Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR

Abstract

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

Fig. 1. TOR regulates nucleolar structures. (A) Rapamycin causes rapid reorganization of nucleolar structures. Exponentially growing wild-type cells (FM391) were treated with rapamycin for different times. The drug carrier methanol was used as a control. Nucleolar structures are visualized by IF with a Nop1 antibody (red). The yeast nuclei were stained with DAPI (blue). (B) Rapamycin does not affect Nop1 protein level. Exponentially growing yeast cells (FM391) were treated with rapamycin for 1 h. Nop1 protein was determined by western blot. Tubulin was used as a loading control. (C) EM analysis of nucleolar structures before and after rapamycin treatment. Exponentially growing yeast cells (FM391) were treated with rapamycin for 1 h and used for EM analysis. The arrowhead indicates the yeast nucleolus. V, vacuole. (D) The effect of rapamycin on nucleolar structure is due to inhibition of TOR. The yeast strain carrying a wild-type TOR1 (SZy998) or a dominant rapamycin-resistant TOR1 allele (TOR1-RR) (SZy997) was treated with rapamycin for 1 h. The morphology and structure of the yeast nucleolus was examined by IF with a Nop1 antibody. (E) Inhibition of protein synthesis is insufficient to cause nucleolar reorganization. Wild-type yeast cells (FM391) were treated with cycloheximide (CHX) for 1 h. Nucleolar structure was analyzed as for (D). (F) Inhibition of rDNA transcription is not sufficient to change nucleolar morphology. Wild-type and rrn3-1 temperature-sensitive mutant (YCC95) strains were switched from permissive temperature (25°C) to restrictive temperature (38°C) for 1 h. Nucleolar structure was determined as for (D).

Fig. 1. TOR regulates nucleolar structures. (A) Rapamycin causes rapid reorganization of nucleolar structures. Exponentially growing wild-type cells (FM391) were treated with rapamycin for different times. The drug carrier methanol was used as a control. Nucleolar structures are visualized by IF with a Nop1 antibody (red). The yeast nuclei were stained with DAPI (blue). (B) Rapamycin does not affect Nop1 protein level. Exponentially growing yeast cells (FM391) were treated with rapamycin for 1 h. Nop1 protein was determined by western blot. Tubulin was used as a loading control. (C) EM analysis of nucleolar structures before and after rapamycin treatment. Exponentially growing yeast cells (FM391) were treated with rapamycin for 1 h and used for EM analysis. The arrowhead indicates the yeast nucleolus. V, vacuole. (D) The effect of rapamycin on nucleolar structure is due to inhibition of TOR. The yeast strain carrying a wild-type TOR1 (SZy998) or a dominant rapamycin-resistant TOR1 allele (TOR1-RR) (SZy997) was treated with rapamycin for 1 h. The morphology and structure of the yeast nucleolus was examined by IF with a Nop1 antibody. (E) Inhibition of protein synthesis is insufficient to cause nucleolar reorganization. Wild-type yeast cells (FM391) were treated with cycloheximide (CHX) for 1 h. Nucleolar structure was analyzed as for (D). (F) Inhibition of rDNA transcription is not sufficient to change nucleolar morphology. Wild-type and rrn3-1 temperature-sensitive mutant (YCC95) strains were switched from permissive temperature (25°C) to restrictive temperature (38°C) for 1 h. Nucleolar structure was determined as for (D).

Fig. 2. Nutrient starvation and rapamycin cause RNA Pol I delocalization from the nucleolus. (A) Nitrogen starvation causes nucleolar reorganization and RNA Pol I delocalization from the nucleolus. Wild-type yeast (FM391) in SC medium was switched to SC minus nitrogen (SC –N) for 1 h. Localization of A190 and A43 was examined by IF with antibodies specific for A190 and A43, respectively. (B) Rapamycin causes RNA Pol I delocalization from the nucleolus. Exponentially growing wild-type yeast cells (FM391) were treated with rapamycin for 1 h. Localization of A190 and A43 was determined as for (A). (C) The structural organization of a yeast rDNA unit and the primers used for ChIP assays and northern blot. ETS, externally transcribed spacer. (D) Rapamycin causes dissociation of A43 from rDNA chromatin. Wild-type yeast (FM391) was treated with rapamycin for 1 h. RNA Pol I association with rDNA chromatin was determined by ChIP with an A43 antibody and by PCR with rDNA primer pairs. CAb, control antibody. (E) Short-term rapamycin treatment does not affect A43 and A190 protein levels. Wild-type yeast (FM391) was treated with rapamycin for 1 h. The levels of A190, A43, Nop1 and Tub1 were determined by western blot.

Fig. 2. Nutrient starvation and rapamycin cause RNA Pol I delocalization from the nucleolus. (A) Nitrogen starvation causes nucleolar reorganization and RNA Pol I delocalization from the nucleolus. Wild-type yeast (FM391) in SC medium was switched to SC minus nitrogen (SC –N) for 1 h. Localization of A190 and A43 was examined by IF with antibodies specific for A190 and A43, respectively. (B) Rapamycin causes RNA Pol I delocalization from the nucleolus. Exponentially growing wild-type yeast cells (FM391) were treated with rapamycin for 1 h. Localization of A190 and A43 was determined as for (A). (C) The structural organization of a yeast rDNA unit and the primers used for ChIP assays and northern blot. ETS, externally transcribed spacer. (D) Rapamycin causes dissociation of A43 from rDNA chromatin. Wild-type yeast (FM391) was treated with rapamycin for 1 h. RNA Pol I association with rDNA chromatin was determined by ChIP with an A43 antibody and by PCR with rDNA primer pairs. CAb, control antibody. (E) Short-term rapamycin treatment does not affect A43 and A190 protein levels. Wild-type yeast (FM391) was treated with rapamycin for 1 h. The levels of A190, A43, Nop1 and Tub1 were determined by western blot.

Fig. 3. Rpd3–Sin3 HDAC is required for nucleolar reorganization. (A) Rapamycin causes rDNA chromatin condensation as judged by FISH of rDNA. Exponentially growing yeast cells (FM391) were treated with rapamycin for 1 h and subjected to FISH analysis. A pseudo yellow color was generated for rDNA for merging with DAPI images. (B) Deletion of the Rpd3–Sin3 HDAC components confers partial rapamycin resistance. Cultures of wild-type and mutant strains (FM391 and derivatives, see Table I) were serially diluted (10-fold), spotted onto SC plates without or with 25 nM rapamycin and incubated for 2 (–Rap) and 5 days (+Rap), respectively. (C) Deletion of the Rpd3–Sin3 HDAC components blocks rapamycin-induced nucleolar reorganization. Wild-type or mutant strains (FM391 and derivatives, see Table I) were treated with rapamycin for 1 h. Nucleolar structures were examined by IF with a Nop1 antibody. (D) Hda1 and Hos1/2/3 are not required for nucleolar reorganization. Wild-type and mutant strains (FM391 and derivatives, see Table I) were treated with rapamycin for 1 h. Nucleolar structures were examined as for (C).

Fig. 4. Rapamycin promotes the association of Rpd3–Sin3 HDAC with rDNA chromatin and causes concomitant histone H4 deacetylation. (A) Rapamycin treatment promotes Rpd3 association with rDNA. Left panel: wild-type (FM392) and rpd3Δ mutant (FM392 rpd3Δ) cells were treated with rapamycin for 1 h. Rpd3 association with rDNA chromatin was determined by ChIP with an Rpd3 antibody. ACT1 and CUP1 were used as controls. CAb, control antibody. Right panel: quantification of Rpd3 binding to rDNA chromatin (three independent experiments). (B) Rapamycin treatment stimulates Sin3 association with rDNA. The Sin3-Myc (TLY446) cells were treated with rapamycin for 1 h. Sin3 association with rDNA was determined by ChIP with a Myc-specific monoclonal antibody (9E10). ACT1 and CUP1 were used as controls. CAb, control antibody. (C) Rapamycin causes histone H4 deacetylation at rDNA chromatin. Wild-type (FM392) and rpd3Δ mutant (FM392 rpd3Δ) cells were treated with rapamycin for 1 h. Histone H4 acetylation at rDNA chromatin was determined by ChIP with an acetylated H4 K5 or K12 antibody. ACT1 and CUP1 were used as controls. CAb, control antibody. (D) Rapamycin has little effect on overall histone H4 K5/12 acetylation. Wild-type (FM392) and rpd3Δ mutant (FM392 rpd3Δ) cells were treated with rapamycin for 1 h. Total acetylated histone H4 was examined by western blot with acetylated histone H4 K5/12 antibodies. Tubulin was used as a loading control.

Fig. 5. Rpd3 and H4 deacetylation are required for regulation of nucleolar organization and RNA Pol I localization. (A) Rpd3 is required for nucleolar reorganization and RNA Pol I delocalization. Wild-type (FM392) and mutant (FM392 rpd3Δ) cells were under rapamycin treatment (top panel) or nitrogen starvation for 1 h (bottom panel). Nucleolar structures were detected by IF with a Nop1 antibody (red). Localization of A43 was examined by IF with an A43-specific antibody (green). (B) The effect of histone H4 lysine mutations on nucleolar organization and RNA Pol I localization. Wild-type H4 (MAY200) and H4 K5,12G (MAY512G) (top panel) and H4 K5,8R (MSY613) and H4 K5,12R (MSY641) (bottom panel) cells were treated with rapamycin for 1 h. The nucleolar structure and the localization of A43 was detected as for (A).

Fig. 5. Rpd3 and H4 deacetylation are required for regulation of nucleolar organization and RNA Pol I localization. (A) Rpd3 is required for nucleolar reorganization and RNA Pol I delocalization. Wild-type (FM392) and mutant (FM392 rpd3Δ) cells were under rapamycin treatment (top panel) or nitrogen starvation for 1 h (bottom panel). Nucleolar structures were detected by IF with a Nop1 antibody (red). Localization of A43 was examined by IF with an A43-specific antibody (green). (B) The effect of histone H4 lysine mutations on nucleolar organization and RNA Pol I localization. Wild-type H4 (MAY200) and H4 K5,12G (MAY512G) (top panel) and H4 K5,8R (MSY613) and H4 K5,12R (MSY641) (bottom panel) cells were treated with rapamycin for 1 h. The nucleolar structure and the localization of A43 was detected as for (A).

Fig. 6. Rpd3 is required for inhibition of rDNA transcription by rapamycin and nutrient starvation. (A) Rpd3 is required for inhibition of rDNA transcription by rapamycin and nitrogen starvation. Wild-type (FM392) and mutant (FM392 rpd3Δ) cells were treated with rapamycin or switched to SC minus nitrogen (SC –N). Aliquots of cells were withdrawn at different times. The nascent rRNA transcript (35S) was detected by northern blot with ³²P-labeled ETS probe. (B) Rapamycin and amino acid starvation cause a decrease of the nucleolar size in mammalian cells. REFs were treated with rapamycin or under amino acid starvation for 24 h. Nucleolar structures were analyzed by IF with a nucleophosmin antibody. The nucleus was stained with DAPI. (C) A model for the regulation of nucleolar structure and function by TOR. See Discussion for details.