Sch9 regulates ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex RPD3L

Abstract

TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.

Figure 1

Stb3, Dot6 and Tod6 are directly phosphorylated by Sch9 in vivo and in vitro. (A–C) Strains of indicated genotype and expressing 3HA–Stb3 (A), Dot6–5HA (B) or Tod6–5HA (C) were grown exponentially in YPD at 30°C and subjected to the indicated treatments. (A) 3HA–Stb3 was immunoprecipitated after denaturing protein extraction and phosphorylation status (anti-R[R/K]xS^*) and abundance (anti-HA) determined by western blot. A strain transformed with empty vector was used as a mock IP control (left lane) (B, C) Dot6–5HA and Tod6–5HA phosphorylation status was determined by migration in SDS–PAGE and anti-HA western blot. (D–G) In vitro kinase assays. GST–Stb3 (D, G), GST–Dot6^wt and GST–Dot6^5A (E) and GST–Tod6^wt and GST–Tod6^6A (F) were tested as substrates for Sch9^3E (Urban et al, 2007) in presence of γ-³²P-ATP (D–F) or unlabelled ATP only (G). Reactions with GST as the substrate (D) or Sch9^kd, a point mutant lacking catalytic activity, as the kinase (D–G) were performed to control for specificity. Reactions were resolved by SDS–PAGE, stained with coomassie (CBB) and ³²P incorporation detected by autoradiography (D–F). Alternatively, reactions were analysed by western blot against the Protein A and GST tags and the phosphorylated R[R/K]x[S/T]^* motif (G).

Figure 2

Sch9 regulates cell growth via Stb3, Dot6, Tod6 and RPD3L. (A) Regulation of Ribi and RP transcription. Strains of the indicated genotype were grown to exponential phase in YPD at 30°C, treated for 30 min with 1NM-PP1 (PP1) or drug vehicle, followed by determination of mRNA-Seq transcriptome profiles. All genes downregulated >1.5-fold by 1NM-PP1 versus drug vehicle in the sch9^as strain are shown, as sorted by magnitude of change. In the right panels, genes belonging to the Ribi or RP regulons or whose promoters contain RRPE or PAC promoter elements or both (R+P) (Jorgensen and Tyers, 2004) are indicated with blue dashes if derepressed preferentially by STB3 deletion or red dashes if derepressed preferentially by DOT6/TOD6 deletion. (B) Regulation of growth rate. SCH9 and sch9^as strains harbouring the indicated gene deletions were grown to exponential phase at 30°C in YPD in the presence of 1NM-PP1 and doubling times calculated from quantitative growth curves. Growth rate effects were recapitulated in spot assays; the phenotypes of the stb3Δ and dot6Δ tod6Δ strains were confirmed by complementation with plasmid-encoded wild-type alleles (Supplementary Figure S3A and B). Data are means of three independent experiments±s.d. ^**P<0.01; ^***P<0.001 versus sch9^as. ^#P<0.05; ^##P<0.01 versus sch9^asstb3Δ. (C, D) Regulation of cell size. Strains of the indicated genotypes were grown as in (B) and cell size distributions determined on a Z2 Coulter counter. Size profiles are shown in two separate panels with the same scales for clarity. Corresponding SCH9^wt control distributions were also determined (Supplementary Figure S3B and C; see Supplementary Table S2 for all quantitative size parameters).

Figure 3

Stb3, Dot6 and Tod6 regulate RNA Pol I transcription initiation and rRNA processing. (A, B) Miller chromatin spreads were prepared from the indicated strains treated for 30 min with 1NM-PP1 or DMSO. (A) Representative electron micrographs of transcribed rDNA genes in Miller spreads. (B) The number of polymerases of each active rDNA repeat was determined and plotted. Averages are marked for each group with a line bounded by triangles. (C) RNA synthesis in the indicated strains following DMSO or 1NM-PP1 treatment was determined by metabolic pulse labelling with ³H-uracil. Total RNA loaded was visualized by staining with ethidium bromide (EtBr). Mature rRNA (25S and 18S) and pre-rRNA (27S and 20S) species are indicated.

Figure 4

Stb3, Dot6 and Tod6 phosphorylation regulates their activity in vivo. (A, B) The indicated strains were transformed with CEN-based plasmids expressing the indicated alleles of STB3, DOT6 or TOD6 from the strong constitutive ADH1 promoter. Cells were then plated in 10-fold dilution series (A) or restruck (B) onto selective synthetic medium and grown for 3 days at 30°C.

Figure 5

Regulation of Tod6 expression level and protein abundance. (A) SCH9 and sch9^as cells expressing Tod6–5HA were grown in YPD at 30°C and subjected to 300 nM 1NM-PP1 treatment as indicated. Proteins were extracted under denaturing conditions and analysed by western blot against the HA epitope; an antibody specific to the Hog1 protein served as loading control. (B) Strains expressing the indicated TOD6–5HA alleles were grown to exponential phase in SC-URA at 30°C and proteins were extracted and analysed as in (A). (C) Quantification of Tod6–5HA versus Hog1 abundance shown in (B) and two other independent experiments. Quantification of TOD6–5HA versus ACT1 mRNA expression was determined from total RNA extracts from the same strains as in (B). The ratio of Tod6–5HA protein to TOD6–HA mRNA was calculated from these values and shown as mean±s.d. of three independent experiments. (D) Endogenous TOD6 versus ACT1 expression levels for the same RNA extracts shown in (C). Values are mean±s.d. of three independent experiments. ^*P<0.05; ^**P>0.01; ^***P<0.001 versus wild-type control.

Figure 6

RPD3L recruitment to Ribi and RP promoters upon Sch9 inhibition in a Stb3- and Dot6/Tod6-dependent manner. (A) Strains of the indicated genotype expressing an Sds3–TAP fusion protein were grown exponentially in YPD at 30°C and treated with 300 nM 1NM-PP1 for 20 min followed by fixation, chromatin extraction and ChIP-Seq analysis. Sds3 (RPD3L)-associated sequences were detected by comparing read counts from the sch9^asSDS3–TAP strain to an sch9^as (untagged) mock control using the MACS algorithm. Peak intensities were calculated in all conditions at these loci (Supplementary File F2) and normalized to the untagged control counts. Peaks showing an upregulation >1.5-fold in the sch9^as strain compared with wild type are shown as sorted by magnitude of change. In the right panels, peaks mapping to Ribi or RP gene promoters are indicated with blue dashes if upregulation upon Sch9 inhibition was preferentially suppressed by STB3 deletion, or with red dashes if upregulation upon Sch9 inhibition was preferentially suppressed by DOT6/TOD6 deletion. (B) Strains of the indicated genotype expressing an Stb3–TAP fusion protein or untagged Stb3 (mock control) were grown, treated and processed for ChIP-Seq analysis as in (A). In all, 70 peaks of Sds3–TAP (RPD3L) binding mapping to Ribi gene promoters were sorted according to their score (Supplementary File F2) and divided into two sets (Ribi 1–35 and 36–70). Each set of peaks were aligned according to their maxima, summed and plotted for each condition (black line; left panels). Total read counts mapping to the aligned loci in the Stb3–TAP ChIP-Seq analyses (red and green) and their mock control (blue) were also plotted. A similar analysis for peaks mapping to RP gene promoters was performed (right panels). The relative position of Rap1 (grey) and Fhl1 (yellow) binding sites in these promoters was evaluated by scoring each loci using previously published position weight matrices of the corresponding motifs (Harbison et al, 2004). (C, D) Stb3 and Dot6/Tod6 cooperate to bind upstream of Ribi genes. sch9^as strains bearing the indicated gene deletions and expressing the indicated TAP-tagged proteins were grown exponentially in YPD at 30°C and treated with 300 nM 1NM-PP1 for 20 min. Cells were then fixed and processed for ChIP–qPCR analysis for the indicated loci. Data are shown as mean±s.d. of four (C) and three (D) independent experiments.

Figure 7

Sch9 regulates ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex RPD3L. Sch9 directly phosphorylates the transcription repressors Stb3, Dot6 and Tod6 to antagonize their ability to recruit the RPD3L histone deacetylase to RP and ribi promoters. The thicker arrows indicate that Stb3 primarily mediates suppression of RP genes while Dot6/Tod6 primarily mediate suppression of ribi genes. The dashed arrow labelled Tod6 is included to highlight the fact that TOD6 belongs to the ribi regulon and thus participates in a feedback loop to maintain homeostasis of ribi gene expression. Please see text for further details.