1. Chromatin Disassembly Mediated by the Histone Chaperone Asf1 Is Essential for Transcriptional Activation of the Yeast PHO5 and PHO8 Genes 
Melissa W Adkins, Susan R Howar, Jessica K Tyler 
Molecular Cell 
Volume 14, Issue 5, Pages (June 2004)
DOI: /j.molcel

2. Figure 1 Asf1p Is Required for Nucleosome Disassembly at PHO5
(A) Schematic of the primer sets used for ChIP analysis of the PHO5 promoter is shown with respect to the nucleosomal organization of the repressed promoter. Light gray circles indicate nucleosomes that are removed during activation of PHO5, and dark gray circles indicate nucleosomes that are not removed. The location of the probe and ClaI site used for restriction enzyme accessibility analysis is shown, and the DNA fragments resulting from an accessible and inaccessible ClaI site following HaeIII digestion to completion are shown.
(B) Restriction enzyme accessibility analysis at the PHO5 promoter. PHO5 was induced in JKT0010 (WT) and MPY0042 (asf1Δ) strains by phosphate depletion. Equal amounts of nuclei from each strain before (+Pi) and after (−Pi) phosphate depletion were subject to digestion with two amounts of ClaI at the location indicated in (A), followed by isolation of the DNA and complete digestion at flanking HaeIII sites. The protected and accessible fragments were detected by Southern analysis with the probe shown in (A).
(C) ChIP analysis of histone H3 levels over the PHO5 promoter. PHO5 was induced in JKT0010 (WT) and MPY0042 (asf1Δ) strains by phosphate depletion, and histone H3 levels were followed at the indicated times following phosphate depletion. The amount of immunoprecipitated DNA was determined by quantitative PCR with primer pairs directed against the PHO5 TATA. As controls, primer sets were also used for PGK1 and COXIII.
(D) Quantitation of histone H3 ChIP analyses of the TATA region of PHO5. Relative histone occupancy was determined by the ratio of immunoprecipitated (IP) TATA product relative to the IP PGK1 product divided by the ratio of input (IN) TATA product relative to the IN PGK1 product. Values shown are averages of three independent experiments; error bars indicate the 95% confidence interval.
(E) ChIP analysis of histone H3 as described in (C), with primers directed against the PHO5 UASp2.
(F) Quantitation of histone H3 ChIP as described in (D) for the UASp2 of PHO5.
(G) ChIP analysis of histone H3 as described in (C), with primers directed against a region adjacent to PHO5.
(H) Quantitation of histone H3 ChIP as described in (D) for the region adjacent to PHO5.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Asf1p Is Required for Induction of PHO5, but Not for Pho4p Recruitment
(A) Time course of PHO5 induction. PHO5 induction was monitored by measuring acid phosphatase activity at the indicated times after transfer of cultures of JKT0010 (WT), MPY0042 (asf1Δ), RDY0012 (cac2Δ), and MAY0038 (gcn5Δ) from a phosphate-containing medium to a phosphate-depleted medium.
(B) Time course of PHO5 transcript levels. RNA was isolated from strains JKT0010 (WT) and MPY0042 (asf1Δ), before and at the indicated times after phosphate depletion. PHO5 transcript levels were determined by quantitative RT-PCR analysis.
(C) Quantitation of PHO5 transcript levels. The average with 95% confidence interval for PHO5 transcript levels, normalized to levels of the RPL3 control from four independent RT-PCR analyses, are shown, with the value before phosphate depletion normalized to 1 for each strain.
(D) Nuclear localization of Pho4p upon PHO5 induction. Representative localization of GFP-tagged Pho4p in strains MAY0054 (WT) and MAY0056 (asf1Δ) grown in phosphate-containing media (+Pi, represses PHO5) and 45 min after shifting the cells to phosphate-depleted media (−Pi, induces PHO5). Cells were counterstained with DAPI.
(E) Schematic showing location of the UASp1 and UASp2 binding sites for Pho4p within the PHO5 promoter.
(F) Binding of Pho4p to UASp1. In vivo DMS footprinting analysis was performed in JKT0010 (WT) and MPY0042 (asf1Δ) strains growing in phosphate-containing media (+Pi) and phosphate-depleted media (−Pi). “M” designates a product that is 6 bp shorter than the position of UASp1 for reference. The sequence spanning UASp1 is shown. The red dot designates the characteristic protection of a G residue in UASp1 from DMS modification that occurs due to Pho4p binding.
(G) Binding of Pho4p to UASp2, as described in (F). “M” designates a product that is 3 bp longer than the position of UASp2 for reference. The sequence spanning UASp2 is shown. The red dot designates the characteristic protection by Pho4p binding in UASp2, while the arrow designates the characteristic enhancement of DMS accessibility when Pho4p binds UASp2.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Indirect End-Labeling Analysis of Chromatin Structure at the PHO5 Promoter
PHO5 was induced in JKT0010 (WT) and MPY0042 (asf1Δ) strains by phosphate depletion. Equal amounts of nuclei from each strain before (+Pi) and after (−Pi) phosphate depletion were subject to digestion with MNase, followed by isolation of the DNA and complete digestion with ApaI. DNA was electrophoresed and Southern blotted with a 32P-labeled probe. For size standards, untreated DNA was digested with ApaI and either BamHI, BstEII, or ClaI and resolved on the same gel as the experimental samples. A 1 hr exposure is shown for the size standards while a 6 hr exposure is shown for the experimental samples. The schematic on the right indicates the positions of the nucleosomes, where dark gray nucleosomes do not get remodeled and light gray nucleosomes (numbered as in Figure 1A) do get remodeled during transcription of PHO5 in WT cells.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Asf1p Mediates Nucleosome Disassembly at PHO5
(A) Time course of PHO5 induction. PHO5 induction was monitored by measuring acid phosphatase activity at the indicated times after transfer of cultures of SH0015 (WT), SH0016 (asf1Δ), and SH0014 (pGALASF1) from a phosphate-containing medium to a phosphate-depleted medium. Two percent galactose was added to all three cultures at 4 hr.
(B) Quantitation of ChIP analysis of histone H3 levels over the PHO5 UASp2. PHO5 was induced in SH0015 (WT), SH0016 (asf1Δ), and SH0014 (pGALASF1) strains by phosphate depletion, and histone H3 levels were followed at the indicated times after phosphate depletion. Two percent galactose was added to all three cultures at 4 hr. The amount of immunoprecipitated DNA was determined as described in Figure 1E. Values shown are averages with the standard deviation of three independent experiments.
(C) Quantitation of ChIP analysis of histone H3 levels over the PHO5 TATA box region. ChIP analysis of histone H3 was performed and quantitated as described above, with primers directed against the PHO5 TATA box.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Asf1p Is Required for Nucleosome Disassembly at PHO8
(A) Schematic of the primer sets used for ChIP analysis of the PHO8 promoter is shown with respect to the nucleosomal organization of the repressed promoter. The light gray circle indicates a nucleosome (−4) that is remodeled during activation of the PHO8 promoter, the two mid gray circles represent unstable nucleosomes (−3, −2), and the dark gray circles represent stable nucleosomes (−1, −5).
(B) PHO8 transcription is not induced in asf1 mutants. Quantitative RT-PCR analysis of PHO8 transcript levels in strains JKT0010 (WT) and MPY0042 (asf1Δ) following phosphate depletion. RPL3 is a control transcript used for normalization.
(C) Quantitation of PHO8 transcript levels. The average with 95% confidence interval for PHO8 transcript levels, normalized to levels of the RPL3 control from four independent RT-PCR analyses are shown, with the value before phosphate depletion normalized to 1 for each strain.
(D) ChIP analysis of H3 levels at nucleosome −4 of PHO8. PHO8 was induced in JKT0010 (WT) and MPY0042 (asf1Δ) strains by phosphate depletion, and histone H3 levels were measured at the indicated times following phosphate depletion. The amount of immunoprecipitated DNA was determined by quantitative PCR with primer pairs directed against PHO8 Nucl 4. As controls, primer sets were also used for PGK1.
(E) Quantitation of histone H3 ChIP analyses of the Nucl −4 region of PHO8. Relative histone occupancy was determined by the ratio of immunoprecipitated (IP) Nucl −4 product relative to the IP PGK1 product divided by the ratio of input (IN) Nucl −4 product relative to the IN PGK1 product. Values shown are averages of three independent experiments; error bars indicate the 95% confidence interval.
(F) ChIP analysis of H3 levels at nucleosome −1 of PHO8, as described in (D).
(G) Quantitation of histone H3 ChIP analyses of the Nucl −1 region of PHO8, as described in (E).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 Nucleosome Reassembly during PHO5 Repression
(A) Time course of PHO5 repression. PHO5 repression was monitored by measuring acid phosphatase activity at the indicated times after transfer of cultures of JKT0010 (WT), RDY0012 (cac2Δ), and SH0014 (pGALASF1) from a phosphate-depleted medium to a phosphate-containing medium.
(B) Quantitation of ChIP analysis of histone H3 levels over the PHO5 promoter. Histone H3 levels were measured by ChIP analysis in strain JKT0010 (WT) growing in phosphate-containing media (+Pi), in phosphate-depleted media (−Pi), and then at the indicated times after shifting to phosphate-containing media again. The amount of immunoprecipitated DNA was determined by quantitative PCR with primer pairs (as described in Figure 2A) directed against the PHO5 TATA.
(C) Quantitation of histone H3 ChIP as described in (B) for the UASp2 of PHO5.
(D) Quantitation of histone H3 ChIP as described in (B) for the adjacent region to PHO5.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Model for Asf1-Mediated Nucleosome Disassembly during Transcriptional Activation and Nucleosome Reassembly during Transcriptional Repression of PHO5
At the top is shown a schematic depicting the repressed PHO5 promoter, where yellow and blue circles represent positioned nucleosomes. The UASp1 and UASp2 binding sites for Pho4p are shown by red boxes. Upon phosphate depletion (inducing conditions), the Pho4p activator relocalizes to the nucleus where it then binds to both the UASp1 and UASp2 binding sites. Next, Asf1p removes the histones (yellow circles) from the PHO5 promoter, allowing transcriptional activation to occur. Upon phosphate addition (repressing conditions), the Pho4p activator is removed, and the nucleosomes are reassembled by an unknown histone chaperone onto the PHO5 promoter to regenerate the repressed promoter.
Molecular Cell  , DOI: ( /j.molcel )