. To this end, we used cdc25-22 cells, which arrested at G2 phase at a restrictive temperature (36°C) and progressed through the cell cycle when shifted to a permissive temperature (25°C). We examined rhp51+ reporter activity in cdc25-22 cells during G2 block (continuous 36°C culture) or after block and release (36°C culture for 4 h followed by a shift to 25°C). We also examined the activity of the cdc18+ promoter using a luciferase reporter vector because cdc18+ is regulated by both G1/S transcription and DNA replication stress. The rhp51+ reporter showed HU-induced activation in wild-type cells under nominal G2 block and block and release conditions (Figure 5A, left panel). In contrast, in cdc25-22 cells, the HU-induced increase in rhp51+ transcription was abolished under G2 block conditions, but was observed under block and release conditions (Figure 5A, right panel). We also examined the activity of Rhp51MCB and Rhp51DRE in synchronized cdc25-22 cells. Rhp51MCB, but not Rhp51DRE, showed HU-induced increases in reporter activity, similar to the full-length promoter (Figure S1). The cycle dependency of HU-induced rhp51+ and cdc18+ transcription is reasonable because HU-induced DNA replication stress should occur, in principle, only during S phase. Similar to the activity of the full-length rhp51+ promoter, cdc18+ reporter activity increased in response to HU treatment under block and release conditions, but not under G2 block conditions (Figure 5B).

Bottom Line: Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways.Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+.Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Kobe University Graduate School of Medicine, Kobe, Japan.

ABSTRACT
DNA replication stress induces the transcriptional activation of rhp51+, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51+ transcription is not understood. The promoter region of rhp51+ contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51+ transcription. The full-length rhp51+ promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51+ promoter abolished the induction of rhp51+ transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51+ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51+ transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51+ transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51+ transcription. The transcription of rhp51+ and cdc18+, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51+ similarly to cdc18+. Collectively, these results suggest that MBF and its regulators mediate rhp51+ transcription in response to DNA replication stress, and underlie rhp51+ transcription at the G1/S transition.

Show MeSH
Related in: MedlinePlus