of state and gene structure, while panel 5 shows enrichment/ depletion for each chromosome arm. B. Karyotype view of the assembled heterochromatic domains defined by the five combi- natorial chromatin states in A. State A: grey; state B: green; state C: purple; state D: blue; state E: orange. The enlarged view of chromosome 4 shows the large fraction of sequences associated with transcriptionally active TSS and elongation (states B, C, D). (PDF) Figure S2 Domains on chromosome 4 supporting strong reporter expression are under control of the Polycomb/trx system. A. Red panels: Enrichment profiles for HP1a (bottom), PC (Polycomb; middle), and their overlay (top) for S2 cells. Blue panels: Enrichment profiles for HP1a, PC, and their overlay for BG3 cells. Genes are shown below in black. Red triangles mark the four domains that support full hsp70-white expression [region 1 near ci (2M-1020; 79,754), region 2 at position 436,655 (7M-201), region 3 near zfh2 (e.g. 2M-371; 522,600), and region 4 within sv (4M-1030; 1,119,408) [10,24]]. X-axis: Position along chromo- some 4 in bp (centromere to the left). Y-axis: Smoothed M-values. B. hsp70-white reporter lines with variegating eye phenotype are excluded from regions associated with PC, and DNase I hypersensitive sites (DHS) are associated with genes in the PC domains. For hsp70-w reporters, red bars denote insertions with red eye phenotype (full expression), while black bars denotes insertions with variegating eyes. (PDF) Figure S3 Distribution of chromosomal proteins and histone marks unique to chromosome 4 in S2 cells. Metagene analysis for the enrichment (averaged smoothed M-values, Y-axis) for selected marks is plotted against position relative to the TSS for a 3 kb scaled metagene (bp, X-axis). The enrichment is examined separately for active (left) and repressed (right) genes in three genomic domains, chromosome 4 (top panel), pericentric hetero- chromatin (middle panel), and euchromatin (bottom panel), with the number of genes for each category illustrated at the right corner. (PDF) Figure S4 Chromosome 4 genes exhibit unique chromatin marks compared to genes in heterochromatin and euchromatin in BG3 cells. Same analysis as shown in Figure 3, now with the same number of genes (N) as present on chromosome 4 randomly chosen from heterochromatin (Hetero) and euchromatin (Eu) as controls. (PDF) Figure S5 Chromosome 4 genes exhibit unique chromatin marks compared to genes in heterochromatin and euchromatin in S2 cells. Same analysis as shown in Figure S3, now with the same number of genes (N) as present on chromosome 4 randomly chosen from heterochromatin (Hetero) and euchromatin (Eu) as controls. (PDF) Figure S6 Heatmap showing the enrichment of select chromo- somal proteins and histone marks at genes on chromosome 4, compared to heterochromatin and euchromatin. A. BG3 cells. B. S2 cells. The region around the TSS and TTS (+/2500 bp) is not scaled, while the gene body is scaled. Therefore, only genes longer than 1 kb are considered here. Enrichment is shown in red, depletion in blue. Eu - euchromatin. Hetero - heterochromatin. (PDF) Figure S7 Histogram of the expected incidence of RNA pol II pausing on chromosome 4. Permutation analysis shows that the Drosophila Chromosome 4 Chromatin Structure low pausing incidence on chromosome 4 is significantly different from that expected based on the pausing occurrence in euchromatin (A. p,3e-5) and pericentric heterochromatin (B. p,0.00024). (PDF) Figure S8 Overlap between genes identified genome-wide as pausing by the GRO-seq analysis and the RNA pol II ChIP-chip analysis. The PI was calculated according to [26] (GRO-seq data; blue) or [31] (RNA pol II ChIP-chip; green). (PDF) Figure S9 Gene features in chromosome 4, pericentric hetero- chromatin, and euchromatin. A. Genes on chromosome 4 are slightly larger than genes in euchromatin and heterochromatin (with a median of 8,001 bp vs. 1,907 bp vs. 1,844 bp). B. Chromosome 4 genes tend to have more exons than genes in euchromatin and pericentric heterochromatin (with a median of 6 vs. 3 vs. 2). C. Expression levels in different genomic domains are similar. Expression magnitude [log10(RPKM+1), Y-axis] is compared between euchromatin, pericentric heterochromatin, and chromosome 4. The expression levels are slightly higher on chromosome 4 compared to euchromatin. D. Longer genes exhibit higher PI, which indicates RNA polymerase is biased toward TSS. The PI was calculated from GRO-seq data [26]. E. Genes with more exons tend to show higher PI. The PI was calculated as in [31] using ChIP-chip data. (PDF) Figure S10 Validation of ChIP-chip HP1a and POF enrichment peaks. A. Fraction of HP1a peaks reduced in HP1a mutants (third instar larvae). 96% of peaks are significantly reduced. X-axis: M- value of HP1a peaks in WT; Y-axis: fraction of peaks reduced in the mutants. B. Fraction of POF peaks reduced in POF mutants (third instar larvae). 79% of peaks are significantly reduced. X- axis: M-value of POF peaks in WT; Y-axis: fraction of peaks reduced in the mutants. (PDF) Figure S11 Effect of HP1a depletion on RNA pol II pausing index. A. Ratio of the PI in mutants lacking HP1a compared to wildtype. The PI is defined as the ratio between the maximum enrichment value around TSS (+/2300 bp) and the medium enrichment values over the gene body (600 bp downstream of TSS to the end of the gene) [31]. 53 of 74 genes show an increase in PI, indicated by a ratio larger than 1. B. Histogram of RNA pol II level fold changes in HP1a mutants (log 2; average per gene) for genes on chromosomes 2, 3, and X (bottom panel) and genes on chromosome 4 (top panel), illustrating a decrease of RNA pol II levels for chromosome 4 genes. C. Relationship between RNA pol II level changes (Y-axis, in log 2) and expression level changes (X- axis, in log 10) of chromosome 4 genes in HP1a mutants compared to wildtype. Data points with x,0 and y,0 correspond to genes where both RNA pol II and expression levels decrease upon HP1a depletion (67 of 84 genes on chromosome 4; r = 0.4, Pearson correlation coefficient). (PDF) Figure S12 HP1a-regulated euchromatic genes respond differ- ently to HP1a depletion than chromosome 4 genes. A. RNA pol II distribution in wildtype (WT) and HP1a mutants is compared for chromosome 4 genes and HP1a-regulated genes identified by Piacentini and colleagues [35]. The promoter region (left) and gene body (right) are examined separately. B. Bar graphs illustrating the same data as in A. C. Expression changes observed in HP1a mutants for HP1a-regulated genes identified by Piacentini et al. compared to chromosome 4 genes. D. Expression PLOS Genetics | www.plosgenetics.org 17 September 2012 | Volume 8 | Issue 9 | e1002954