Genome-wide screening for genes associated with valproic acid sensitivity in fission yeast

Abstract

We have been studying the action mechanisms of valproic acid (VPA) in fission yeast Schizosaccharomyces pombe by developing a genetic screen for mutants that show hypersensitivity to VPA. In the present study, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 148 deletion strains to be VPA sensitive. Of the 148 strains, 93 strains also showed sensitivity to another aliphatic acids HDAC inhibitor, sodium butyrate (SB), and 55 strains showed sensitivity to VPA but not to SB. Interestingly, we found that both VPA and SB treatment induced a marked increase in the transcription activity of Atf1 in wild-type cells. However, in clr6-1, a mutant allele the clr6(+) gene encoding class I HDAC, neither VPA- nor SB induced the activation of Atf1 transcription activity. We also found that VPA, but not SB, caused an increase in cytoplasmic Ca(2+) level. We further found that the cytoplasmic Ca(2+) increase was caused by Ca(2+) influx from extracellular medium via Cch1-Yam8 channel complex. Altogether, our present study indicates that VPA and SB play similar but distinct roles in multiple physiological processes in fission yeast.

Figure 1. Representative growth pattern of the S. pombe deletion mutants in the presence of VPA or SB.

The log-phase wild-type (wt) and deletion cells as indicated were streaked onto YPDA plates with or without 5 mM VPA or 30 mM SB, and incubated at 27°C for 6 days.

Figure 2. VPA and SB may function as HDAC inhibitor in fission yeast.

(A) The Venn diagrams of VPA- or SB- sensitive strains. The SB-sensitive strains were completely included in VPA-sensitive strains. Of the 148 VPA-sensitive strains, 93 strains also showed sensitivity to SB, the other 55 strains only showed sensitivity to VPA. (B) Immunoblot analysis of histone acetylation. The wild-type cells and clr6-1 mutants were cultured in YPD at 27°C for 10 hours to exponential phase. Then the cell extracts were subjected to electrophoresis using 11% polyacrylamide gel and were immunoblotted using Acetyl-histone H4 antibody set (Ac K5; Ac K8; Ac K12) to detect histone H4 acetylation. Endogenous levels of total histone H4 protein was used as a loading control and was immunoblotted using anti-histone H4 antibodies. (C) VPA and SB treatment increased histone H4 acetylation. The exponentially growing wild-type cells were divided into three equal portions. One portion is left without treatment and the other three portions were treated with 4 mM VPA, 60 mM SB or 20 µg/ml TSA for 20 minutes, respectively. Then the protein is extracted and immunoblotted as described in Figure 2A.

Figure 3. VPA and SB increased Atf1 transcriptional activity in a Clr6-dependent manner.

(A) VPA or SB treatment markedly increased Atf1 transcription activity in wild-type cells. The wild-type cells harboring the multicopy plasmid (3XCRE::luc(R2.2) report plasmid) were grown to exponential phase, and assayed as described in Materials and Methods. The luminescence was followed for 5 hours. The data represent the accumulated value ratio of each sample (VPA or SB treatment) to the basal (EMM). Standard deviations are from three independent experiments, and each sample was analyzed in triplicate. Mean±S.D. (n = 6). P<0.05. (B) VPA or SB treatment failed to increase Atf1 transcription activity in clr6-1 mutant cells. The experiments were performed as described in Figure 3A, except clr6-1 mutant cells were monitored instead of the wild-type cells. Mean±S.D. (n = 6). P<0.01.

Figure 4. VPA, but not SB, caused an increase in the cytoplasmic Ca²⁺ level.

(A) VPA caused an increase in the cytoplasmic Ca²⁺ level. The wild-type cells harboring adh1-GFP-19-AEQ (pKB6892) were grown to exponential phase, and then the cells were collected and treated as described in Materials and Methods. A 10µl volume of EMM or 10X stock of various concentration of VPA (A) or SB (B) was added into the 96-well plate, and the cells were delivered to the wells via the luminometer pump. The aequorin luminescence was followed for 4 hours. The luminescence, given as relative light units (RLU) s⁻¹, is plotted versus time. The data are representative of six independent experiments. (B) SB didn’t cause an increase in the cytoplasmic Ca²⁺ level. The experiments were performed as described in Figure 4A except that SB was used as stimulant instead of VPA. The data are representative of six independent experiments.

Figure 5. VPA triggers Ca²⁺ influx via the Cch1-Yam8 channel complex.

(A) The increase in the cytoplasmic Ca²⁺ level is derived from the extracellular medium. The experiment was performed as described in Figure 4, expect that prior to the addition of 6 mM VPA, various concentrations of BAPTA (0.5, 1 and 2 mM) were added to chelate Ca²⁺ in EMM medium. The data are representative of six independent experiments. (B) The cch1 deletion abolished VPA-induced Ca²⁺ influx. The Δcch1 cells harboring pKB6892 were cultured and assayed as described in the legend of Figure 4A. The data are representative of six independent experiments. (C) The yam8 deletion abolished VPA-induced Ca²⁺ influx. The Δyam8 cells harboring pKB6892 were cultured and assayed as described in the legend of Figure 4A. The data are representative of six independent experiments. (D) The VPA-induced Ca²⁺ influx was observed in Δpmk1. The Δpmk1 cells harboring pKB6892 were cultured and assayed as described in Figure 4A. The data are representative of six independent experiments.