changes observed in HP1a mutants comparing chromosome 3R genes to chromosome 4 genes. (PDF) Figure S13 Effect of POF depletion on RNA pol II. A. Ratio of the pausing index in pof mutants and 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]. 63 of 74 genes on chromosome 4 show an increased PI. B. Histogram of RNA pol II level fold changes in pof mutants (log 2; average per gene) for genes on chromosomes 2, 3, and X (upper) and genes on chromosome 4 (lower), illustrating a decrease of RNA pol II levels for chromosome 4 genes. (PDF) Figure S14 Relationship between protein enrichment on chromosome 4 genes and their PI change observed in mutant lacking HP1a. A. Heatmap illustrating protein enrichment (red – enrichment, blue – depletion) for genes are ordered by their PI changes in HP1a mutants (in grey below enrichment panel). Data source: Third instar larvae. B. Histogram illustrating the correlation between the enrichment of select proteins at chromo- some 4 genes with the PI change in HP1a mutants. (PDF) Figure S15 Residual HP1a in pof and egg mutants is associated with repeated sequences. A. Histogram showing the distance between residual HP1a and repeats in pof mutants. The observed distance is significantly smaller than the expected distance based on permutation analysis (38 bp vs. 132 bp, p,0.001). B. Histogram illustrating the distance between residual HP1a and repeats in egg mutants. The observed distance is significantly smaller than the expected distance based on permutation analysis (19 bp vs. 135 bp, p,0.001). (PDF) Figure S16 Heterochromatic marks are maintained at wildtype levels in the ,70 kb of assembled sequence adjacent to the centromere in larvae lacking POF (pof D119). ChIP results from wildtype and mutant larvae are compared. The first panel shows RNA-seq data from wildtype larvae. X-axis: position along chromosome 4 in bp; Y-axis: ChIP enrichment for HP1a (top), H3K9me2 (middle), and H3K9me3 (bottom). (PDF) Table S1 Description of ChIP datasets. Overlap of top 40% peaks – for the replicate experiments, overlap between the top 40% of peaks is calculated, with peaks calls based on p-value enrichment scores. Overlap based on size-adjusted threshold – for replicate experiments, overlap between the peaks is determined with peaks calls based on p-value enrichment scores and adjusted for the number of peaks called in each experiment. Correlation coefficient r – correlation coefficient is calculated from the log10 p- value enrichment scores of the two replicate experiments. When more than two replicate datasets were available, the average correlation coefficient is reported. (XLS) Table S2 Description of ChIP datasets from mutants. Overlap of top 40% peaks – for the replicate experiments, overlap between the top 40% of peaks is calculated, with peaks calls based on p- value enrichment scores. Overlap based on size-adjusted threshold – for replicate experiments, overlap between the peaks is determined with peaks calls based on p-value enrichment scores and adjusted for the number of peaks called in each experiment. Correlation coefficient r – correlation coefficient is calculated from Drosophila Chromosome 4 Chromatin Structure the log10 p-value enrichment scores of the two replicate experiments. When more than two replicate datasets were available, the average correlation coefficient is reported. (XLSX) Table S3 Polymerase pausing is rare on chromosome 4. Genes with paused RNA pol II have been identified by two methods. A. Polymerase pausing determined by GRO-seq analysis in S2 cells (Method 1). B. Genes with paused polymerase are identified solely based on ChIP-chip mapping of RNA pol II ([31]; Method 2). The percentage of paused genes is reported relative to the number of genes associated with RNA pol II. Note: In the table below, in A, the analysis is carried out on a per transcript basis. Thus, the total number of ‘‘genes’’ is inflated as some genes have multiple different transcripts and TSSs. (DOCX) Table S4 Developmental control genes are not depleted among chromosome 4 genes. GO term analysis demonstrates that the following terms are significantly enriched among chromosome 4 genes (p,0.05). No terms are significantly depleted. (DOCX) Table S5 The frequency of GAGA factor motifs, but not the pause button sequence, is underrepresented in chromosome 4 promoters. PB - Pause button. GAGA factor (TRL) binding motif – TRL motif. Inverted GAGA factor binding motif – iTRL motif. (DOCX) Table S6 The average Tm of 9-mer sequences downstream of TSSs on chromosome 4 is significantly lower than on other chromosomes. The minimum (min.), median, mean, and maxi- mum (max.) melting temperature (Tm, uC) for 9-mer sequences in the first 100 bp downstream of all unique TSSs in the D. melanogaster genome are compared by chromosome arm. (DOCX) Table S7 HP1a and H3K9me2/3 enriched regions on chromo- some 4 in wildtype, pof D119, Su(var)205, and egg10.1a mutant third instar larvae. Significantly enriched regions based on smoothed M-value profiles (FDR = 1e-3) are compared between wildtype and mutant. Note that the magnitude of the enriched peaks is not considered. POF+ region: POF enriched region in wildtype; POF- region: No POF enrichment in wildtype. HP1a+ region: HP1a enriched region in wildtype; HP1a- region: No HP1a enrichment in wildtype. (DOCX) Acknowledgments We thank A. Gorchakov and E. Bishop for helpful discussions and insightful comments on the manuscript. We would like to acknowledge our technicians, D. Acevedo, S. Gadel, C. Kennedy, S. Marchetti, and D. McCabe. We would like to particularly thank W. Leung for assistance with the Tm analysis. Arrays were processed at the Rutgers BRTC. The RNA- seq data from wildtype tissues/cells were produced by the Drosophila transcriptome modENCODE group (lead-PI S. Celniker; UC – Berkeley). We acknowledge the Drosophila Genomics Resource Center for cell lines and other materials received. We also thank our many colleagues in the Drosophila community who have generously donated antibodies to our project. We thank the Genome Technology Access Center (GTAC) in the Department of Genetics at Washington University School of Medicine for high-throughput sequencing and help with genomic analysis. Author Contributions Conceived and designed the experiments: NCR TG MIK VP GHK PJP SCRE. Performed the experiments: NCR TG AAA DA HG AM AP YBS. Analyzed the data: NCR TG YLJ. Contributed reagents/materials/ analysis tools: PVK MYT. Wrote the paper: NCR YLJ TG PJP SCRE. PLOS Genetics | www.plosgenetics.org 18 September 2012 | Volume 8 | Issue 9 | e1002954