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

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

Activities of the full-length <i>rhp51</i>⁺ and 3xMCB reporters in Δ<i>yox1</i> and Δ<i>nrm1</i> cells treated with HU.

<p>Wild-type (wt) cells, Δ<i>yox1</i> cells, and Δ<i>nrm1</i> cells transformed with the full-length <i>rhp51</i>⁺ (A, “Rhp51”) or 3xMCB (B) reporter were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. The reporter activity at 300 min was analyzed and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. <i>n</i> = 3 for each group. *<i>P</i><0.05 and ***<i>P</i><0.001 compared with the vehicle condition for the respective genotype using one-way ANOVA followed by Tukey's test. ###<i>P</i><0.001 compared with wild-type cells treated with the same HU concentration using one-way ANOVA followed by Tukey's test.</p

Activities of the full-length <i>rhp51</i>⁺ and 3xMCB reporters in Δ<i>chk1</i>, Δ<i>cds1</i>, Δ<i>chk1</i>Δ<i>cds1</i>, and Δ<i>rad3</i> cells treated with HU.

<p>Cells transformed with the full-length <i>rhp51</i>⁺ (A, “Rhp51”) or 3xMCB (B) reporter were treated with HU at 1 mM, 2 mM, or 4 mM or with vehicle, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. The reporter activity at 300 min was analyzed and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. <i>n</i> = 3 for each group. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 compared with the vehicle condition for the respective genotype using one-way ANOVA. #<i>P</i><0.05, ##<i>P</i><0.01, and ###<i>P</i><0.001 compared with wild-type cells treated with the same HU concentration using one-way ANOVA. +<i>P</i><0.05 and ++<i>P</i><0.01 compared with Δ<i>chk1</i> cells treated with the same HU concentration using one-way ANOVA.</p

Induction of DNA replication stress upon HU treatment.

<p><i>A</i>. Mobility shift detection of phosphorylated Cds1 upon HU treatment. Wild-type cells (“wt”; KP456) and cells expressing Flag-tagged Cds1 (“Cds1-Flag”; KP6609) were cultured to mid-log phase at 27°C in EMM supplemented with 225 mg/L leucine and uracil. The cells were divided into four equal volumes and incubated with HU at a final concentration of 1 mM, 2 mM, or 4 mM for 4 h. Cell lysates were subjected to Phos-tag SDS-PAGE, and Flag-tagged Cds1 was detected by immunoblotting with an anti-FLAG antibody. Because the migration of phosphorylated proteins is slower than that of non-phosphorylated proteins, the black and white arrows likely indicate the non-phosphorylated and phosphorylated forms of Cds1, respectively. Note that these signals were not detected in lysates from wild-type cells. <i>B.</i> Effect of HU treatment on cell length. Wild-type cells were cultured to mid-log phase at 27°C in EMM. The cells were divided into four equal volumes and incubated with HU at a final concentration of 1 mM, 2 mM, or 4 mM for 8 h. The cells were collected, stained with Calcofluor White, and observed under a fluorescence microscope. Scale bar, 10 µm. The cell lengths at each respective HU concentration were averaged, and the results are shown in the graph. <i>n</i> = 31 for each group. ***<i>P</i><0.001 compared with the vehicle condition using one-way ANOVA followed by Tukey's test (<i>F</i>_(3,120)  = 37.49, <i>P</i><0.0001).</p

The MluI Cell Cycle Box (MCB) Motifs, but Not Damage-Responsive Elements (DREs), Are Responsible for the Transcriptional Induction of the <i>rhp51</i>⁺ Gene in Response to DNA Replication Stress

<div><p>DNA replication stress induces the transcriptional activation of <i>rhp51</i>⁺, a fission yeast <i>recA</i> homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates <i>rhp51</i>⁺ transcription is not understood. The promoter region of <i>rhp51</i>⁺ 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 <i>rhp51</i>⁺ transcription. The full-length <i>rhp51</i>⁺ 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 <i>rhp51</i>⁺ promoter abolished the induction of <i>rhp51</i>⁺ transcription by DNA replication stress. Consistent with a role for MCB motifs in <i>rhp51</i>⁺ transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded <i>rhp51</i>⁺ 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 <i>rhp51</i>⁺ 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 <i>rhp51</i>⁺ transcription. The transcription of <i>rhp51</i>⁺ and <i>cdc18</i>⁺, an MBF-dependent G1/S gene, peaked simultaneously in synchronized <i>cdc25-22</i> cells. Furthermore, DNA replication stress maintained transcription of <i>rhp51</i>⁺ similarly to <i>cdc18</i>⁺. Collectively, these results suggest that MBF and its regulators mediate <i>rhp51</i>⁺ transcription in response to DNA replication stress, and underlie <i>rhp51</i>⁺ transcription at the G1/S transition.</p></div

<i>rhp51</i>⁺ and <i>cdc18</i>⁺ transcription in synchronized <i>cdc25-22</i> cells treated with HU.

<p>Wild-type cells transformed with the full-length <i>rhp51</i>⁺ (A) or <i>cdc18</i>⁺ (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. The reporter activity at 300 min was analyzed and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111936#pone-0111936-g002" target="_blank">Figure 2B</a>. <i>n</i> = 8 and 4 for each group in the block condition and the block and release condition, respectively. ***<i>P</i><0.001 compared with the vehicle condition for the respective genotype and temperature condition using one-way ANOVA. ##<i>P</i><0.01 and ###<i>P</i><0.001 compared with G2 block at the same HU concentration using one-way ANOVA.</p

Real-time monitoring of <i>rhp51</i>⁺ gene transcription in wild-type cells treated with HU.

<p><i>A</i>. A schematic diagram of luciferase reporter vectors containing the full-length <i>rhp51</i>⁺ promoter or <i>rhp51</i>⁺ promoter deletion mutants. Two DRE decamers are located between bp −234 to −225 (DRE1) and bp −213 to −204 (DRE2) relative to the translation initiation site of the <i>rhp51</i>⁺ promoter. Two MCB motifs are located between bp −192 to −187 (MCB1) and bp −183 to −178 (MCB2) in the <i>rhp51</i>⁺ promoter. The following regions of the <i>rhp51</i>⁺ promoter were inserted upstream of the open reading frame of luciferase: the full-length promoter ranging from bp −345 to −14 (pKB8310, designated Rhp51), a fragment from bp −201 to −14 containing two MCB motifs (pKB8608, designated Rhp51^MCB), a fragment from bp −345 to −202 containing two DREs (pKB8606, designated Rhp51^DRE), and the full-length promoter from which the two MCB motifs at bp −192 to −178 were deleted (pKB8929, designated Rhp51^ΔMCB). <i>B</i>. Effect of HU on promoter activation. Wild-type cells transformed with the full-length <i>rhp51</i>⁺ (Rhp51), Rhp51^MCB, Rhp51^DRE, or Rhp51^ΔMCB reporter were incubated with luciferin and then treated with HU (1 mM to 4 mM) for real-time monitoring of luciferase activity. Relative light units (RLU) were normalized to the values from wild-type cells harboring the full-length <i>rhp51</i>⁺ reporter plasmid at 300 min without HU treatment. Representative traces of real-time monitoring are shown in the upper graphs. The lower graphs show the normalized RLU averaged across independent samples at 300 min in cells harboring the indicated reporter plasmids. <i>n</i> = 4 for each group. *<i>P</i><0.05 and ***<i>P</i><0.001 compared with the vehicle condition for the respective reporter using one-way ANOVA followed by Tukey's test.</p

<i>Schizosaccharomyces pombe</i> haploid strains used in this study.

<p><i>Schizosaccharomyces pombe</i> haploid strains used in this study.</p