Drosophila Chromosome 4 Chromatin Structure Figure 1. The chromatin composition of D. melanogaster chromosome 4 shows distinct patterns of enrichment. A. Enrichment levels for all histone marks/chromosomal proteins (green asterisks indicate newly reported marks) are shown for the five main combinatorial chromatin states within heterochromatin as defined in Riddle et al [15]. Panel 1- histone marks; panel 2- chromosomal proteins; panel 3- repeat enrichment and expression status; panel 4- the fraction of chromatin within each state associated with various structural features of genes; panel 5- enrichment/ depletion of states for each chromosome arm, with the numbers to the right reflecting the percentage of the heterochromatin assigned to each state. Chromatin source: BG3 cells. B. Karyotype view of the assembled heterochromatic domains defined by the five combinatorial chromatin states in A. The distal 1.2 Mb region of chromosome 4 exhibits a higher density of transcriptionally active genes (states B, C, D) and polycomb-dominated domains (state E). Chromatin source: BG3 cells. C. hsp70-white transgenes leading to a red eye phenotype are preferentially found in state E. Triangles mapped onto the expanded chromosome 4 indicate the insertion sites for hsp70-white transgenes exhibiting a red (red triangle) or variegating (dotted triangle) eye phenotype [9]. D. Browser shot illustrating the relationship between histone marks and chromosomal proteins on chromosome 4. The silent gene CG1909 shows correlations typical of pericentric heterochromatin (dashed blue box; enriched H3K9me2, H3K9me3, HP1a; state A), while the expressed gene Ephrin shows the patterns typical for chromosome 4 active genes (dashed red box; upstream promoter regions, depleted for H1 and H4, state D; regions immediately downstream from TSSs, enriched for H3K4me2/3 and depleted of H3K9me2/3, state C; and regions across the body of the gene, enriched for H3K36me3, H3K9me3, HP1a and POF, state B). Silent gene zfh shows the chromatin pattern typical for genes under the regulation of Polycomb system, enriched in H3K27me3 and depleted of HP1a. doi:10.1371/journal.pgen.1002954.g001 of other chromosomes, chromosome 4 contains less chromatin in state A, which represents the classical heterochromatin enriched for H3K9me2, H3K9me3, HP1a, HP2, SU(VAR)3-7, and SU(VAR)3-9 (Figure 1A, panel 5). Mapping these chromatin states across the karyotype (Figure 1B and 1C) and at higher resolution across chromosome 4 (Figure 1D) suggests a distinct domain with a higher gene (exon) density in the distal portion of chromosome 4. Interestingly, we do not see any evidence for euchromatic domains, as defined by depletion of H3K9me2/H3K9me3/HP1a and association with activation marks. Such domains had been suggested by the full expression of an hsp70-white transgene reporter (red eye phenotype) at certain sites [10,24]. Rather, we observe a strong correlation between these permissive sites and regions regulated by the Polycomb (PcG) system (Figure 1C and Figure S2). PcG regulated genes can be associated with a number of alternative chromatin states, including a repressive state (enriched for Polycomb [PC]), an active state (enriched for ASH1 and TRX), and a void state (lacking PC, ASH1, and TRX) [25]. The insertion sites of all red-eyed reporters correspond to four regions that in some cell types lack H3K9 methylation and HP1a, but contain H3K27me3 and PC. In contrast, none of the 24 hsp70-white reporter lines with a variegating eye phenotype are found in regions associated with PC in the cells and tissues examined to date (Figure S2B). While it is unknown which PcG chromatin state is present at the insertion sites of red-eyed reporter lines in the developing eye, our results confirm that HP1a and PC occupy separate domains on chromosome 4 and suggest that the domains regulated by the Polycomb system are transcription- permissive for hsp70-white reporters in the critical cell type. In contrast, the bulk of the genes on chromosome 4 are associated with HP1a, a well-established mark of heterochromatin, correlated with silencing. To explore how these genes function, we looked further at the distribution of chromosomal proteins associated with these genes. Active genes on chromosome 4 are characterized by a distinct combination of POF, H3K36me3, HP1a, and H3K9me2/3 Previous work by us and by others has indicated that HP1a correlates well with H3K9me2 and H3K9me3 in pericentric heterochromatin [14,15]. However, H3K9me2 and H3K9me3 have distinct distributions on chromosome 4 (Figure 1A, compare states A–E), leading us to re-examine the correlation of these marks as well as a few others in chromosome 4 and pericentric heterochromatin. While pericentric heterochromatin maintains the expected association among silencing marks, we find that HP1a and H3K9me3 correlate positively with active marks POF and H3K36me3 on chromosome 4 (Figure 2). Other marks associated with silencing (H3K9me2, SU(VAR)3-9, SU(VAR)3-7, and HP2) show little or no correlation on the chromosome 4. Given that chromosome 4 is distinguished from pericentric heterochromatin by its higher gene density, we hypothesized that the change in the correlation patterns is related to genes specifically. Thus, we examined the ‘‘metagene’’ profiles for active and silent genes on chromosome 4, within pericentric heterochro- matin, and in euchromatin. (Active and silent genes were defined by RNA-seq data, as described in Materials and Methods.) Indeed, the correlated histone modifications and proteins noted above map together only on chromosome 4, enriched over the body of active genes, in contrast to what is observed at other active loci (Figure 3, results from BG3 cells; see Figure S3 for data from S2 cells). This difference is not due to the relatively small number of genes present on chromosome 4, but is also seen when the same number of genes are compared for chromosome 4, heterochromatin, and euchromatin (metagenes in Figure S4 and S5, heatmaps in Figure S6). H3K9me2 is the only mark on chromosome 4 preferentially associated with repressed gene bodies. The high levels of POF and HP1a associated with transcribed genes on chromosome 4 confirm prior findings by Johannson and colleagues [17]. The enrichment of H3K9me3 in these regions of active transcription is unexpected and suggests a unique mechanism regulating H3K9 methylation on chromosome 4. Chromosome 4 genes rarely display RNA polymerase pausing As previously reported, silencing marks are depleted at the TSSs [15]. Figure 3 compares the chromatin composition at the TSS and the gene body for chromosome 4 genes. The distinctive enrichment patterns observed for TSSs and gene bodies suggested a possible role for this chromatin structure in regulation at the TSS. Given the anticipated difficulty in transcribing through a region with HP1a and H3K9me3, we considered changes in polymerase dynamics, such as pausing, to be likely affected. For a significant number of active genes, RNA pol II initiates transcription but pauses after 25–50 nt, remaining there until pausing is relieved. We investigated polymerase association with genes and polymerase pausing on chromosome 4 using global run- on followed by sequencing (GRO-seq) with data from S2 cells produced by Larschan and colleagues [26]. First, we compared the association of polymerase with genes in euchromatin, pericentric heterochromatin, and chromosome 4. RNA-seq data derived from steady state mRNA revealed that, while pericentric heterochro- matin has a lower gene density, the fraction of active genes is roughly the same between heterochromatin (pericentric hetero- chromatin and chromosome 4) and euchromatin (54% vs. 52% in PLOS Genetics | www.plosgenetics.org 4 September 2012 | Volume 8 | Issue 9 | e1002954