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Results: 7

1.
Figure 5.

Figure 5. From: The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

The H3K56ac pathway regulates rRNA processing. (A) Defects in Asf1 cause accumulation of non-processed rRNA. Wild-type, asf1Δ, uaf30Δ and hmo1Δ were grown to log phase, total RNA was extracted, cDNA was synthesized and qPCR performed with the indicated primer sets. The average and standard deviation of four independent experiments are shown with significance determined by t-test. **P < 0.05; ****P < 0.005. (B) H3K56ac regulates rRNA processing. The histone H3 wild-type and H3K56A mutant were grown to log phase and processed as described in (a) to measure rRNA levels. The average and standard deviation of at least three or more independent experiments is shown and significance was determined by t-test. **P < 0.05.

Hongfeng Chen, et al. Nucleic Acids Res. 2012 August;40(14):6534-6546.
2.
Figure 3.

Figure 3. From: The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

H3K56ac defects in tco89Δ cannot be rescued by increased Tor1 or Sch9 kinase activity but are rescued by loss of the Sirtuin deacetylases Hst3 or Hst4. (A) Reexpression of Tco89 fully rescues global H3K56ac in tco89Δ cells. Wild-type and tco89Δ cells were transformed with either an empty vector or vector expressing Tco89 as a C-terminal mono-Flag fusion and grown in SC-Ura media to select for plasmid maintenance. An amount of 30 µg of WCE were prepared and analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. (B) Increased TORC1 kinase activity does not rescue global H3K56ac in tco89Δ cells. Experiment was performed as in (A) except cells were transformed with control vector or vectors expressing HA-tagged wild-type Tor1 or the mutant Tor1A1957V kinase that has increased kinase activity (44). (C) TORC1 regulation of H3K56ac is independent of the downstream Sch9 kinase. The experiment was performed as in (A) except cells were transformed with control vector or vectors expressing HA-tagged wild-type Sch9 or the Sch92D3E mutant that is active independent of upstream TORC1 activity (9). (D) H3K56ac is rescued in tco89Δ by inactivation of Hst3 or Hst4. Wild-type, tco89Δ, tco89Δ hst3Δ and tco89Δ hst4Δ strains were grown to log phase before preparing WCE . 30 µg of WCE were resolved by SDS–PAGE and analyzed by α-H3K56ac or α-H3 immunoblot. (E) Global inhibition of Sirtuin activity rescues H3K56ac in tco89Δ. Wild-type and tco89Δ cultures were grown to log phase and then mock treated or treated with 25 mM nicotinamide for 1 h before harvesting and preparing WCE. Samples were analyzed as in (D).

Hongfeng Chen, et al. Nucleic Acids Res. 2012 August;40(14):6534-6546.
3.
Figure 1.

Figure 1. From: The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

The H3K56ac pathway is required for TORC1-regulated growth. (A) H3 lysine to alanine mutants exhibit variable sensitivity to rapamycin. Wild-type (WT) and the indicated histone H3 lysine to alanine mutants were grown overnight at 30°C and then cell density for each strain was measured by taking the OD600. Equivalent numbers of cells were serially diluted 5-fold and replica spotted to control YPD or YPD plates containing 25 nM rapamycin and incubated at 30°C for 4 days before photographing. (B) Acetylation of H3K56 is key in TORC1-regulated growth. Experiment was performed as in (A) except photographs were taken at 3 days to highlight the growth difference between the H3K56Q and WT strains. (C) H3K56ac regulators are selectively required for TORC1-dependent cell growth. The experiment was performed as in (A) with the WT and indicated gene deletion mutants. Photographs were taken after 4 days at 30°C. (D) H3K56ac regulators do not undergo cell-cycle arrest after an inhibitory rapamycin treatment. The indicated yeast strains were cultured to log phase and then equal numbers of cells were pelleted, 5-fold serially diluted and spotted to YPD plates. The remaining cultures were treated with 200 nM rapamycin for 5.5 h and then equal numbers of cells were pelleted, washed and 5-fold serially diluted before spotting to YPD plates. Photographs were taken after incubation at 30°C for 3 days.

Hongfeng Chen, et al. Nucleic Acids Res. 2012 August;40(14):6534-6546.
4.
Figure 6.

Figure 6. From: The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

The H3K56ac pathway regulates rDNA transcription. (A) Asf1 coassociates with RNA Pol I. WCE from the indicated log phase cultures were prepared and α−Myc immunoprecipitations were performed using 750 µg of WCE. Samples were resolved by 10% SDS–PAGE, immunoblotted with α−HA antibody and then membranes were stripped and reprobed with α−Myc antibody. Input samples represent 30 µg of each extract. The asterisk denotes a cross-reactive protein found in all the samples. (B) Asf1 localizes to the rDNA promoter. ChIP experiments using α-HA antibody and either no tag control or Asf1-6XHA expressing cells were performed using the rDNA promoter primer set indicated in Figure 3A. The Asf1-specific signal was expressed as fold increase over the no tag signal which was set to 1. The average and standard deviation of three independent experiments are presented and significance was determined by t-test. **P < 0.05. (C) Asf1 regulates RNA Pol levels on the rDNA. α-Myc ChIP was performed using no tag control, Rpa190-Myc or Rpa190-Myc asf1Δ strains and the level of RNA Pol I determined at the indicated positions on the rDNA. The wild-type Rpa190-Myc signal was set to 1 and the no tag control and asf1Δ signals were expressed relative to this signal. The average and standard deviation of three or more independent experiments are shown with significance determined by t-test. ***P < 0.01. (D) Loss of H3K56ac reduces RNA Pol I recruitment to the rDNA. No tag control, histone H3 wild-type or H3K56A strains expressing Rpa190-9XMyc were used in α−Myc ChIP as described in (C). The histone H3 wild-type strain was set to 1 for each primer set and the no tag control and H3K56A mutant expressed relative to it. The average and standard deviation of four to five independent experiments are shown with significance determined by t-test. **P < 0.05; ***P < 0.01.

Hongfeng Chen, et al. Nucleic Acids Res. 2012 August;40(14):6534-6546.
5.
Figure 4.

Figure 4. From: The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

H3K56ac regulators localize to the rDNA. (A) Simplified schematic of the rDNA locus and the relative locations of primers used in qPCR. Note that the ITS1 specific primers span the A2 site cleaved by the SSU processome during rRNA processing. Detailed description of rRNA processing reactions involved in rRNA maturation are described previously (49). (B) H3K56ac localizes to rDNA but is not decreased in tco89Δ. ChIP using α−H3K56ac or α−H3 antibodies and primers to the promoter, 18S, ITS1 or 25S rDNA sequences. Data are the average and standard deviation of a minimum of two or more independent experiments. (C) tco89Δ does not decrease rRNA levels. Total RNA was prepared from wild-type (WT) and tco89Δ cells grown to log phase and cDNA was synthesized using 1 µg of RNA and random hexamer primers. qPCR was performed with the indicated primer sets, the WT signal was set to 1 and tco89Δ expressed relative to WT The average and standard deviation of two independent experiments are shown. (D) H3K56ac is significantly reduced in WT cells upon rapamycin treatment. WT cells were grown to log phase and either mock treated or treated with an inhibitory (200 nM) concentration of rapamycin for 1 h. WCE were prepared and 30 µg analyzed by SDS–PAGE and α-H3K56ac and α-H3 immunoblotting. (E) Rapamycin mediated inhibition of TORC1 signaling reduces H3K56ac on the rDNA. Experiment was performed as in (D) except samples were processed for α-H3K56ac or α-H3 ChIP. The regions of the rDNA are indicated analyzed are indicated. The data are the average and standard deviation of three or more independent experiments. **P < 0.05; ***P < 0.01; ****P < 0.005.

Hongfeng Chen, et al. Nucleic Acids Res. 2012 August;40(14):6534-6546.
6.
Figure 2.

Figure 2. From: The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

The TORC1 subunit Tco89 is required for H3K56ac. (A) Asf1 mutations exhibit negative genetic interactions only with Tco89, but not Tor1, mutants. Wild-type and the indicated single and double gene deletion mutants were grown overnight and then equal numbers of cells were 5-fold serially diluted and spotted to YPD or SC media. Plates were incubated at 30°C and photographs taken after 2 days. (B) Disruption of the EGO complex or TORC1 reduces global H3K56ac. Wild-type or the indicated gene deletion mutants were cultured in YPD to log phase, WCE were prepared and 30 µg resolved by 15% SDS–PAGE. Samples were transferred to PVDF membrane and immunoblotted with α-H3K56ac or α-H3 specific antibodies. (C) Quantitation of immunoblots. α−H3K56ac and α−H3 immunoblots were quantified using ImageJ software and expressed as a ratio of H3K56ac over total H3. The average and standard deviation of a minimum of at least three or more independent experiments for each mutant are plotted and statistical significance determined by t-test. **P < 0.05; ****P < 0.005. (D) H3K56ac reduction in tco89Δ is not due to a significant disruption in cell-cycle progression. Wild-type, tor1Δ, tco89Δ and asf1Δ were grown to an OD600 = 0.8 before staining with SYTOX Green and performing flow-cytometry. The histograms for each strain are presented as well as the percentage of cells in G1, S and G2 phase.

Hongfeng Chen, et al. Nucleic Acids Res. 2012 August;40(14):6534-6546.
7.
Figure 7.

Figure 7. From: The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

H3K56ac regulates rDNA chromatin and rDNA transcriptional deregulation sensitizes cells to rapamycin in asf1Δ. (A) Hmo1 rDNA binding is reduced in H3K56A mutants. α-HA ChIP was performed at the rDNA with no tag control, histone H3 wild-type and H3K56A mutants expressing Hmo1-6XHA. The no tag control and H3K56A signals were expressed relative to the histone H3 wild-type signal. The average and standard deviation of three independent experiments are shown with significance determined by t-test. **P < 0.05; ****P < 0.005. (B) H3K56ac is necessary for SSU processome recruitment. Experiment was performed as in (A) except strains used were either a no tag control or expressed the SSU processome subunit Utp9 as a 6XHA fusion in the histone H3 wild-type or H3K56A background. Data are the average and standard deviation of four independent experiments with significance determined by t-test. **P < 0.05; ***P < 0.01. (C) Deregulation of rDNA transcription in tco89Δ, asf1Δ and hmo1Δ mutants sensitizes cells to TORC1 inhibition. Wild-type or the indicated mutants were transformed with control vector or a vector expressing the rDNA from a galactose-inducible promoter. Cultures were grown in SC-Ura media overnight to select for plasmid maintenance and then equal numbers of cells were 5-fold serially diluted and spotted to YPGalactose in the absence or presence of 1 or 10 nM rapamycin. Plates were incubated at 30°C for 3–6 days before photographing. (D) A speculative model for the role of H3K56ac in nutrient regulated TORC1 signaling. TORC1 signaling may regulate histone H3K56ac on a non-chromatin bound pool of histone H3 and/or at a subset of genomic locations in part, but not necessarily exclusively, through suppression of Hst3 or Hst4 activity. At the rDNA specifically, the H3K56ac pathway is required for creating an optimal chromatin environment that allows binding of both Hmo1 and the SSU processome to promote the high level RNA Pol I transcription and rRNA processing necessary for cell growth and proliferation.

Hongfeng Chen, et al. Nucleic Acids Res. 2012 August;40(14):6534-6546.

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