Table 1.
S. pombe strains used in this study.
Figure 1.
Deletion of gaf1+ causes reduced growth on nitrogen-starved medium.
Cells grown to mid-log phase in EMM2 for 18 h were serially diluted 5-fold and spotted on agar plates. The plates were incubated at 30°C for 3 d. (A) Cells of WT (972) and gaf1Δ (KL230) strains were spotted on EMM, EMM-N, and EMM-G agar plates. (B) Cells of WT(ED665)/pREP42, gaf1Δ(KL210)/pREP42, WT(ED665)/pREP42-Gaf1, and gaf1Δ(KL210)/pREP-Gaf1 strains were spotted onto EMM-N plates with (+B1) or without (−B1) 20 µM thiamine. WT denotes the wild-type (gaf1+).
Figure 2.
Deletion of gaf1+ causes accelerated G1-arrest under nitrogen-starved conditions.
Cells grown to mid-log phase in EMM2 for 18 h were shifted to EMM-N medium, and their DNA contents were monitored by FACS analysis at intervals. The FACS data represent typical examples of three independent experiments. (A) Cells of heterothallic (h−) WT (972) and gaf1Δ (KL230) strains were shifted to EMM-N medium. (B) Cells of homothallic (h90) gaf1Δ(KL211)/pREP-Gaf1 strain were shifted to EMM-N medium with (+B1) or without (−B1) 20 µM thiamine. WT denotes the wild-type (gaf1+).
Table 2.
Mating and sporulation frequency of S. pombe strains.
Figure 3.
Overexpression of gaf1+ results in reduction of sporulation efficiency.
Cells and colonies of the homothallic (h90) strains, WT(JY4)/pREP42, gaf1Δ(KL211)/pREP42, WT(JY4)/pREP-Gaf1, and gaf1Δ(KL211)/pREP-Gaf1, were analyzed for sporulation by DIC microscopy and iodine staining, respectively. (A) Cells were grown to mid-log phase in EMM2 for 18 h and shifted to EMM-N medium. Samples were taken from the cultures at intervals, and the morphological characteristics of cells were observed by DIC microscopy. Bar, 10 µm. (B) Cells were grown to mid-log phase in EMM2 for 18 h and spotted onto EMM-N plates with (+B1) or without (−B1) 20 µM thiamine. Sporulation was monitored by iodine vapor staining of colonies after 3-d incubation at 30°C. The FM value presented under each panel was determined by observing the cells under a DIC microscope. The values represent the average of at least three independent assays carried out in triplicate. WT denotes the wild-type (gaf1+).
Figure 4.
Effect of gaf1Δ mutation and nitrogen starvation on the global gene expression profiles of S. pombe.
RNA samples from the nitrogen-starved (−N) and unstarved (+N) cells of wild-type (gaf1+, ED668) and gaf1Δ (KL216) strains were used for the transcriptome analysis with the GeneChip Yeast Genome 2.0 Array. A Venn diagram is constructed from the lists of the genes up-regulated (≥1.5-fold, p<0.05) in unstarved gaf1Δ cells (Group −G, Table S1), nitrogen-starved wild-type cells (Group −N, Table S2), and nitrogen-starved gaf1Δ cells (Group −N/−G, Table S3). The overlapping and non-overlapping portions of the three groups (Group −G, −N, and −N/−G) are designated as Subgroup A to G. The lists of the genes included in Subgroup A–G are provided in Tables S4, S5, S6, S7, S8 S9, and S10, respectively, in the Supporting Information.
Table 3.
List of the Ste11 target genes up-regulated by gaf1Δ mutation.
Figure 5.
Deletion of gaf1+ results in accelerated induction of ste11+ expression under nitrogen-starved conditions.
(A) Northern blot analysis of ste11+ and gaf1+ mRNA from wild-type and gaf1Δ cells exposed to nitrogen starvation. Cells of wild-type (972) and gaf1Δ (KL230) strains pre-grown in EMM2 (+N) were shifted to EMM-N (−N) and cultured with constant shaking. At indicated time points, cells were harvested and washed twice with distilled water, and total RNAs were extracted from the cells. RNA blots were hybridized with 32P-labeled PCR-amplified gaf1+ and ste11+ probes. For internal control, all blots were stripped and subsequently rehybridized with 32P-labeled actin-specific probe (act1+). (B) β-Galactosidase reporter assay for analysis of ste11+ expression in wild-type and gaf1Δ cells subjected to nitrogen starvation. Cells of wild-type (ED665) and gaf1Δ (KL240) strains carrying pJLC-Ste11(p)-LacZ were cultivated to mid-log phase in EMM2 (+N) and shifted to EMM-N (−N). At indicated time points, cells were harvested and washed twice with distilled water, and the level of ste11+ expression was estimated by measuring the activity of β-galactosidase in each sample. Values are the mean ± standard error of three independent experiments carried out in triplicate, n = 9. *, p<0.01; **, p<0.05 (two-tailed Student's t-test, versus wild-type). WT denotes the wild-type (gaf1+).
Figure 6.
Gaf1 protein specifically binds to the GATA binding motif of ste11+ promoter in vitro.
(A) Schematic diagram of the promoter region of ste11+. The open- and closed-ovals represent binding sites for Rst2 (UASst) and Ste11 (TR1 and TR2), respectively. The major transcription start site (position number +1) is designated by a hinged arrow. The regions contained in the upstream probe PA (−834∼−225) and the downstream probe PB (−231∼+10) are shown as bars. The dark square represents the GATA motif spanning from −385 to −352 for which wild-type (PW) and mutant (PM) double-stranded oligonucleotide probes were designed. Restriction sites: Sp, SphI; N, NdeI; RV, EcoRV. (B) Search for Gaf1-binding region in ste11+ promoter by EMSA. GST-Gaf1 protein (1 µg) was incubated with labeled PA probe containing the 0.6-kb upstream region of ste11+ promoter in the absence (lanes 2 and 5) or presence of excess cold competitor, PA (lane 3, 50-fold; lane 4, 100-fold) or PB (lane 6, 50-fold; lane 7, 100-fold). (C) Analysis of Gaf1 binding to the GATA motif of ste11+ promoter by EMSA. GST-Gaf1 protein (lane 3, 0.1 µg; lanes 4–6, 1 µg) was incubated with labeled PA probe in the absence (lanes 3 and 4) or presence of 100-fold excess of cold competitor PM (lanes 5) or PW (lanes 6). (D) Mutational dissection of the Gaf1-binding GATA motif of ste11+ promoter by EMSA. Varying amounts of GST-Gaf1 protein (lane 3, 0.1 µg; lane 4, 0.2 µg; lane 5, 0.5 µg; lanes 6, 7, 11, and 12, 1 µg) were incubated with labeled PW oligonucleotide probe in the absence (lanes 3 and 4) or presence of 100-fold excess of cold competitor PM (lanes 5) or PW (lanes 6). Mock reaction mixtures without GST-Gaf1 or with GST protein were used as negative controls in (B)–(D). The closed and open arrow heads in (B)–(D) represent shifted bands and free probes, respectively.
Figure 7.
Schematic diagram showing the proposed function of Gaf1 in the nitrogen-signaling pathways in S. pombe.
Nitrogen starvation causes induction of gaf1+ expression, and Gaf1, in turn, represses the expression of ste11+ via direct interaction with its promoter. It has been previously known that nitrogen starvation leads to the activation of Rst2 via the cAMP-dependent PKA pathway [14], [15] as well as Atf1-Pcr1 via the Sty1 MAPK pathway [3], [5], [6], [14]–[16], [47]–[49], consequently resulting in induction of ste11+ expression. In addition, phosphodiesterase is most likely stimulated by PKA activity to create a feedback mechanism [50]. The pathway addressed in this study is shown in thick lines, and other paths previously determined are shown in thin lines. Activation and inhibition are indicated by arrows and crossing bars, respectively. Dotted lines indicate pathways remained to be fully determined.