(A) Nucleosomes are first cross-linked to DNA using formaldehyde. Cross-linked chromatin is digested to mononucleosomes with micrococcal nuclease. Mononucleosomal digests are immunoprecipitated using an antibody specific to a particular histone modification, and immunoprecipitated DNA is isolated and labelled with Cy5. DNA is also isolated from the same nuclease titration step prior to immunoprecipitation, labelled with Cy3, and mixed with Cy5-labeled immunoprecipitated DNA. Labelled DNA is then hybridized to a tiled microarray covering half a megabase of yeast genome.
(B) Example of raw data. Data are shown for all modifications tested, along with PolII data. Red (green) indicates enrichment (depletion), while grey indicates missing data. Data from probes found in linker regions are not shown. Each row represents median data from multiple replicates with one antibody, as indicated (PanAc refers to a nonspecific antibody to acetyl-lysine, which we used to measure bulk acetylation). “Nucleosomes” shows positions of nucleosomes previously described [29], with dark brown for well-positioned nucleosomes, very light brown for linkers, and intermediate brown for delocalized nucleosomes. “ORFs” shows locations of annotated genes. Data shown are for Chromosome III coordinates 58,900 to 72,100.

(A) H4K16Ac aligned by ATG. In this representation, the horizontal axis represents location relative to the downstream gene's start codon, and each horizontal line represents one PolII-driven gene. Each cell in the resulting matrix corresponds to the acetylation level at a given microarray probe for one tail position. Red (green) cells mark hyper-acetylated (hypo-acetylated) probes. Non-nucleosomal probes are blackened. We clustered the promoters using a probabilistic agglomerative clustering algorithm (see Materials and Methods). Arrow indicates annotated ATG.
(B) H4K16 aligned by transcriptional start site, as in (A), except that arrow indicates TSS (identified in [29]) and data before and after the TSS are aligned by the first nucleosome in that direction.
(C) Relationship of histone modification patterns to transcription level. Genes were split into three groups based on PolII enrichment, and averaged data for these groups are shown as indicated, aligned as in (B). Transcription level is indicated by red triangles to the left of each set of three rows.

(A) Schematic of annotation scheme for nucleosomes based on their position relative to transcribed units. Intergenic nucleosomes were assigned to the following categories: promoter region (anything upstream of a coding region), nucleosome immediately upstream to the TSS (“distal”), and the nucleosome immediately downstream of the TSS (“proximal”). Transcribed regions were separated into 5′, middle, and 3′ CDSs. Finally, to capture features of chromatin not associated with PolII genes, we independently classified nucleosomes associated with ARS sequences, tRNA genes, and Null (any other intergenic region).
(B) Hierarchical clustering of 2,288 nucleosomes. Left panel: each row corresponds to a single nucleosome, and each column to a particular modification. Red (green) denotes hyper-acetylation (hypo-acetylation) in the first nine columns and relative level of methylation in the last three columns. Rows are sorted according to the dendogram built during clustering. PolII shows the PolII occupancy of the gene associated with the nucleosome in question. Right panel: each row corresponds to a nucleosome (matching the left panel), and each column corresponds to an annotation of the nucleosome according to the scheme of (A). A blue cell denotes a positive annotation of the nucleosome with the appropriate column label. Numbers indicate examples of clusters, as follows: (1) nucleosomes enriched for H3K9Ac, H3K14Ac, and H3K4Me3 that are mostly upstream of transcribed regions; (2) strongly hypo-acetylated nucleosomes, mostly at upstream regions or 3′ of coding regions; (3) nucleosomes acetylated at H4K8 and K16, and H2B K16 that are almost exclusively at the middle and 3′-ends of coding regions; and (4) hyper-acetylated and methylated nucleosomes that are mostly found at the 5′-end of coding regions.
(C) The Pearson correlations of the 12 modification levels between different probes show that there are two tightly correlated groups of acetylations at specific residues. The first group consists of H2A K7; H3K9, K14, and K18; and H4K5 and K12. The second group consists of H2B K16; and H4K8 and K16. Mono- and di-methylation of H3K4 are correlated with the second group, while tri-methylation of H3K4 is correlated with the first group.
(D) The percent of variance captured by using different number of components. The x-axis denotes the number of components, and the y-axis denotes the percent of the variance in the data explained by each components (blue bars) as well as the cumulative percentage explained (red bars).
(E) Representation of all nucleosomes in two-dimensional modification space. In the left panel, each point represents a nucleosome plotted according to the relative level of the first principal component (x-axis) and second principal component (y-axis) for the modification pattern. The right panel is a three-dimensional plot showing density of points along the plane.

(A) Analysis of differential modification for each class of nucleosomes. Rows correspond to specific modifications, and columns correspond to genomic locations. Each cell is coloured by the average modification level of nucleosomes with this annotation. Non-significant (using false discovery rate of 95% on t-test p-values) cells are blackened.
(B) Promoter nucleosomes (orange) significantly differ from coding region nucleosomes (pink) in their histone modifications pattern. The left panel shows the two types of nucleosomes as points in the plane, where the x-axis represents the level of the first principal component, and the y-axis represents the second principal component. The right panel shows the density within each class.
(C) Distinction between nucleosomes in transcribed regions. Colours denote 5′-end (red), middle (green), and 3′- end (blue) nucleosomes. Visualization is as described in (B).

(A) Modification patterns of nucleosomes associated with actively transcribed genes. Genes with high levels of PolII occupancy were grouped, and the modification data for the indicated nucleosome types were averaged.
(B) Modification patterns of nucleosomes associated with poorly transcribed genes, grouped as in (A), except that genes with low levels of PolII were selected.
(C) Schematic view of yeast chromatin architecture. Cartoon view showing chromatin structure of an arbitrary yeast gene. Yeast genes are typically characterized by an upstream nucleosome-free region, which serves as the transcriptional start site [29]. Surrounding this nucleosome-free region are two nucleosomes that exhibit low levels of acetylation at H2BK16, H4K8, and H4K16, and that carry Htz1 in place of the canonical H2A (unpublished data). The remaining acetylations occur in a gradient from 5′ to 3′ over actively transcribed genes. Similarly, actively transcribed genes exhibit a gradient of H3K4 methylation, with trimethylation occurring at the 5′- ends of genes, and di- and mono-methylation occurring over the middle of the coding region. Nucleosomes are coloured to emphasize the different average modification patterns at each indicated location.

(A) Classification plot of nucleosomes in 5′-coding regions according to PolII occupancy. A classifier was trained to distinguish between nucleosomes with high and low PolII occupancy, and evaluated using leave-one-out cross-validation. Each row corresponds to one nucleosome. Nucleosomes are split into three groups associated with genes corresponding to high, intermediate, and low PolII occupancy level (from top to bottom, respectively). The left 12 columns denote modification patterns of each nucleosome. Modifications with significant differences between high and low nucleosomes are marked with the p-value determined by t-test. Colours denote relative acetylation/methylation levels. The rightmost three columns correspond to the classifier's prediction of transcription, the expression level (mRNA abundance; see Materials and Methods) and the PolII occupancy of genes. The average accuracy of random classification was 60.71%, with a standard deviation of 4.3%. Accuracy of classifier was 75.38% (p < 0.0001).
(B) Classification plot of TSS proximal nucleosomes, labelled as in (A). The average accuracy of random classification was 62.45%, with a standard deviation of 4.75%. Accuracy of classifier was 72.8% (p = 0.0004).
(C) Classification plot of TSS distal nucleosomes; as in (A). The average accuracy of random classification was 65.79%, with a standard deviation of 4.22%. Accuracy of classifier was 58.4% (p = 0.9333).

Analysis of differential modification of nucleosomes associated with various transcriptional regulators. Promoter nucleosomes located near binding sites of the indicated factors were tested for enrichment of all modifications relative to the overall promoter modification pattern. Each cell is coloured by the average modification level of nucleosomes with this annotation. Non-significant cells (using false discovery rate of 95% on t-test p-values) are blackened. Localization data are taken from the indicated studies [39–42].

(A) H3K4Me3 boundaries occur across TSSs. The x-axis represents the level of H3K4Me3 for a given nucleosome, and the y-axis represents the level of this modification for the preceding nucleosome. Pairs of nucleosomes flanking the TSS for a gene on the W strand are plotted as blue squares, and pairs flanking TSSs for genes on the C strand are plotted as red squares. Remaining nucleosome pairs are plotted as grey circles.
(B) Example of a punctate nucleosome. Histone modification plotted as in Figure 1B for a subset of histone modifications. Arrow indicates a nucleosome whose modification pattern differs significantly for H3K4Me3 from nucleosomes to either side. Gene names are as labelled.
(C) Example of a punctate nucleosome, labelled as in (B).