Drosophila Chromosome 4 Chromatin Structure Figure 6. Lack of POF leads to large-scale changes in HP1a and H3K9me2/3 and demonstrates that HP1a on chromosome 4 consists of POF-dependent and -independent pools. A. Mutations in POF alter H3K9me2, H3K9me3 and HP1a enrichment on chromosome 4. Enrichment levels (M-values) are shown for H3K9me2, H3K9me3, and HP1a on chromosome 4 (left) and in pericentric heterochromatin (right) in wildtype (dark color) and pof D119 homozygous mutant (light color) third instar larvae. Error bars: Standard error of the mean (SEM). B. Browser shots illustrating the loss of HP1a on chromosome 4 (top) in pof D119 homozygous mutant third instar larvae and the retention of high levels of HP1a in pericentric heterochromatin (bottom panel). The M-value scale (Y-axis) is identical for wildtype and mutant but differs between marks (0–3 for H3K9me2 and H3K9me3; 0–4 for HP1a). C. Metagene plots showing H3K9me3 and H3K9me2 levels are reduced mainly over active genes on chromosome 4 in the mutant. Genes on chromosome 4 were divided into transcriptionally active (left column) and transcriptionally silent (right) based on RNA-seq data. D. The changes in H3K9me2, H3K9me3, and HP1a enrichment induced by the pof mutation correlate with gene features on chromosome 4. Changes in H3K9me2/me3 and HP1a enrichment (Y-axis: smoothed M-values) are examined separately for TSSs of actively transcribed genes, gene bodies of active genes, and silent regions on chromosome 4. Error bars: SEM. doi:10.1371/journal.pgen.1002954.g006 S15A, p,0.001). (Note that our assay is restricted to uniquely mapping sequences and does not score identical repeats. Due to the incomplete genome assembly, it is formally possible that some of the sequences attributed to chromosome 4 also exist in the unassembled portions of the Drosophila genome.) This finding indicates that there are two mechanisms recruiting HP1a to chromosome 4: the majority of the HP1a recruitment is POF- dependent, but a significant amount of HP1a recruitment is targeted to repeat clusters, and this recruitment is independent of POF. H3K9me2 and H3K9me3 levels are reduced on the transcribed genes of chromosome 4 in pof D119 mutants ChIP-chip data also reveal abnormal H3K9 methylation patterns in pof mutant larvae on chromosome 4 (Figure 6 and Table S7). In wildtype, H3K9me2 and H3K9me3 are enriched along chromosome 4, with H3K9me3 enriched most strongly over transcribed gene bodies, mimicking POF and HP1a. In pof mutants, the extent of the H3K9me2 and H3K9me3 enriched domains on chromosome 4 is reduced chromosome-wide by 40% and 59%, respectively, regardless of wildtype POF enrichment levels in these domains (POF+/POF2; Table S7). The enrichment levels of H3K9me2 and H3K9me3 on chromosome 4 are decreased by 36% and 40%, respectively (Figure 6A), whereas in pericentric heterochromatin they are slightly increased in the pof mutant (Figure 6A). H3K9me2 and H3K9me3 enrichment levels are significantly decreased in actively transcribed regions (37% and 49% reductions), with little change observed in silent regions (p.0.05, Figure 6C and 6D). These results indicate that POF has a positive effect on H3K9me2/3 enrichment only on chromosome 4 and specifically in transcribed domains. The regions of H3K9me2/me3 enrichment that remain in the pof mutant correlate well with the remaining HP1a enrichment regions, with 82.8% of the HP1a enriched sequences found embedded in H3K9me2 regions, and 98% of the HP1a enriched regions overlapping with H3K9me3 regions. This positive correlation resembles that observed in pericentric heterochromatin; one now sees higher levels of HP1a, H3K9me2, and H3K9me3 over the intergenic and silent gene regions. The overlap suggests that the remaining H3K9 methylation might serve as a ‘‘seed’’ to recruit the residual HP1a observed in repeat-rich regions of chromosome 4, but that the recruitment of HP1a to the body of active genes requires POF. Alternatively, it is possible that HP1a is directly recruited to repetitive sequences, and can then recruit the PLOS Genetics | www.plosgenetics.org 10 September 2012 | Volume 8 | Issue 9 | e1002954