1. Nucleosomes Unfold Completely at a Transcriptionally Active Promoter
Hinrich Boeger, Joachim Griesenbeck, J.Seth Strattan, Roger D. Kornberg 
Molecular Cell 
Volume 11, Issue 6, Pages (June 2003)
DOI: /S (03)

2. Figure 1
DNase I Digestion Pattern of the PHO5 Promoter in Isolated Nuclei
Digestion was performed with nuclei from PHO80 (YS18, yM2.1) and pho80Δ (yM7.8, yM8.14) cells, with (yM2.1, yM8.14) and without RS elements (YS18, yM7.8) inserted at the PHO5 locus, with 0.5, 1.5, 5, or 10 U/ml DNase I for 30 min. DNA was extracted, digested with ApaI, electrophoresed in a 1.5% agarose gel, blotted, and hybridized with the probe indicated. Markers were provided by gel lanes containing genomic DNA digested with ApaI and BamHI (B) or with ApaI and ClaI (C). Positions of promoter nucleosomes and of the upstream RS element are indicated by ovals and by a box containing an arrowhead, respectively.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Restriction Endonuclease Accessibility of the PHO5 Promoter in Isolated Nuclei and in Purified Chromatin Circles
(A) Nuclei isolated from PHO80 (yM2.1) and pho80Δ (yM8.14) strains, designated R and A, respectively, were digested with 50 or 200 U of the restriction endonuclease indicated for 1 hr. DNA was extracted, digested with HaeIII, electrophoresed in a 1.5% agarose gel, blotted, and hybridized with the probe indicated. An arrowhead at the left indicates the position of DNA uncut by the restriction endonuclease indicated. Positions of promoter nucleosomes and of the upstream RS element are diagrammed as in Figure 1, with a solid black circle for UASp1, a gray circle for UASp2, a white circle for the TATA box, and an arrow to indicate the transcription start site.
(B) Chromatin circles (50 attomol DNA) purified from PHO80 (yM2.1) and pho80Δ (yM8.14) strains were digested with 5 or 20 U of the restriction endonuclease indicated in 100 μl for 30 min at 37°C. The DNA was extracted, digested with EcoRV, electrophoresed in a 2% agarose gel, blotted, and hybridized with an open reading frame probe. An arrowhead at the left indicates the position of uncut DNA as in (A).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Topological Analysis of PHO5 Chromatin Circles
Topoisomers were resolved in agarose gels containing 15 μg chloroquine/ml. Under these conditions, all topoisomers were positively supercoiled, so those with larger linking numbers exhibited higher electrophoretic mobilities. Blot hybridization was performed with a probe spanning the entire PHO5 gene.
(A) Topoisomer distributions of PHO5 chromatin circles from PHO80 (yM2.1) and pho80Δ (yM8.14) strains, designated R and A, respectively. As a control, naked circles, designated D, were relaxed by treatment with topoisomerase. An arrowhead indicates the position of the mean of the topoisomer distribution, computed from the profile of radioactivity in the blot (as shown in [C]). An asterisk indicates the band due to nicked circular DNA.
(B) Topoisomer distributions of PHO5 chromatin circles from PHO80 (yM18.17) and pho80Δ (yM19.2) strains lacking the PHO5 TATA box, designated R and A, respectively.
(C) Chromatin circles (0.15 ng of DNA) purified from PHO80 (yM18.17) and pho80Δ (yM19.2) strains, designated R and A, respectively, were incubated with 1.5 μg of competitor DNA in 25 μl for 30 min at 37°C and subsequently relaxed with topoisomerase. Controls (data not shown) were performed to verify that topoisomerase was active on isolated chromatin circles. Radioactivity profiles of topoisomer distributions of in vitro relaxed circles are shown on the right (in vitro). For comparison, profiles of distributions shown in (B) are presented on the left (in vivo). An arrowhead indicates the distribution center. Δ<Lk> is the linking difference between activated and repressed circles.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Fragmentation of PHO5 Chromatin by Micrococcal Nuclease and Sedimentation Analysis
(A) Nuclei isolated from PHO80 (yM2.1) or pho80Δ (yM8.14) cells were subjected to partial digestion with micrococcal nuclease, and chromatin fragments released from the nuclei were sedimented in a 5%–30% maltose gradient. DNA extracted from gradient fractions (numbered on the abscissa) was analyzed by gel electrophoresis and blot hybridization with probes spanning the LYS2 gene and the N-1, N-2, and N-3 regions of the PHO5 promoter. Arrows indicate the direction of sedimentation. M, Di, and T indicate the bands in the gel due to nucleosome monomer, dimer, and trimer DNA.
(B) Peak positions of nucleosome monomers, dimers, trimers, and a 146 bp naked DNA molecule (D) are plotted against their known sedimentation coefficients (S20,w). Numbers of gradient fractions are indicated on the abscissa. An arrow indicates the position in the gradient expected for a histone tetramer particle, based upon its reported sedimentation coefficient.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Fragmentation of PHO5 Chromatin by Restriction Endonucleases and Sedimentation Analysis
The procedure was as in Figure 4. The DNA concentration relative to that at the peak of the distribution, determined from the radioactivity in the blot, is plotted for each gradient fraction on the ordinate. The numbers of the fractions are indicated on the abscissa. The distributions derived from PHO80 and pho80Δ nuclei are blue and green, respectively. Reference distributions are brown. The reference distribution shown in (B), (D), and (F), labeled C (for core particle), was obtained by reconstitution of a core particle on a 140 bp DNA fragment in vitro. The two peaks in these distributions identify the positions of core particles and naked DNA (asterisk) in the gradients.
(A) Distribution of the N-2 region released as a 220 bp fragment from PHO80 (N-2R) and pho80Δ (N-2A) nuclei by BstEII/HaeII digestion. (A′) The distribution of the N-2 region released by MNase digestion from PHO80 nuclei (CN-2) and the distribution of the naked 220 bp N-2 DNA fragment (D-2220) are shown for reference.
(B) Distribution of the N-3 region released as a 140 bp fragment from pho80Δ nuclei (N-3A) by BamHI/HaeII digestion. The distribution of the N-3 region released from PHO80 nuclei (CN-3) by MNase digestion is shown for reference.
(C) Distribution of the N-1 region released as a 216 bp fragment from PHO80 (N-1R) and pho80Δ (N-1A) nuclei by BstEII/HaeIII digestion. (C′) The distribution of the N-1 region released by MNase digestion from PHO80 nuclei (CN-1) and the distribution of the naked 216 bp N-1 DNA fragment (D-1216) are shown for reference.
(D) Distribution of the N-1 region released as a 146 bp fragment from pho80Δ nuclei (N-1A) by ClaI/HaeIII digestion.
(E) Distribution of the N-1 region released as a 316 bp fragment from pho80Δ nuclei (N-1A) by ClaI/HaeIII digestion. The distribution of the N-1 region released by MNase nuclei (CN-1) and the distribution of the naked 316 bp N-1 DNA fragment (D-1316) are shown for reference.
(F) Distribution N-1A results from the superposition of distributions N-1A (146 bp, [D]) and N-1A (316 bp, [E]).
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Shift in Position of PHO5 Promoter Nucleosome N-1 upon Transcriptional Activation
(A) PHO5 chromatin circles (150 attomoles of DNA) purified from PHO80 and pho80Δ cells were digested with 0.6, 1.2, and 1.8 U of micrococcal nuclease in 200 μl for 5 min at 37°C. DNA was extracted, digested with PflMI, electrophoresed in an agarose gel, blotted, and hybridized with a probe from part of the PHO5 open reading frame. The inferred positions of nucleosomes and the location of the upstream RS element are diagrammed as in Figure 1, and UASp1, UASp2, and the TATA box are represented as in Figure 2. The shift in position of N-1 upon transcriptional activation is indicated by the partial overlap of open and diagonally lined ovals on the right, corresponding to the repressed and activated states, respectively. Cloned PHO5 DNA was digested with combinations of PflMI and the restriction enzymes indicated on the right to provide markers. The arrow indicates the band due to cleavage at the ClaI site in N-2 but not that in N-1.
(B) Upper panel: Nuclei isolated from PHO80 (yM18.17) and pho80Δ (yM19.2) strains, designated R and A, respectively, were digested with 50 and 200 U of ClaI. DNA was extracted, digested with BamHI/PstI, electrophoresed in an agarose gel, blotted, and hybridized with a probe spanning the open reading frame. The dart indicates the band due to cleavage at the site in N-1. Lower panel: Nuclei isolated from PHO80 (yM3.2) and pho80Δ (yM9.7) strains, designated R and A, respectively, were digested with 50 and 200 U of SalI. DNA was processed as for the upper panel, except that the secondary digestion was with XbaI and the hybridization probe was from the N-3 region. An arrowhead at the left indicates the position of DNA uncut by the restriction endonuclease indicated. The shift of position of N-1 upon transcriptional activation is diagrammed as in (A).
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 Kinetics of Micrococcal Nuclease Digestion
(A) Nuclei isolated from PHO80 (yM2.1) or pho80Δ (yM8.14) cells, designated R and A, respectively, were digested in 200 μl with 1, 5, 10, or 20 U/ml micrococcal nuclease for 20 min. DNA was extracted, electrophoresed in an agarose gel, blotted, and hybridized with a probe spanning the open reading frame (ORF) or the domain of nucleosomes N-1 to N-3 (Pro).
(B) PHO5 circles (150 attomol of DNA) purified from PHO80 (yM2.1) or pho80Δ (yM8.14) cells, designated R and A, respectively, were digested in 200 μl with 0.5, 1.5, and 4.5 U of micrococcal nuclease for 5 min at 37°C. DNA was extracted, electrophoresed in an agarose gel, blotted, and hybridized with probes for the N-1, N-2, and N-3 regions.
(C) The total hybridization signal S was measured by integrating the profile of radioactivity in each lane of (A) and (B). The ratio of values S for activated, A, and repressed, R, states is plotted against the nuclease concentration [E] (in arbitrary units).
Molecular Cell  , DOI: ( /S (03) )

9. Figure 8
Association of Histones with the PHO5 Promoter, Assessed by ChIP Analysis with His-Tagged Histone H2B
(A) Chromatin circles from repressed (yM40.1) or activated (yM25.1) cells expressing His-tagged H2B, designated R and A, respectively, were crosslinked and processed as for ChIP analysis except with the use of a TALON affinity matrix for binding the tagged histone H2B. The fraction of input DNA retained on the affinity matrix was measured by real-time quantitative PCR and is graphed for PHO5 promoter circles and for an unlinked LEU2 control circle. The heights of the bars correspond to the mean values calculated from at least nine ChIP analyses for each strain. Error bars indicate the sample standard deviation. Untagged strains expressed only wild-type histone H2B.
(B) Accessibility of the ClaI site in repressed, R, and activated, A, PHO5 promoter chromatin from H2B-tagged strains was compared as in Figure 2. The arrowhead marks the uncut promoter.
Molecular Cell  , DOI: ( /S (03) )