1. Volume 23, Issue 2, Pages 207-217 (July 2006)
Nucleocytosolic Acetyl-Coenzyme A Synthetase Is Required for Histone Acetylation and Global Transcription 
Hidekazu Takahashi, J. Michael McCaffery, Rafael A. Irizarry, Jef D. Boeke 
Molecular Cell 
Volume 23, Issue 2, Pages (July 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Localization of Acetyl-CoA Production Pathways in Yeast
(A) A summary of acetyl-CoA metabolism in yeast. PDH, pyruvate dehydrogenase complex. The three-step PDH bypass pathway, essential on glucose, is indicated by thick arrows. Mitochondrial and peroxisomal enzymes are shown in green and blue, respectively. The enzyme with unknown localization (ACS2) is shown in red. The multiple steps through which glucose and glycerol are converted into pyruvate are omitted and indicated by wavy lines.
(B) Expression and purification of GST-Acs2p from bacteria, BB5153. The purified proteins were separated by 4%–20% SDS-PAGE and stained with Coomassie G-250.
(C) Immunoblotting of total yeast proteins with anti-Acs2p antibody. The total proteins from BY4741, YHT125, and YHT228-3 were separated by 4%–20% SDS-PAGE and immunoblotted with anti-Acs2p antibody.
(D and E) Immunofluorescence of native Acs2p. BY4741 was stained with anti-Acs2p (D) and a DNA staining reagent, TOPRO3 (E).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Immuno-EM Analysis of Acs2p
The “n,” “ne,” and “m” represent nucleus, nuclear envelope, and mitochondrion, respectively. The bar corresponds to 0.1 μm.
(A) Wt yeast (BY4741) cultured in rich media with 2% glucose (YPD).
(B) Wt cultured in rich media with 2% each glycerol and ethanol (YPGE). The inset shows a magnified view.
(C) The acs2Δ mutant (YHT228) precultured in YPGE to A600 of 0.3 and then glucose was added to 2% and the cells were grown an additional 2 hr.
(D) The acs2Δ mutant cultured in YPGE.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Immunoblotting of Crude Extracts of Acetyl-CoA Synthesis Mutants
(A) The indicated single deletion mutants in YPD medium.
(B) The above mutants in combination with acs2-Ts1 in YPD.
(C) The single deletion mutants in YPGE medium.
(D) The mutants in combination with acs2-Ts1 in minimal medium with 2% each glycerol and ethanol without uracil (SCGE − uracil).
The columns are labeled as follows: 1, wt (YHT432); 2, acs1Δ (YHT125); 3, acs2Δ (YHT228); 4, pda1Δ (YHT204); 5, pot1Δ (YHT446); 6, wt (YHT651); 7, acs2-Ts1 (YHT652); 8, acs1Δ acs2-Ts1 (YHT729); 9, acs1Δ acs2-Ts1 pda1Δ (YHT730); 10, acs1Δ acs2-Ts1 pda1Δ pot1Δ (YHT751); 11, rpf2-Ts1 (YHT772); P, positive control extracts (YHT712 for H2B K16Ac and YHT714 for the rest); and N, negative control extracts (YHT713 for H2B K16Ac, YHT715 for H3 K9Ac, YHT716 for H3 K56Ac, YHT717 for H4 K5Ac, and YHT720 for H4 K16Ac). Note that some extra false bands were seen in both positive and negative controls, but not in the other strains because these controls are prepared from other strain backgrounds.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Time Course of Loss of Histone Acetylation after Inactivation of Acs2p
(A) Cell survival rate, or colony forming units per cell plated (y axis) versus time after temperature shift (x axis). Open circle, wt (YHT651); closed circle, acs2-Ts1 (YHT652). Standard deviation is shown as vertical bars.
(B) Stability of Acs2p after temperature shift in wt (YHT651) and acs2-Ts1 (YHT652). Top, the total crude extract proteins blotted on a PVDF membrane were stained. Bottom, anti-Acs2p immunoblotting of the same membrane.
(C) Time course of the histone acetylation level in the acs2-Ts1 mutant (YHT652) after the temperature shift. Ratios of the anti-acetyl H3/H4 N-terminal antibody signals relative to the control antibody signals were plotted as relative values (%) against time (x axis).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Nucleocytosolic and Mitochondrial Acetyl-CoA Pools Are Distinct
(A) Schematic representation of the ACS constructs transformed into the acs2-Ts1 strain. ACS2pr, ACS2 promoter; SeACS, Salmonella enterica ACS; GFP, green fluorescent protein; NLS, nuclear localization signal; NES, nuclear export signal; MTS, mitochondria targeting signal 1, YHT818; 2, YHT814; 3, YHT830; 4, YHT817; 5, YHT816; and 6, YHT819.
(B) Immunoblotting of crude extracts from acs2-Ts1 strains harboring constructs shown in (A).
(C) Subcellular localization of Acs-GFP proteins in acs2-Ts1 mutants.
(D and E) Growth of acs2-Ts1 mutants (D) or wt cells (E) transformed with constructs shown in (A) on SC-leucine-uracil with 2% glucose at 25°C or 37°C.
(F and G) Immunoblotting of crude extracts from acs2-Ts1 strains harboring constructs shown in (A), shifted to 37°C for 1 hr (F) or cultured at 25°C (G).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 ACS2 and Global Gene Expression
(A) The MA (MvA) plot of the expression levels of 5716 yeast ORFs. x and y axes show log2 mean signal intensities and log2 fold signal change (Fc), respectively, between wt and the acs2-Ts1 mutant. Genes upregulated in the acs2-Ts1 mutant have positive Fc values. Red lines show 0.1 and 99.9 percentile Fc values in the null set for significance assessment.
(B) Plot of distance (kilobases) from telomeres (x axis) and Fc (y axis). A lowess (local weighted regression) curve fit to the data is shown in red. Dotted lines exhibit point-wise 99% confidence intervals.
(C) ACS2 governs chromatin function through histone acetylation. “Ac” denotes “acetyl-” and “CoA” denotes “coenzyme A.” Acs2p produces acetyl-CoA, the acetyl donor used by HATs. Acetate can be produced from carbon metabolism and/or from histone deacetylation reaction by HDACs. Note that acetyl-CoA produced by Acs2p is perhaps utilized for other processes such as N-terminal protein acetylation and fatty acid synthesis.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions